Antimicrobial properties of phenolic compounds from berries

R. Puupponen-Pimia
1
, L. Nohynek
1
, C. Meier
1
, M. Ka hko nen
2
, M. Heinonen
2
,
A. Hopia
2
and K.-M. Oksman-Caldentey
1
1
VTT Biotechnology, and
2
University of Helsinki, Department of Applied Chemistry and Microbiology, Food
Chemistry Division, University of Helsinki, Finland
540/9/00: received 6 September 2000, revised 22 November 2000 and accepted 28 November 2000
R. PUUPPONEN- PI MI A

, L. NOHYNEK, C. MEI ER, M. KA

HKO

NEN, M. HEI NONEN, A. HOPI A AND

K. - M. OKSMAN- CALDENTEY. 2001.
Aims: To investigate the antimicrobial properties of phenolic compounds present in Finnish
berries against probiotic bacteria and other intestinal bacteria, including pathogenic species.
Methods and Results: Antimicrobial activity of pure phenolic compounds representing
avonoids and phenolic acids, and eight extracts from common Finnish berries, was measured
against selected Gram-positive and Gram-negative bacterial species, including probiotic
bacteria and the intestinal pathogen Salmonella. Antimicrobial activity was screened by an agar
diffusion method and bacterial growth was measured in liquid culture as a more accurate assay.
Myricetin inhibited the growth of all lactic acid bacteria derived from the human
gastrointestinal tract ora but it did not affect the Salmonella strain. In general, berry extracts
inhibited the growth of Gram-negative but not Gram-positive bacteria. These variations may
reect differences in cell surface structures between Gram-negative and Gram-positive
bacteria. Cloudberry, raspberry and strawberry extracts were strong inhibitors of Salmonella.
Sea buckthorn berry and blackcurrant showed the least activity against Gram-negative bacteria.
Conclusions: Different bacterial species exhibit different sensitivities towards phenolics.
Signicance and Impact of the Study: These properties can be utilized in functional food
development and in food preservative purposes.
I NTRODUCTI ON
Flavonoids are common substances in the daily diet. These
polyphenolic compounds are widely found in various types
of edible plants, especially in vegetables, fruits, tea and wine.
Over 4000 different avonoids have been described and they
are categorized into several subgroups. Flavonols (quercetin,
myricetin and kaempferol) and avones (apigenin and
luteolin) are the most common phenolics in plant-based
foods. Flavanones are typically present in citrus fruit, and
avanols in green tea. Berries, which are traditionally a part
of the Finnish diet, are good sources of avonols while the
predominating group of avonoids, especially in red berries,
is anthocyanins (Fig. 1) (Heinonen et al. 1998; Hakkinen
et al. 1999a).
Dietary avonoids have attracted much interest recently
because in vitro and in vivo studies suggest that they have a
variety of benecial biological properties, which may play an
important role in the maintenance of human health. The
avonoids are potent antioxidants, free radical scavengers
and metal chelators; they inhibit lipid peroxidation
and exhibit various physiological activities including
anti-inammatory, antiallergic, anticarcinogenic, antihyper-
tensive, antiarthritic and antimicrobial activities. Epidemi-
ological studies have indicated that high avonoid
consumption is associated with reduced risk of chronic
diseases like cardiovascular diseases (Middleton and Kan-
daswami 1994; Hertog et al. 1995). However, little is known
about the absorption and subsequent distribution, metabo-
lism and excretion of avonoids in humans.
The intestinal microora consists of approximately 10
14
bacteria of over 400 species. This microora and its
consistency apparently play central roles in the metabolism
and bioavailability of plant phenols such as avonoids, the
importance of which is largely unknown so far. By
increasing the number and activity of probiotic bacteria
Correspondence to: Dr Riitta Puupponen-Pimia, VTT Biotechnology, PO Box
1500 (Tietotie 2), FIN-02044 VTT, Finland (e-mail: riitta.puupponen-
pimia@vtt.).
2001 The Society for Applied Microbiology
Journal of Applied Microbiology 2001, 90, 494507
Apigenin
Luteolin
Kaempferol
Quercetin
Myricetin
Rutin
Isoquercitrin
Naringenin
Hesperetin
(+) Catechin
(-) Epigallocatechin
Pelargonidin
Cyanidin
Delphinidin
Peonidin
Malvidin
Kuromanin
Flavones
Flavonols
Flavanones
Flavanols (Catechins)
Anthocyanidins
R
1
R
2
H
OH
H
OH
OH
H
OH
H
OH
OH
OH
H
OH
OH
OCH
3
OCH
3
OH
H
H
H
H
OH
OH
H
OH
OCH
3
H
OH
H
H
OH
H
OCH
3
H
Cinnamic acid
3-Coumaric acid
Caffeic acid
Ferulic acid
H
H
H
H
H
OH
OH
OH
H
H
OH
OCH
3
R
1
R
1
R
2
R
1
R
2
R
1
R
2
R
1
R
2
R
2
R
3
HO
O
O OH
R
1
R
2
OH
5
7
4
2
3
4'
HO
O
O HO
OH
4
2
3
R
3
HO
O
O HO
4
2
3
HO
O
HO
4
2
3
OH
OH
HO
O
HO
R
3
OH
+
COOH
5'
6'
R
1
R
2
R
1
R
2
R
1
R
2
R
1
R
2
R
1
R
2
R
3
Chlorogenic acid
HO
OH
OH
O
HO
H
H
H
O
OH
OH
O
A C
B
Phenolic acids
H
H
H
rutinose
-D-glucose
R
3
H
H
H
H
H
glucose
R
3
Fig. 1 Phenolic compounds present in edible plants
ANTI MI CROBI AL PHENOLI C COMPOUNDS 495
2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 494507
such as Lactobacillus and Bidobacterium in the colon, it has
been possible to ease the symptoms of lactose intolerance, to
heal and prevent diarrhoeal diseases and to stimulate the
immune response (Salminen and Saxelin 1996). Interactions
between plant phenolics and probiotics and the gastrointes-
tinal ora remain poorly characterized. Many plant phenols
are known to possess antimicrobial properties, so they might
change the composition of the colonic ora in an unexpected
way. On the other hand, microbial glucosidases and
glucuronidases in the colon can possibly affect the bioactiv-
ity of glycosylated compounds by deconjugating or other-
wise modifying them.
The aim of this study was to investigate the antimicrobial
properties of phenolic compounds present in Finnish berries
against probiotic bacteria and other intestinal bacteria,
including pathogenic species. Such knowledge is important
for the development of health-promoting functional foods
containing both probiotic bacteria and plant material, such
as berries.
MATERI ALS AND METHODS
Bacterial strains and culture conditions
The bacterial strains used as test organisms are listed in
Table 1. Excluding Lactobacillus plantarum VTT E-78076,
all the other Lactobacillus strains have potential probiotic
properties; most of the strains originate from the human
gastrointestinal tract. Escherichia coli strain CM 871 (trpE65,
uvrA155, recA56, lexA) is a DNA repair-decient derivative
from E. coli WP2 (Tweats et al. 1981). Salmonella enterica
sv. Typhimurium SH5014 is a rough mutant strain (Stocker
et al. 1979; LPS chemotype Rb2). The E. coli strains and
Typhimurium SH5014 strain represented Gram-negative
bacteria. All other strains were Gram-positive.
Lactobacillus strains, Enterococcus faecalis VTT E-93203
T
and Bidobacterium lactis VTT E-94508 were cultured in
MRS broth (de Man, Rogosa, Sharpe-Broth, Oxoid) or on
MRS agar (de Man, Rogosa, Sharpe-Agar, Oxoid). Cys-
teine (005% w/v) was added to MRS media for B. lactis.
Lactobacillus strains and B. lactis were grown anaerobically
at 37C, except Lact. plantarum VTT E-78076, which was
grown aerobically at 30C. Enterococcus faecalis was cul-
tured aerobically at 37C. Escherichia coli strains and
Typhimurium SH5014 were cultured aerobically at 37C
in Nutrient Broth (Difco) with agitation, or on Nutrient
Agar (Difco). Frozen stock cultures were maintained at
70C. Before experimental use, cultures were transferred to
solid or liquid media and incubated for 12 days. Cultures
were then subcultured in liquid media, incubated for 12
24 h, and used as the source of inoculum for each
experiment.
Chemicals
Apigenin, caffeic acid, (+)-catechin, chlorogenic acid,
3-coumaric acid, cyanidin chloride, delphinidin chloride,
ferulic acid, isoquercitrin, kaempferol, cyanidin-3-glucoside
(kuromanin), luteolin, myricetin, pelargonidin chloride,
quercetin dihydrate, rutin trihydrate and trans-cinnamic
acid (Fig. 1) were purchased from Extrasynthese. Folin-
Ciocalteu's phenol reagent and sodium carbonate were from
Merck. All organic solvents were of HPLC grade.
Preparation and analysis of phenolic berry
extracts
Phenolic extracts from blueberry (Vaccinium myrtillus),
raspberry (Rubus idaeus, var. Ottawa), lingonberry (Vaccini-
um vitis-idaea), blackcurrant (Ribes nigrum, var. O

jeby),
Table 1 Bacterial strains used in the study
Bacterial strains and catalogue numbers Origin Received from
Bidobacterium lactis VTT-E-94508 (Bb-12) Milk product B. Grenov, Chr., Hansen, Denmark
Enterococcus faecalis VTT E-93203
T
(DSM 20478) Adult intestine
Escherichia coli 50 VTT E-94564
T
(ATCC 11775) Urine of cystitis patient
E. coli CM 871 B. A. Bridge, England
Lactobacillus crispatus VTT E-96725 (M247) Human faeces L. Morelli, UCSC, Italy
Lactobacillus johnsonii VTT E-97797 (LJ1) Human faeces S. Blum, Nestle Research Center, Switzerland
Lactobacillus paracasei F19 VTT E-94510 (LMG P-17806) Human small intestine R. Fonden, Arla R & D, Sweden
Lactobacillus plantarum VTT E-78076 British beer
Lactobacillus reuteri VTT E-97849 (ATCC 55730) Breast milk I. Casas, Biogaia, Biologics, USA
Lactobacillus rhamnosus VTT E-97800 Human faeces Own isolation, VTT
Lact. rhamnosus GG VTT E-96666 (ATCC 53103) Human faeces
Salmonella enterica ser. Typhimurium SH5014 I. Helander, VTT
ATCC: American Type Culture Collection (USA); DSM: DSM2-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Germany).
496 R. PUUPPONEN- PI MI A

ET AL.
2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 494507
cloudberry (Rubus chamaemorus), cranberry (Vaccinium oxy-
coccus), sea buckthorn berry (Hippophae rhamnoides) and
strawberry (Fragaria ananassa Senga Sengana) were pre-
pared according to Kahkonen et al. 1999. Briey, ground
lyophilized berry material (500 mg) was weighed into a
centrifuge tube, 10 ml of aqueous 70% acetone were added
and the sample was homogenized (Ultra-Turrax) for 1 min.
Tubes were centrifuged (3000 g, 15 min) and the clear
supernatant uid was collected. The procedure was repeated
with another 10 ml of solvent. The supernatant uids were
combined and taken to dryness. The solid residue was
dissolved in water. Solid-phase extraction (SPE) was used to
remove sugars in the berry extracts (Heinonen et al. 1998)
prior to lyophilization of the extract.
The berry extracts were analysed for total phenolics
spectrophotometrically (Lambda Bio-UV/VIS spectropho-
tometer, Perkin Elmer, Germany) by the FolinCiocalteau
procedure (Kahkonen et al. 1999). The total phenolic
content was expressed as gallic acid equivalents (GAE) in
milligrams per gram of dry material. The phenolic compo-
sition of the berry extracts was analysed by HPLC as
modied from Lamuela-Raventos and Waterhouse (1994).
The HPLC system consisted of three Waters 501-series
pumps, a Waters Pump Control Module, a Waters WISP
700 autosampler equipped with a cooling device (Waters,
Milford, MA), column oven (Merck T-6300) and a Waters
PDA 996 diode array detector, all controlled by a Millen-
nium computer programme. The column was a Nova-Pak
C-18 (39 150 mm, particle size 4 lm) from Waters with a
precolumn (Spherisorb S5 ODS 22 20 mm, particle size
5 lm) housed at 40C. The gradient programme consisted
of 50 mmol l
1
dihydrogen ammonium phosphate adjusted
to pH 26 with orthophosphoric acid (solvent A), 20%
solvent A with 80% acetonitrile (solvent B) and 02 mol l
1
orthophosphoric acid adjusted with ammonia to pH 15
(solvent C) (Table 2).
The phenolic compounds were divided into four classes,
identied according to their spectral properties, and quan-
tied as follows: avan-3-ols as catechin equivalents at
280 nm, hydroxycinnamates as caffeic acid equivalents at
320 nm, avonols as rutin at 365 nm, and anthocyanins as
cyanidin-3-glucoside equivalents at 520 nm.
Tests for antimicrobial activity
Two methods were used to investigate antimicrobial effects
of phenolic compounds: the agar diffusion assay with a soft
agar overlay and measurement of bacterial growth in liquid
culture. For agar diffusion assays, all reagents and extracts
were dissolved in methanol. Lyophilized berry extracts were
used as such in liquid culture experiments.
Agar diffusion assay. Soft agar containing 45 mg ml
1
Bacto-agar (Difco) in liquid growth medium was cooled to
42C after autoclaving, inoculated with liquid overnight
culture to a cell density of 5 10
5
cfu ml
1
, and plates
containing 20 ml of agar media were overlaid with 5 ml of
this inoculated soft agar. After boring wells (7 mm in
diameter) in the agar, the plates were kept at room
temperature for 30 min and then at 4C for 25 h to allow
liquid discharge into wells. The pure test compounds and
the berry extracts in different concentrations (see Tables 4
and 5) were dissolved in methanol and pipetted into the agar
wells (50 ll). Apigenin, luteolin and cyanidin-3-glucoside
were tested at lower concentrations owing to the poor
solubility of these substances in methanol. Polymyxin B
sulphate (90 lg in 50 ll) was used as a positive control for
Typhimurium, and streptomycin (150 and 600 lg in 50 ll)
for all other strains. The negative control was methanol (50
ll). The diameter of the inhibition zone was measured at
24 h and 48 h. All determinations were made in triplicate.
Bacterial growth curve measurement. In liquid culture
experiments, 10 ml of fresh growth medium were inoculated
with 1 or 5% of overnight culture. Lyophilized berry
extracts or pure phenolic compounds as such were added to
the culture media to give nal concentrations of 05, 1 or
5 mg ml
1
. The cultures were shaken well and incubated as
described above. Bacterial growth was followed by taking
samples from the cultures 56 times during an incubation
period of 934 h, depending on the strain's growth rate. The
samples were diluted with peptone saline (Maximal Recov-
ery Diluent, Lab M) and the proper dilutions were plated.
The plates were incubated as above and the bacterial counts
were recorded. The inhibitory (or stimulatory) effects of test
compounds on the bacteria were measured by comparing the
control growth curves with those obtained from cultures
with berry extracts or pure phenolic compounds.
Table 2 The HPLC gradient programme for phenolic composi-
tion analysis
Time (min) Solvent A (%) Solvent B (%) Solvent C (%)
000 100 0 0
500 100 0 0
1500 96 4 0
2500 92 8 0
2501 0 8 92
4500 0 20 80
5000 0 30 70
5500 0 40 60
6500 0 80 20
7000 0 80 20
7500 100 0 0
ANTI MI CROBI AL PHENOLI C COMPOUNDS 497
2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 494507
RESULTS
Phenolic composition of the berry extracts
The amounts of total phenolics and the composition of the
phenolic compounds (expressed as mg g
1
phenolic extract)
in berry extracts are shown in Table 3. The spectrophoto-
metric total phenolic measurement resulted in higher
amounts of phenolics than the HPLC analysis of the four
phenolic subclasses. This could be partly due to dissimilar
responses of different phenolic compounds in the Folin
Ciocalteu procedure, but it also indicates the presence of
unidentied phenolic compounds in the extracts, for
example, benzoic acids and tannins. Especially in cloudberry,
raspberry and strawberry, only 10% or less of the total
phenolics were identied as anthocyanins, avonols,
hydroxy-cinnamates or avanols. In the blueberry extract,
more than 80% of total phenolics belonged to the above-
mentioned groups, anthocyanins being the major group.
Antimicrobial activity by the agar diffusion method
The antimicrobial activity of 17 pure phenolic compounds
representing 13 avonoids and four phenolic acids, and, in
addition, eight berry extracts, was rst measured by agar
diffusion, which is suitable for semi-quantitative estimation.
Due to the scarcity of material, the antimicrobial properties
of cranberry, cloudberry and sea buckthorn berry extracts
were only tested against the most interesting bacterial
strains, especially Gram-negative bacteria. The results are
presented in Table 4 for pure compounds and in Table 5 for
berry extracts.
Sensitivity to the compounds was found to differ signi-
cantly among the test organisms. Lactic acid bacteria
(LAB), in general, were more resistant than the other
bacteria to pure phenolic compounds. Escherichia coli CM
871 exhibited marked sensitivity to phenolic compounds.
Myricetin was the only compound which showed strong
inhibitory effects on the growth of all LAB strains of human
origin. Lactobacillus plantarum, a beer isolate, was resistant
to myricetin at all test concentrations, as well as to all other
pure phenolic compounds. Also, growth of E. coli strains
was inhibited by myricetin. Myricetin seemed to retard the
growth of both Ent. faecalis and B. lactis, although no clear-
cut inhibition zones in the agar diffusion assay were
detected. Typhimurium was not affected by myricetin.
The phenolic acids (cinnamic acid, 3-coumaric acid, caffeic
acid, ferulic acid and chlorogenic acid) showed activity only
against Gram-negative bacteria at high concentrations (500
lg well
1
). Chlorogenic acid was the weakest inhibitor,
exhibiting activity only against E. coli CM 871.
Luteolin was slightly inhibitory to Gram-positive LAB
but not to Gram-negative bacteria. No clear-cut inhibition
zones were detected, but the density of bacteria (cfu) in agar
plates containing luteolin was signicantly less than in
control cultures, indicating weak bacteriostatic effects.
The anthocyanidins pelargonidin, delphinidin and cyani-
din, as well as cyanidin-3-glucoside, only inhibited growth of
E. coli CM 871 and had no effect on other bacterial strains.
All berry extracts possessed strong antimicrobial activity
against Gram-negative bacteria. At higher concentrations,
raspberry, cloudberry and blueberry extracts also inhibited
the growth of some LAB strains. Raspberry exhibited the
strongest antimicrobial activity, followed by cloudberry and
blueberry. Sea buckthorn berry and blackcurrant showed the
least antimicrobial activity. Among the seven LAB species
tested, Lact. rhamnosus VTT E-97800 and GG VTT
E-96666 were the most sensitive to the berry extracts, while
Lact. johnsonii and Lact. crispatus were the least sensitive.
Strawberry extract was active against Ent. faecalis. All berry
extracts except sea buckthorn berry and blackcurrant
showed antimicrobial activity against Typhimurium.
Bacterial growth curve measurement
For more accurate determination of antimicrobial activity,
liquid culture experiments were performed. The antimi-
crobial activity of selected pure phenolic compounds and
eight berry extracts on bacterial strains was studied by
measuring the differences in bacterial growth curves in
liquid cultures fortied with the test compounds. The
Berry extract Anthocyanins Flavonols OH-cinnamates Flavan-3-ols Total phenolics
Blackcurrant 106 10 7 7 410
Blueberry 260 6 23 5 360
Cranberry 117 59 43 ND 330
Cloudberry 09 8 10 1 470
Lingonberry 23 10 6 6 280
Raspberry 24 2 4 3 470
Sea buckthorn berry ND 35 2 3 230
Strawberry 34 3 8 1 460
ND, not detected.
Table 3 Phenolic composition of berry
extracts
498 R. PUUPPONEN- PI MI A

ET AL.
2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 494507
Table 4 Antimicrobial activity of pure phenolic compounds by the agar diffusion method. ( ) No inhibition, ( ) no clear inhibition, but bacteriostatic
effects
A
N
T
I
M
I
C
R
O
B
I
A
L
P
H
E
N
O
L
I
C
C
O
M
P
O
U
N
D
S
4
9
9

2
0
0
1
T
h
e
S
o
c
i
e
t
y
f
o
r
A
p
p
l
i
e
d
M
i
c
r
o
b
i
o
l
o
g
y
,
J
o
u
r
n
a
l
o
f
A
p
p
l
i
e
d
M
i
c
r
o
b
i
o
l
o
g
y
,
9
0
,
4
9
4

5
0
7
Table 4 (Contd.)
5
0
0
R
.
P
U
U
P
P
O
N
E
N
-
P
I
M
I
A

E
T
A
L
.

2
0
0
1
T
h
e
S
o
c
i
e
t
y
f
o
r
A
p
p
l
i
e
d
M
i
c
r
o
b
i
o
l
o
g
y
,
J
o
u
r
n
a
l
o
f
A
p
p
l
i
e
d
M
i
c
r
o
b
i
o
l
o
g
y
,
9
0
,
4
9
4

5
0
7
Table 5 Antimicrobial activity of berry extracts by the agar diffusion method. ( ) No inhibition, ( ) no clear inhibition, but bacteriostatic effects, (h) not tested
A
N
T
I
M
I
C
R
O
B
I
A
L
P
H
E
N
O
L
I
C
C
O
M
P
O
U
N
D
S
5
0
1

2
0
0
1
T
h
e
S
o
c
i
e
t
y
f
o
r
A
p
p
l
i
e
d
M
i
c
r
o
b
i
o
l
o
g
y
,
J
o
u
r
n
a
l
o
f
A
p
p
l
i
e
d
M
i
c
r
o
b
i
o
l
o
g
y
,
9
0
,
4
9
4

5
0
7
Table 6 Antimicrobial activity of selected pure phenolic compounds and berry extracts in liquid culture. ( ) No inhibition: plate counts differ by <5 10
1
; ( ) clear
inhibition: plate counts differ by 5 10
1
5 10
2
; ( ) strong inhibition: plate counts differ by 5 10
2
5 10
4
; (j) very strong inhibition: plate counts differ by >5 10
4
; (h) not
tested
5
0
2
R
.
P
U
U
P
P
O
N
E
N
-
P
I
M
I
A

E
T
A
L
.

2
0
0
1
T
h
e
S
o
c
i
e
t
y
f
o
r
A
p
p
l
i
e
d
M
i
c
r
o
b
i
o
l
o
g
y
,
J
o
u
r
n
a
l
o
f
A
p
p
l
i
e
d
M
i
c
r
o
b
i
o
l
o
g
y
,
9
0
,
4
9
4

5
0
7
10
7
10
8
10
9
10
10
0 5 10 15 20 25 30
Incubation time (h)
c
f
u
m
l

1
c
f
u
m
l

1
c
f
u
m
l

1
c
f
u
m
l

1
c
f
u
m
l

1
c
f
u
m
l

1
10
7
10
8
10
9
10
10
0 10 20 30 40
Incubation time (h)
10
6
10
7
10
8
10
9
0 5 10 15 20 25 30
Incubation time (h)
10
6
10
7
10
8
10
9
10
10
0 2 4 6 8 10
Incubation time (h)
10
6
10
7
10
8
10
9
10
10
0 5 10 15 20 25 30
Incubation time (h)
10
5
10
6
10
7
10
8
10
9
0 5 10 15 20 25 30
Incubation time (h)
(a)
(b)
(c)
(e) (f)
(d)
ANTI MI CROBI AL PHENOLI C COMPOUNDS 503
2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 494507
results are presented in Table 6 and selected growth curves
are shown in Fig. 2.
In general, the results were in agreement with those
obtained in the agar diffusion experiments. The growth of
Lactobacillus strains was not inhibited by any of the berry
extracts at low concentrations (1 mg ml
1
). However, when
ve times higher concentrations of raspberry and blueberry
extracts were used, growth of tested Lactobacillus strains was
clearly inhibited by raspberry extract (Fig. 2a) and slightly
inhibited by blueberry extract (data not shown). Bidobac-
terium lactis was slightly inhibited by raspberry, strawberry
and cloudberry extracts (1 mg ml
1
), and Ent. faecalis by
blueberry, raspberry and strawberry extracts (Table 6).
Other extracts tested had no effects on these strains
(Fig. 2b, c).
Escherichia coli strain50 was sensitive to all phenolic extracts
except blackcurrant (Fig. 2d, Table 6). In the presence of
05 mg ml
1
or 10 mg ml
1
raspberry extract, the number of
viable cells remained approximately one logarithm lower than
in the control culture (Fig. 2e). Growth of the DNA repair
mutant strain E. coli CM 871 was strongly inhibited by all
eight berry extracts; in the presence of 05 mg ml
1
raspberry
extract, the number of viable cells decreased by two
logarithms, and in the presence of 10 mg ml
1
raspberry
extract, the number of viable E. coli CM 871 cells decreased
rapidly beneath the detection limit (10
5
cfu ml
1
) (Fig. 2f).
Growth of Typhimuriumwas totally inhibited by cloudberry,
raspberry and strawberry extracts (1 mg ml
1
). Cloudberry
extract exerted the strongest inhibitory effect, as indicated by
the rapid inhibition of the Typhimurium culture in the
presence of cloudberry extract (Fig. 2g).
Fig. 2 Selected growth curves of test bacteria. (a) Lactobacillus
paracasei E-94510: () control growth curve, (j) blueberry
1 mg ml
1
, (h) 5 mg ml
1
, (m) raspberry 1 mg ml
1
, (D) 5 mg ml
1
.
(b) Bidobacterium lactis Bb-12: () control growth curve, (j)
blueberry 1 mg ml
1
, (s) lingonberry 1 mg ml
1
, () blackcurrent
1 mg ml
1
, (m) raspberry 1 mg ml
1
. (c) Enterococcus faecalis E-93203:
() control growth curve, (j) cranberry 1 mg ml
1
, (D) buckthorn
berry 1 mg ml
1
, () cloudberry 1 mg ml
1
. (d) Escherichia coli 50: ()
control growth curve, () cloudberry 1 mg ml
1
, (j) blueberry 1 g ml
1
,
(h) cranberry 1 mg ml
1
, (m) buckthorn berry 1 mg ml
1
, (D)
raspberry 1 mg ml
1
. (e) Escherichia coli 50: () control growth curve,
(D) raspberry 05 mg ml
1
, (m) raspberry 1 mg ml
1
. (f) Escherichia coli
CM 871: () control growth curve, (D) raspberry 05 mg ml
1
, (m)
raspberry 1 mg ml
1
. (g) Salmonella enterica sv. Typhimurium: ()
control growth curve, (j) cranberry 1 mg ml
1
, (m) buckthorn berry
1 mg ml
1
, () cloudberry 1 mg ml
1
. (h) Lactobacillus rhamnosus E-
96666: () control growth curve, (j) myricetin 05 mg ml
1
, (D) rutin
05 mg ml
1
, () catechin 05mg ml
1
, (s) chlorogenic acid 05 mg ml
1
.
(i) Escherichia coli CM 871: () control growth curve, (j) myricetin
05 mg ml
1
, (D) rutin 05 mg ml
1
, () catechin 05 mg ml
1
, (s)
chlorogenic acid 05 mg ml
1
b
10
4
10
5
10
6
10
7
10
8
10
9
0 2 4 6 8 10
Incubation time (h)
10
7
10
8
10
9
10
10
0 5 10 15 20 25 30
Incubation time (h)
10
6
10
7
10
8
10
9
0 5 10 15 20 25 30
Incubation time (h)
c
f
u
m
l

1
c
f
u
m
l

1
c
f
u
m
l

1
(g)
(h)
(i)
504 R. PUUPPONEN- PI MI A

ET AL.
2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 494507
In summary, in liquid culture, cloudberry showed the
strongest antimicrobial activity against Gram-negative bac-
teria. Raspberry exhibited almost similar activity, whereas
blackcurrant was least active (Table 6). The results are in
accord with those obtained in the agar diffusion assay,
although the strong inhibitory effects of cloudberry were
particularly evident in liquid cultures. Blackcurrant slightly
stimulated the growth of Lact. rhamnosus VTT E-97800
and GG VTT E-96666, and Lact. paracasei (data not
shown).
The pure phenolic compound myricetin (05 mg ml
1
)
slightly inhibited the growth of Lact. rhamnosus VTT E-
97800, Lact. rhamnosus GG VTT E-96666 (Fig. 2h), Ent.
faecalis and B. lactis, whereas E. coli CM 871 was strongly
inhibited (Fig. 2i) (Table 6). Lactobacillus rhamnosus VTT
E-97800 was also weakly inhibited by rutin, and Lact.
rhamnosus GG VTT E-96666 by quercetin (Table 6). The
results are in agreement with agar diffusion data, although
the inhibitory effects of myricetin were generally more
prominent in agar diffusion than in liquid cultures.
DI SCUSSI ON
Antimicrobial activity of 17 pure phenolic compounds
representing avonoids and phenolic acids, and eight berry
extracts, was measured against selected Gram-positive and
Gram-negative bacteria, including probiotic bacteria and an
intestinal pathogen. The results showed that different
bacterial species exhibit different sensitivities towards
phenolics. In addition, different strains of the same bacterial
species showed differences in sensitivity to one avonoid. In
general, berry extracts inhibited Gram-negative but not
Gram-positive bacteria. These variations may reect differ-
ences in cell surface structures between Gram-negative and
Gram-positive bacteria. In particular, the outer membrane
of Gram-negative bacteria functions as a preventive barrier
against hydrophobic compounds (Helander et al. 1998).
Among the Gram-negative test bacteria, the DNA repair
mutant strain E. coli CM 871 was particularly strongly
affected by phenolic compounds. This strain lacks the
repairing mechanism of DNA and is therefore expected to
be more sensitive than strain E. coli 50 to damage caused by
mutagenic agents. The latter strain represented in this study
a normal E. coli strain from the human gastrointestinal ora.
The antimicrobial effect of phenolics on E. coli CM 871 may
be an indication of the potential mutagenic activity of these
compounds. Anthocyanidins as a group of compounds, and
quercetin and chlorogenic acid as single compounds,
inhibited the growth of E. coli CM 871, with no inhibition
of E. coli 50. Therefore, it can be hypothesized that the main
reason for the antibacterial activity of these compounds is
their reaction with DNA. On the other hand, phenolic acids
showed activity against all Gram-negative test bacteria. In
these cases, other mechanisms are also apparently involved
in the antimicrobial action, genotoxicity being one (Stam-
mati et al. 1999). Helander et al. (1998) have studied the
action of plant-derived essential oil components on Gram-
negative bacteria. They found that small phenolic com-
pounds such as carvacrol and thymol inhibited E. coli and
Salmonella. Inhibitory effects involved the disruptive action
of these compounds on the outer membrane.
On the basis of the present results, the degree of
hydroxylation might affect the antimicrobial activity of pure
phenolic compounds. The avonol myricetin, as a pure
compound, clearly inhibited the growth of all LAB of
human gastrointestinal tract origin, as well as the Gram-
positive Ent. faecalis and B. lactis. The other avonols tested,
quercetin and kaempferol, are more lipophilic in nature (one
and two hydroxyl groups less in the B ring than in
myricetin, respectively), and they showed no inhibition
against the above bacteria. The avone luteolin was
bacteriostatic against some of the tested LAB as well as
against Ent. faecalis and B. lactis. No such effects were found
with the avone apigenin, which has one hydroxyl group less
in the B ring. The results showed that the number of
hydroxyl groups in the B ring in avonols and avones is
associated with the antimicrobial activity against LAB. No
other structureactivity relationship was found. Padmavati
et al. (1997) studied the antimicrobial effects of avonoids
on major rice pathogens. The tested avonoids differed in
their hydroxylation patterns in the B and C rings. They
showed, contrary to the present results, that the non-polar
avonoids were the most effective compounds, showing
appreciable inhibition of spore germination of Pyricularia
oryzea (the fungal blast pathogen). The surface of the
eukaryotic spore, however, differs considerably from the cell
wall of active bacterial cells, and may therefore affect the
response towards phenolic compounds. In the present
experiments, myricetin did not affect the growth of
Typhimurium and E. coli 50. Typhimurium is a rough-
type mutant with a truncated lipopolysaccharide component
of the outer membrane (Stocker et al. 1979). However, the
outer membrane of the mutant strain is not functionally
impaired.
The berry extracts mainly inhibited the growth of Gram-
negative bacteria but had no effect on Gram-positive
bacteria. The antimicrobial activities of the naturally-
occurring phenolics from olives, tea and wine have been
widely studied (Ruiz-Barba et al. 1990; Vivas et al. 1997;
Chou et al. 1999). However, there is very little information
about the antimicrobial capacity of phenolics present in
berries, except in cranberry. In our studies, cranberry and
blueberry extracts rich in anthocyanins inhibited Gram-
negative bacteria. The antibacterial properties of cranberry
juice have been known for a long time (Clague and Fellers
1934; Moen 1962; Kinney and Blount 1979), and the effect
ANTI MI CROBI AL PHENOLI C COMPOUNDS 505
2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 494507
may be associated with the inhibition of E. coli adherence to
mucosal surfaces (Sobota 1984; Schmidt and Sobota 1988);
Howell et al. (1998) recently suggested that proanthocyani-
dins (condensed tannins) were responsible for this. Similar
inhibitory activities were reported for blueberry juice by
Ofek et al. (1996). The present results agree with these
observations.
Similar to other berry extracts, lingonberry extract
showed activity only against Gram-negative bacteria. Annuk
et al. (1999) have studied the antimicrobial activity of
aqueous extracts of bearberry (Arctostaphylos uva-ursi (L.)
Spreng., Ericaceae) and cowberry (Vaccinium vitis-idaea L.,
Ericaceae) against the Gram-negative pathogen Helicobacter
pylori. Tannic acid seemed to be the responsible component.
According to Holopainen et al. (1988), extracts of the aerial
parts of bearberry and lingonberry were active against the
Gram-negatives E. coli and Proteus vulgaris. The activity is
known to be due to the phenolic glycosides arbutin and
metylarbutin. Lingonberries are also rich in benzoic acid, a
commonly used antimicrobial agent in foods.
In the liquid culture experiments, strawberry extract
strongly inhibited the growth of Typhimurium and E. coli
CM 871. Pratt et al. (1960) and Powers et al. (1960) studied
the antimicrobial effects of anthocyanins and anthocyanidins
from grapes and strawberries against several bacterial
strains. Delphinidin-3-monoglycoside, pelargonidin-
3-monoglycoside and malvidin-3,5-diglycoside were the
most effective compounds. They concluded that the gluco-
sides were hydrolysed and the aglycone was the active
fraction. Also, anthocyanin extracts from the leaves and
pericarp of the pigmented rice cultivar, Purpleputtu, showed
inhibition of the Gram-negative species Xanthomonas, and
the pigments have been characterized as cyanidin and
peonidin glycosides (Reddy et al. 1995). Anthocyanidins in
the agar diffusion experiments inhibited the growth of
mutant E. coli strain CM 871 but had no effect on the other
test bacteria. These results suggest that compounds other
than anthocyanidins in the strawberry extract are mainly
responsible for the inhibition of Typhimurium.
In the present study, cloudberry, raspberry and straw-
berry extracts were the strongest inhibitors of Gram-
negative bacteria, especially Typhimurium. Recently, Rauha
et al. (2000) studied the antimicrobial effects of several berry
extracts. They also found that the widest bactericidal
activity was expressed by berries belonging to the genus
Rubus (cloudberry and raspberry). Hakkinen et al. (1999a)
detected selected avonoids and phenolic acids from 19
berries by HPLC. They found that ellagic acid was the main
phenolic compound in the hydrolysed berry extracts of the
genera Rubus and Fragaria (strawberry). Ellagic acid is a
hydrolysis product from ellagitannins, which, together with
gallotannins, form the predominant group of tannins in
these berries; thus, it is not present in fresh berries or
unhydrolysed berry extracts (Macheix et al. 1990). Ellagic
acid has been reported to exhibit a dose-dependent inhib-
itory effect (IC
50
1 mmol l
1
) on Helicobacter pylori
isolated from peptic ulcer patients (Chung 1998). Also,
ellagitannin extracts inhibit a range of pathogenic organisms
including Vibrio cholerae, Shigella dysenteriae and Campylo-
bacter spp. (Scalbert 1991; Silva et al. 1997). It can be
hypothesized that ellagitannins could be one of the compo-
nents in cloudberries, raspberries and strawberries causing
the inhibition against Salmonella.
Hakkinen et al. (1999b) recently determined the contents of
the avonols quercetin, myricetin and kaempferol in 25 edible
berries. Cranberry and blueberry contained the highest
concentration of myricetin, 108 and 71 mg kg
1
, respectively.
Interestingly, the present results showed that although
myricetin itself was a strong inhibitor, berries rich in
myricetin were not. In addition, compared with the high
antimicrobial activity of berry extracts against Gram-negative
strains, these bacteria, excluding the mutant strain E. coli CM
871, were not inhibited by pure phenolic compounds. These
results suggest that the inhibitory effects of berry extracts may
not be due to simple phenolics but to more complex phenolic
polymers such as ellagitannins, tannins and proanthocyani-
dins. The antimicrobial activity of berry extracts is evidently a
synergistic effect of various phenolic compounds, many of
which are still unidentied. Also, other bioactive compounds
in plant extracts, alone or in combination with phenols, might
be responsible for the antimicrobial effects.
In conclusion, phenolic extracts of eight berries commonly
consumed in Finland inhibited the growth of selected
Gram-negative bacteria and were not active against Gram-
positive LAB. Cloudberry, raspberry and strawberry
extracts were strong inhibitors of the intestinal pathogen
Salm. enterica. The antimicrobial effects of berry extracts
against Gram-negative bacteria decreased in the following
order: cloudberry > raspberry > strawberry > lingonberry >
blueberry > cranberry > sea buckthorn berry > blackcur-
rant. In further investigations, fractionation of phenolic
berry extracts will be carried out in order to identify the
components responsible for antimicrobial activity. In addi-
tion, the possible role of berry seeds in antimicrobial action
will be studied. Antimicrobial effects of berry extracts
against a selection of Salmonella strains, and also against
other intestinal pathogens, are under investigation, and the
synergistic effects of plant phenols are being studied. The
results of these studies will be used in functional food
development and for food preservative purposes.
ACKNOWLEDGEMENTS
The authors thank Professor Veli Kauppinen for his ideas
and valuable advice. Professor Atte von Wright, and Drs
Maria Saarela and Ilkka Helander, are also thanked for
506 R. PUUPPONEN- PI MI A

ET AL.
2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 494507
useful discussions. The skilful technical assistance of Tuuli
Teikari and Niina Torttila is gratefully acknowledged. This
study was nancially supported by Tekes, the National
Technology Agency.
REFERENCES
Annuk, H., Hirmo, S., Turi, E., Mikelsaar, M., Arak, E. and
Wadstrom, T. (1999) Effect on cell surface hydrophobicity and
susceptibility of Helicobacter pylorii to medicinal plant extracts.
FEMS Microbiology Letters 172, 4145.
Chou, C.-C., Lin, L.-L. and Chung, K.-T. (1999) Antimicrobial
activity of tea as affected by the degree of fermentation and
manufacturing season. International Journal of Food Microbiology 48,
125130.
Chung, J.G. (1998) Inhibitory actions of ellagic acid on growth and
arylamine N-acetyltransferase activity in strains of Helicobacter pylori
from peptic ulcer patients. Microbios 93, 115127.
Clague, J.A. and Fellers, C.R. (1934) Relation of benzoic acid content
and other constituents of cranberries to keeping quality. Plant
Physiology 9, 631636.
Hakkinen, S.H., Heinonen, M., Karenlampi, S., Mykkanen, H.,
Ruuskanen, J. and Torronen, R. (1999a) Screening of selected
avonoids and phenolic acids in 19 berries. Food Research Interna-
tional 32, 345353.
Hakkinen, S.H., Karenlampi, S.O., Heinonen, I.M., Mykkanen, H.M.
and Torronen, A.R. (1999b) Content of the avonols quercetin,
myricetin, and kaempherol in 25 edible berries. Journal of Agricul-
tural and Food Chemistry 47, 22742279.
Heinonen, I.M., Meyer, A.S. and Frankel, E.N. (1998) Antioxidant
activity of berry phenolics on human low-density lipoprotein and
liposome oxidation. Journal of Agricultural and Food Chemistry 46,
41074112.
Helander, L.M., Alakomi, H.-L., Latva-Kala, K. et al. (1998)
Characterization of the action of selected essential oil components
on Gram-negative bacteria. Journal of Agricultural and Food
Chemistry 46, 35903595.
Hertog, M.G.L., Kromhout, D., Aravanis, C. et al. (1995) Flavonoid
intake and long-term risk of coronary heart disease and cancer in
seven countries study. Archives of Internal Medicine 155, 381386.
Holopainen, M., Jahodar, L., Seppanen-Laakso, T., Laakso, I. and
Kauppinen, V. (1988) Antimicrobial activity of some Finnish
Ericaceous plants. Acta Pharmaceutica Fennica 97, 197202.
Howell, A.B., Vorsa, N., Marderosian, A.D. and Foo, L.Y. (1998)
Inhibition of the adherence of P-mbriated Escherichia coli to uro-
epithelial surfaces by proanthycyanidin extracts from cranberries.
New England Journal of Medicine 339, 10851086.
Kahkonen, M.P., Hopia, A.I., Vuorela, H.J. et al. (1999) Antioxidant
activity of plant extracts containing phenolic compounds. Journal of
Agricultural and Food Chemistry 47, 39543962.
Kinney, A.B. and Blount, M. (1979) Effect of cranberry juice on
urinary pH. Nursing Research 28, 287290.
Lamuela-Raventos, R.M. and Waterhouse, A.L. (1994) A direct HPLC
separation of wine phenolics. American Journal of Enology and
Viticulture 45, 15.
Macheix, J.-J., Fleuriet, A. and Billot, J. (1990) Fruit phenolics. CRC
press, Boca Raton, FL.
Middleton, E. Jr and Kandaswami, C. (1994) The impact of plant
avonoids on mammalian biology: implications for immunity,
inammation and cancer. In The Flavonoids. Advances in Research
Since 1986 ed. Harborne, J.B. pp. 619652. London: Chapman &Hall.
Moen, D.V. (1962) Observations of the effectiveness of cranberry juice
in urinary infections. Wisconsin Medical Journal 61, 282283.
Ofek, I., Goldhar, J. and Sharon, N. (1996) Anti-Escherichia coli
adhesin activity of cranberry and blueberry juices. In Toward Anti-
Adhesion Therapy for Microbial Diseases ed. Kahane, I. and Ofek, I.
pp. 179183. New York: Plenum Press.
Padmavati, M., Sakthivel, N., Thara, K.V. and Reddy, A.R. (1997)
Differential sensitivity of rice pathogens to growth inhibition by
avonoids. Phytochemistry 46, 499502.
Powers, J.J., Somaatmadja, D., Pratt, D.E. and Hamdy, M.K. (1960)
Anthocyanins. II. Action of anthocyanin pigments and related
compounds on the growth of certain microorganisms. Food
Technology 14, 626632.
Pratt, D.E., Powers, J.J. and Somaatmadja, D. (1960) Anthocyanins. I.
The inuence of strawberry and grape anthocyanins on the growth
of certain bacteria. Food Research 25, 2632.
Rauha, J.-P., Remes, S., Heinonen, M. et al. (2000) Antimicrobial
effects of Finnish plant extracts containing avonoids and other
phenolic compounds. International Journal of Food Microbiology 56,
312.
Reddy, V.S., Dash, S. and Reddy, A.R. (1995) Anthocyanin pathway in
rice (Oryza sativa L.): identication of a mutant showing dominant
inhibition of anthocyanins in leaf and accumulation of proanthocy-
anidins in pericarp. Theoretical and Applied Genetics 91, 301312.
Ruiz-Barba, J.L., Rios-Sanchez, R.M., Fedriani-Iriso, C., Olias, J.M.,
Rios, J.L. and Jimenez-Diaz, R. (1990) Bactericidal effect of phenolic
compounds from green olives on Lactobacillus plantarum. Systematic
and Applied Microbiology 13, 199205.
Salminen, S. and Saxelin, M. (1996) Comparison of successful
probiotic strains. Nutrition Today 31 (Suppl. 1), 32S34S.
Scalbert, A. (1991) Antimicrobial properties of tannins. Phytochemistry
30, 38753883.
Schmidt, D.R. and Sobota, A.E. (1988) An examination of the anti-
adherence activity of cranberry juice on urinary and nonurinary
bacterial isolates. Microbios 55, 173181.
Silva, O., Duarte, A., Pimentel, M. et al. (1997) Antimicrobial activity of
Terminalia macroptera root. Journal of Ethnopharmacology 57, 203207.
Sobota, A.E. (1984) Inhibition of bacterial adherence by cranberry
juice: potential use for the treatment of urinary tract infections.
Journal of Urology 131, 10131016.
Stammati, A., Bonsi, P., Zucco, F., Moezelaar, R., Alakomi, H.-L. and
von Wright, A. (1999) Toxicity of selected plant volatiles in
microbial and mammalian short-term assays. Food and Chemical
Toxicology 37, 813823.
Stocker, B.A.D., Nurminen, M. and Makela, P.H. (1979) Mutants
defective in the 33-K outer membrane protein of Salmonella
typhimurium. Journal of Bacteriology 139, 376383.
Tweats, D.J., Green, M.H.L. and Muriel, W.J. (1981) A differential
killing assay for mutagens and carcinogens based on an improved
repair-decient strain of Escherichia coli. Carcinogenesis 2, 189194.
Vivas, N., Lonvaud-Funel, A. and Glories, Y. (1997) Effects of
phenolic acids and anthocyanins on growth, viability and malolactic
activity of a lactic acid bacterium. Food Microbiology 14, 291300.
ANTI MI CROBI AL PHENOLI C COMPOUNDS 507
2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 494507