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Process Biochemistry 39 (2004) 19391946

Antimicrobial activity against select food-borne pathogens by phenolic


antioxidants enriched in cranberry pomace by solid-state bioprocessing
using the food grade fungus Rhizopus oligosporus
D.A. Vattem, Y.-T. Lin, R.G. Labbe, K. Shetty
Laboratory of Food Biotechnology, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA
Received 4 August 2003; accepted 19 September 2003

Abstract
Listeria monocytogenes, Vibrio parahaemolyticus and Escherichia coli 0157:H7 are important food-borne pathogens. Control of these
pathogens using synthetic antimicrobials such as currently approved antibiotics is challenging due to potential development of resistance. A
profile of antimicrobials compared to a single compound could be potentially more effective. Cranberry pomace is a byproduct of the cranberry
processing industry. Solid-state bioprocessing of cranberry pomace using the food grade fungus Rhizopus oligosporus improves phenolic and
antioxidant profiles. We hypothesize that these phytochemicals mobilized during pomace bioprocessing could improve the antimicrobial functionality. The objective of this research was to use the food grade fungus R. oligosporus to release phenolic aglycones and relate its antioxidant
functionality and diphenyl mobilization to antimicrobial activity against L. monocytogenes, V. parahaemolyticus and E. coli 0157:H7. Bioprocessing of pomace was done for 20 days with R. oligosporus. Total phenolics in water extracts were assayed using a Folin-Ciocalteu method.
The antioxidant potential was measured using a -carotene oxidation system (APF) and 1,1-diphenyl-2-picrylhydrazyl-radical (DPPH) system. Changes in phenolic profiles were analyzed using HPLC. Antimicrobial activities of the extracts during growth were tested using standard
disk assays. Soluble phenolics, antioxidant activity and ellagic acid concentrations were enriched by bioprocessing and antimicrobial activity
of the extracts against L. monocytogenes and V. parahaemolyticus correlated with highest soluble phenolics and APF in the same extracts. For
E. coli 0157:H7 inhibition correlated with the extracts corresponding to highest DPPH and ellagic acid concentration. The bioprocessing-based
antimicrobial activity depended on different phenolic functional properties of the extracts. Sensitivity towards soluble phenolics reflected in
DPPH activity suggested inhibition by the disruption of the membrane by hyperacidification. Whereas, sensitivity to APF and ellagic acid
suggests potential antimicrobial activity by membrane-transport disruption. The variation in sensitivity of pathogens has implications for designing new food grade antimicrobials. This bioprocessing strategy can be an innovative approach to produce broad spectrum antimicrobials
against important food-borne pathogens.
2003 Elsevier Ltd. All rights reserved.
Keywords: Solid-state bioprocessing; Antimicrobial activity; Listeria monocytogenes; Vibrio parahaemolyticus; Escherichia coli 0157:H7; Rhizopus
oligosporus

1. Introduction
Each year, about 76 million cases of food-borne disease
occur in the United States. Most of these cases are mild and
cause symptoms for only 1 or 2 days. But some of these infections, estimated at 325,000, result in hospital stays. About
5000 cases a year prove to be fatal [1]. There are several organisms responsible for the incidence of food-borne diseases

Corresponding author. Tel.: +1-413-545-1022;


fax: +1-413-545-1262.
E-mail address: kalidas@foodsci.umass.edu (K. Shetty).

0032-9592/$ see front matter 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2003.09.032

and the three important ones are Listeria monocytogenes,


Vibrio parahaemolyticus and Escherichia coli 0157:H7 [2].
L. monocytogenes causes listeriosis, a serious disease for
pregnant women and their unborn babies, newborns and
adults with weakened immune systems [2]. It is found in soil
and water, dairy products, including soft cheeses, and in raw
and undercooked meat, poultry, seafood and related produce.
V. parahaemolyticus has been associated with the consumption of raw, improperly cooked, or cooked, recontaminated
fish and shellfish. Diarrhea, abdominal cramps, nausea,
vomiting, headache, fever, and chills may be associated
with infections caused by this organism. E. coli 0157:H7 is
a bacterium that can produce potent toxins. It is found in

1940

D.A. Vattem et al. / Process Biochemistry 39 (2004) 19391946

meat, especially undercooked or raw hamburger, and raw


milk. Humans typically get sick when they consume food or
water that has been contaminated with microscopic amounts
of cow feces. Some of the symptoms of the diseases caused
by this pathogen are severe and bloody diarrhea and painful
abdominal cramps, and in severe cases, can cause temporary
anemia, profuse bleeding and kidney failure, and even death.
Several antimicrobials have been developed over the years
to control these microorganisms. However, the development
of antimicrobial resistance and the relatively narrow spectrum of the antimicrobials [36] have had limited success
and the microbial contamination of food still poses an important public health and economic challenge. Cranberry
and its products have traditionally been used to treat urinary tract infections and disorders of the intestinal tract [7]
which are now known to be caused by infections linked to
E. coli O157:H7 [8]. Fruit and berry phenolics have recently
been shown to have antimicrobial activity [9]. We believe
naturally occurring antimicrobials in cranberry will prove
to be an effective additional strategy to control food-borne
pathogens. It is our hypothesis that the profile of phytochemicals that are present in natural sources confer a broad
spectrum antimicrobial capacity and potentially limit the development of antimicrobial resistance because of their possible differences in modes of action. Cranberry pomace is
the byproduct of the cranberry juice processing industry.
Disposal of pomace into soil or landfills poses significant
environmental and ecological problems because of its relatively low pH [10]. In previous research we reported the
enrichment of phenolic antioxidants in cranberry pomace by
its solid-state bioprocessing using a food grade fungi Rhizopus oligosporus [11]. In this paper we have investigated
the effect of solid-state bioprocessing on antimicrobial activity of cranberry pomace against three important food-borne
pathogens.

2. Materials and methods


2.1. Solid-state bioprocessing
2.1.1. Microorganism
R. oligosporus was isolated from un-pasteurized Tempeh product. The Tempeh product was kindly provided
by Life-Life Foods Co., Greenfield, MA. The fungus was
maintained on PDA slants and Petri plates at 4 C and
sub-cultured monthly. The fungus was revived by transferring onto PDA plate and cultured at room temperature 10
days before use.
2.1.2. Media and cultivation
Freshly pressed cranberry pomace was obtained from
Veryfine Inc., Westford, MA, and was vacuum-dried and
stored in a refrigerator before use. One hundred and
twenty-five milliliters of Erlenmeyer flasks containing 10 g
cranberry pomace 0.5 g of CaCO3 , 20 ml water and 0.5 g of

NH4 NO3 or 2 ml fish protein hydrolysate (FPH) as the supplemental nitrogen source were used for solid-state bioprocessing. FPH was obtained from Ocean Crest (Gloucester,
MA) as herring waste containing 0.6575 g ml1 of soluble
solids. The media contained in flasks were autoclaved at
121 C for 20 min and the spores from one PDA plate were
inoculated into eight flasks. Flasks were incubated at 28 C
for 16 days.
2.1.3. Water extraction
One hundred milliliters of distilled water was added
to a fungus-pomace flask. The culture was homogenized
for 1 min using a Waring blender and then centrifuged at
15,000 g at 4 C for 20 min. The supernatant was then
filtered through a Whatman No. 1 filter paper.
2.1.4. Ethanol extraction
One hundred milliliters of 95% ethanol was added to
a fungus-pomace flask. The culture was homogenized for
1 min using Waring blender and then centrifuged at 15,000
g at 4 C for 20 min. The supernatant was then filtered
through a Whatman No. 1 filter paper.
2.1.5. Total phenolics assay [12]
One milliliter of extract was transferred into a test tube and
mixed with 1 ml of 95% ethanol and 5 ml of distilled water.
To each sample 0.5 ml of 50% (v/v) Folin-Ciocalteu reagent
was added and mixed. After 5 min, 1 ml of 5% Na2 CO3
was added to the reaction mixture and allowed to stand for
60 min. The absorbance was read at 725 nm. The absorbance
values were converted to total phenolics and expressed in
milligrams equivalents of gallic acid per 10 g dry weight
(dw) of the sample. Standard curves were established using
various concentrations of gallic acid in 95% ethanol.
2.1.6. Determination of antioxidant activity
2.1.6.1. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) inhibition system [13]. To 3 ml of 60 M DPPH in ethanol,
500 l of fungus bioprocessed cranberry pomace extracts
were added, the decrease in absorbance was monitored at
517 nm until a constant reading was obtained. The readings
were compared with the controls, which contained 500 l of
95% ethanol instead of the extract. The inhibition (%) was
calculated by:


AControl
AExtract
517
517
inhibition (%) =
100
AControl
517
2.1.6.2. -Carotene oxidation model system [14]. One
milliliter of 200 g ml1 of -carotene in chloroform was
pipetted into a round-bottomed flask. The chloroform was
evaporated using a rotary evaporator under vacuum at
40 C for 5 min. The -carotene adhered to the sides of
the flask were scraped and dissolved with 20 l of purified
linoleic acid and 184 l of Tween 40 emulsifier. To this,
50 ml of 50 mM H2 O2 was added and shaken vigorously

D.A. Vattem et al. / Process Biochemistry 39 (2004) 19391946

2.2.1. Microorganism and media


L. monocytogenes: tryptic soy broth/agar with 1.5% yeast
extract (TSY);
V. parahaemolyticus: tryptic soy broth/agar with 2%
sodium chloride (TSS);
E. coli 0157:H7: tryptic soy broth/agar (TS).
2.2.2. Extracts and antimicrobial assay
Water and ethanol extracts were concentrated 10 times by
evaporation in a boiling water bath or in an oven at 100 C.
L. monocytogenes was grown overnight at 37 C in TSY
broth. Hundred microliters of the culture was smeared on
Petri plates containing TSY agar. Three to four sterile disks
of high grade cellulose of diameter 10 mm were placed on
the Petri plates and well spaced out. Hundred to two hundred microliters of the concentrated extract was added to
the disc and incubated overnight at 37 C. Controls were
run for water and ethanol after subjecting them to the same
treatment as the extracts. The diameter of clear zone (no
growth) around the disk in the bacterial lawn was measured
using a ruler and was used to represent the antimicrobial
activity of the extracts.

140

450

120

400
350

100

300

80

250

60

200
150

40

100
20

Total Phenolics
Ellagic acid

Ellagic acid (g/g)

Total Phenolics (mg/10g)

The water extractable phenolics for both the nitrogen


sources showed a similar trend (Figs. 1A and 2A). The
amounts of free phenolics for both nitrogen sources were
constant until day 8 and reached a maximum by day 10 before gradually decreasing for the remaining days of the bioprocessing. This increase was however, higher for the FPH
supplemented compared to when NH4 NO3 was used as nitrogen source.
The changes in ethanol soluble phenolics were quite different compared to water soluble phenolics over the course

50

0
0

(A)

10

12

14

16

Day of fermentation
120

1.4
1.3

100

1.2
1.1

80

1.0

60

0.9
0.8

40

0.7

APF
DPPH

0.6

20

0.5

DPPH Inhibition (%)

2.2. Antimicrobial assay

3.1. Changes in soluble phenolic content and diphenyls

Antioxidant PF

2.1.7. HPLC analysis of ellagic acid [15,16]


Two milliliters of cranberry pomace-fungal extracts were
filtered through a 0.2 m filter. Five microliters of sample
was injected using Agilent ALS 1100 autosampler into a
Agilent 1100 series HPLC (Agilent Technologies, Palo Alto,
CA) equipped with VWD 1100 variable wavelength detector. The solvents used for gradient elution were (A) 10 mM
phosphoric acid (pH 2.5) and (B) 100% methanol. The
methanol concentration was increased to 60% for the first
8 min and to 100% for the 7 min, then decreased to 0% for
the next 3 min and was maintained for the next 7 min (total
run time, 25 min). The analytical column used was Agilent
Zorbax SB-C18, 250 mm 4.6 mm i.d., with packing material of 5 m particle size at a flow rate of 1 ml min1 at ambient temperature. During each run the chromatogram was
recorded at 306 nm and integrated using Agilent Chemstation enhanced integrator. Pure ellagic acid (purchased from
Sigma Chemical Co., St. Louis, MO) in 100% methanol
were used to calibrate the standard curve and retention times.

3. Results and discussion

0
0

(B)

10

12

14

16

Day of fermentation

2.0
L. monocytogenes
V. parahemolyticus
E. coli

1.6

Diameter (cm)

until a uniform emulsion was obtained. Aliquots (5 ml) of


this emulsion were transferred to each test tube containing
100 l of extract. The samples were vortexed for 1 min and
incubated at 50 C for 30 min. Subsequently, absorbance
readings were recorded at 470 nm and compared to a control which had 100 l of ethanol in place of the extract. The
antioxidant activity was expressed as protection factor (PF)
and was calculated as follows:
 Sample 
A470
antioxidant protection factor (APF) =
AControl
470

1941

NH4NO3

1.2
0.8
0.4
0.0
0

(C)

10

12

14

16

Day of fermentation

Fig. 1. Effect of solid-state bioprocessing on: (A) total phenolics and


ellagic acid; (B) antioxidant capacity and (C) antimicrobial activity in
water extracts of NH4 NO3 supplemented cranberry pomace.

D.A. Vattem et al. / Process Biochemistry 39 (2004) 19391946

300

100

250

80

200
60

150

40

100
Total Phenolics
Ellagic acid

20

50

0
2

(A)

10

12

14

150

100

100
50

Total Phenolics
Ellagic acid

80

1.1
1.0

60

0.9
40

0.8
APF
DPPH

20

Antioxidant PF

1.2

DPPH Inhibition (%)

100

1.3

10

12

14

16

Day of fermentation
1.5

98

1.4

96

1.3

94

1.2

92

1.1

90

1.0

88

0.9

86

0.8

84

0.7

82

APF
DPPH

0.6

80
78

0
2

0.5

0.5
0

50
0

(A)

1.4

Antioxidant PF

200

120

0.6

250

150

16

1.5

10

12

14

16

(B)

10

12

14

16

Day of fermentation

Day of fermentation

(B)

2.0

2.0
L. monocytogenes
V. parahemolyticus
E. coli

NH4NO3

FPH

Diameter (cm)

1.6

Diameter (cm)

300
200

Day of fermentation

0.7

350

250

0
0

400

300

Ellagic acid (g/g)

120

350

DPPH Inhibition (%)

400
Total Phenolics (mg/10g)

140

Ellagic acid (g/g)

Total Phenolics (mg/10g)

1942

1.2
0.8

L. monocytogenes
V. parahemolyticus
E. coli

1.6
1.2
0.8
0.4

0.4
0.0
0

0.0
0

(C)

10

12

14

16

Day of fermentation

Fig. 2. Effect of solid-state bioprocessing on: (A) total phenolics and


ellagic acid; (B) antioxidant capacity and (C) antimicrobial activity in
water extracts of FPH supplemented cranberry pomace.

of fungal bioprocessing. (Figs. 3A and 4A). The NH4 NO3


supplemented extract showed a gradual increase in total phenolics until day 10 after which it rapidly decreased. When
FPH was used as a nitrogen source the ethanol extractable
phenolics did not show any significant increase. However,
they decreased gradually towards the end of the growth
period.
HPLC analysis showed that ellagic acid was the major
diphenyl that was mobilized in the water and ethanol extracts for both the nitrogen sources during the course of fungal growth. In both the treatments after an initial increase
there was a sharp decrease in ellagic acid content. When

(C)

10

12

14

16

Day of fermentation

Fig. 3. Effect of solid-state bioprocessing on: (A) total phenolics and


ellagic acid; (B) antioxidant capacity and (C) antimicrobial activity in
ethanol extracts of NH4 NO3 supplemented cranberry pomace.

NH4 NO3 was used as a nitrogen source, the ellagic acid


content increased linearly five-fold by day 12 before sharply
decreasing in the water extracts (Figs. 1A and 2A). In the
water extracts when FPH was used as nitrogen source the
ellagic acid showed two sharp peaks of increase, on day 2
the ellagic acid increased over three-fold and then increased
to over four-fold by day 12 compared to initial values.
The changes in the trends of ellagic acid content of ethanol
extracts were similar for both the nitrogen sources (Figs. 3A
and 4A). When NH4 NO3 used as a nitrogen source the ellagic acid increased gradually until day 8 by 25% and then
decreased rapidly before increasing by 27% by day 14. However, when FPH was used as a nitrogen source the ellagic

350

250

300
250

200

200
150
150
100

100

50

50

Total Phenolics
Ellagic acid

0
0

10

12

14

16

Day of fermentation

(A)

Antioxidant PF

1.5

98

1.4

96

1.3

94

1.2

92

1.1

90

1.0

88

0.9

86

0.8

84

0.7

82

APF
DPPH

0.6

80

0.5

78
0

10

12

16

Day of fermentation

(B)

2.0

Diameter (cm)

14

FPH
L. monocytogenes
V. parahemolyticus
E. coli

1.6
1.2
0.8

3.2. Antioxidant activity


The antioxidant activity of the extracts over the course of
fungal growth was monitored using the -carotene antioxidant protection factor (APF) measurement and by the DPPH
radical inhibition (DRI) assay.
The APF of the water extract for both the nitrogen sources
showed similar trends (Figs. 1B and 2B). The highest antioxidant protection was reached on day 10 with an increase of
25% for FPH and 20% for NH4 NO3 compared to the initial
values. Changes in the APF for ethanol extracts were also
similar for both the nitrogen sources. Both showed two peaks
of increase in APF during the course of growth (Figs. 3B
and 4B). On day 4 the APF increased by 13 and 16% for
NH4 NO3 and FPH nitrogen sources, respectively. Following
this stage the APF rapidly decreased until day 8 before increasing again by 19 and 11% compared to the initial values
before stabilizing by day 12.
When NH4 NO3 was used as a nitrogen source, the DRI
capacity in the water extracts increased 5% by day 8 compared to the initial value before rapidly decreasing. There
was no significant increase in the DRI capacity when FPH
was used as a nitrogen source, but on day 10 the DRI capacity decreased below the initial values (Figs. 1B and 2B). In
the ethanol extracts of the fungal bioprocessed pomace there
was no significant increase in the DRI capacity for both the
NH4 NO3 and FPH supplemented pomace. The DRI capacity of the extracts however, decreased rapidly after day 10
(Figs. 3B and 4B).
3.3. Antimicrobial activity (Fig. 5)

0.4
0.0
0

(C)

1943

acid initially increased by 15% by day 4 before rapidly decreasing and again increasing to the same value by day 12.
Ellagic acid (g/g)

300

DPPH Inhibition (%)

Total Phenolics (mg/10g)

D.A. Vattem et al. / Process Biochemistry 39 (2004) 19391946

10

12

14

16

Day of fermentation

Fig. 4. Effect of solid-state bioprocessing on: (A) total phenolics and


ellagic acid; (B) antioxidant capacity and (C) antimicrobial activity in
ethanol extracts of FPH supplemented cranberry pomace.

3.3.1. Water extracts (Figs. 1C and 2C)


Antimicrobial activities of the extracts were studied at
different time points during the fungal bioprocessing. For
the water extracts of the NH4 NO3 treated pomace the antimicrobial activity against L. monocytogenes and V. parahaemolyticus was observed only for the extracts on days 8,

Fig. 5. Antimicrobial plate assays against: (A) L. monocytogenes; (B) V. parahaemolyticus and (C) E. coli O157:H7. Zone of clearing is due to inhibition
of bacterial growth and corresponds to antimicrobial activity of the Rhizopus oligosporus bioprocessed cranberry pomace extracts.

1944

D.A. Vattem et al. / Process Biochemistry 39 (2004) 19391946

10 and 12 with the extracts on days 10 and 12 showing maximum activity against L. monocytogenes and day 12 extract
against V. parahaemolyticus. The water extracts on days 4,
6, 10 and 12 showed antimicrobial activity against E. coli
0157:H7 with extracts on days 4 and 6 showing slightly
higher activity than that on days 10 and 12. Fewer extracts
had antimicrobial capacity over the course of fungal bioprocessing when FPH was used as a nitrogen supplement.
The extracts on days 2, 10 and 12 had antimicrobial activity
against L. monocytogenes with the extract on day 12 having
highest activity. Only the water extract on day 10 had antimicrobial activity against V. parahaemolyticus. Both the extracts on days 6 and 10 showed antimicrobial activity against
E. coli 0157:H7.
3.3.2. Ethanol extracts (Figs. 3C and 4C)
When NH4 NO3 was used as a nitrogen supplement only
the extracts on days 4, 8 and 10 had antimicrobial activity
against L. monocytogenes with highest activity observed in
the day 10 extracts. Highest antimicrobial activity against V.
parahaemolyticus was also observed in the day 10 extracts
with extracts on days 2 and 4 also showing some antimicrobial activity. The extracts on days 4 and 10 had highest
antimicrobial activity against E. coli O157:H7. The antimicrobial activity of the ethanol extracts of FPH supplemented
pomace had highest activity against V. parahaemolyticus on
day 10. Only the extract on day 4 had antimicrobial activity against L. monocytogenes. The extracts on days 4,
6 and 8 had similar antimicrobial activity against E. coli
O157:H7.

4. Discussion
4.1. Antimicrobial activity and its correlation to phenolic
content, ellagic acid content and antioxidant activity of the
pomace
When antimicrobial activities of the extracts were studied at different time points during the fungal growth, they
correlated well with the changes in total phenolic content
over the course of fungal growth. In the water extracts this
correlation was especially strong for L. monocytogenes (R2 :
0.650.70) and V. parahaemolyticus (R2 : 0.620.71) compared to E. coli O157:H7 (R2 : 0.50) showing that increase
in phenolic content during bioprocessing of the pomace increased the antimicrobial activity of the extracts. For the
water extracts of pomace supplemented with NH4 NO3 the
correlation of antimicrobial activity with ellagic acid was
strong for all the three microorganisms tested. For L. monocytogenes, V. parahaemolyticus and E. coli O157:H7 the correlation coefficients were 0.80, 0.78 and 0.66, respectively.
No such correlation to phenolic content or ellagic acid content was observed in the ethanol extracts. The antimicrobial
activities of the extracts were also correlated with the antioxidant activity of the extracts over the duration of fun-

gal growth. It was observed that the antimicrobial activity


of the extracts correlated strongly with both DRI (0.65) capacity and APF (0.85). Whereas for E. coli O157:H7 only
the extracts having high APF had high antimicrobial activity and this correlation ranged from 0.67 for water extracts
to 0.84 for ethanol extracts. The antioxidant activity of only
the ethanol extracts of the pomace correlated with their antimicrobial activity against L. monocytogenes.
Phenolics measured by the Folin-Ciocalteu assay are
essentially simple soluble phenolics. Correlation of antimicrobial activities to phenolic content, DPPH radical inhibition, APF and ellagic acid provides insights into potentially
different mechanisms of action of these antimicrobials.
Soluble phenolics are thought to exert their antimicrobial effect by causing hyperacidification at the plasma membrane
interface of the microorganism, which potentially results in
disruption of the H+ -ATPase required for ATP synthesis.
Different sensitivities of the microorganisms against the
phenolic content in the extracts may be because of the difference in their membrane structure and associated cell wall
differences between gram positive and gram negative bacterial species. Therefore, hyperacidification of the membrane
and disruption of H+ -ATPase activity of the bacterial cell
membrane may be a probable mechanism by which the phenolics released from cranberry pomace is likely inhibiting
the growth of gram positive L. monocytogenes because of its
simpler membrane structure, making it easy for the phenolics to exert their localized protonation effects. All three microorganisms were sensitive to the presence of ellagic acid
in the extracts, this may be because of the partial hydrophobicity of ellagic acid compared to simple phenolics. Partial
hydrophobicity of the ellagic acid would allow it to act efficiently at the membranewater interface of the bacteria, and
therefore ellagic acid can possibly stack itself on the membrane and destabilize it, resulting in membrane disruption or
destabilization. This kind of mechanism of action is especially required for inhibiting Gram negative bacteria such as
V. parahaemolyticus and E. coli O157:H7 because of the external lipopolysaccharide layer present around its cell which
confers more hydrophobicity. This makes it unfavourable
for simple phenolics to exert their hyperacidification effect. The release of hydrophobic phenolic antioxidants and
ellagic acid enrichment is thought to occur through the
molecular rearrangement of simple phenolics and hydrolysis
of complex polyphenols such as tannins by fungal enzyme
systems [11]. Tannins and hydrolyzed tannins are known to
exhibit inhibitory effects on microorganisms [17]. Tannins
and their hydrolyzed products have been shown to inhibit
the growth of microorganism by sequestering metal ions
critical for the microbial growth and metabolism [18,19] or
by inhibiting critical functions of the bacterial membrane
such as ion channels and proteolytic activity [20]. Higher
sensitivity of V. parahaemolyticus to ellagic acid compared
to E. coli O157:H7 could be because of the better mechanisms available in E. coli O157:H7 to manage acidic stress
compared to V. parahaemolyticus. Ellagic acid might also

D.A. Vattem et al. / Process Biochemistry 39 (2004) 19391946

be disrupting some key ion channels in the membrane of V.


parahaemolyticus which confer the bacteria its salt tolerance
and help maintain its cellular homeostasis under conditions
of high salt concentration. Disruption of such a mechanism
in the bacterial cell membrane may be fatal to the microorganism. The sensitivity of all the microorganisms to ellagic
acid and diphenyls is also reflected in their relatively higher
correlation to the antioxidant capacity as measured by APF
compared to the DRI. The formation of hydrophobic antioxidants and ellagic acid mobilization during the solid-state bioprocessing of cranberry pomace has been discussed in detail
previously [11]. For a phenolic to function effectively as an
antimicrobial it should be able to function at the lipidwater
interface and therefore has to be partially hydrophobic.
Phenolic mobilizations occurring during the solid-state
bioprocessing of pomace have different hydrophobicities
depending on the duration of fungal bioprocessing. The
nature of the antioxidants however can be deduced by
their abilities to behave as antioxidants in DRI assay and
-carotene antioxidant assays. The -carotene system potentially quantifies the ability of the antioxidant to function
at a lipidwater interface and therefore, the compound has
to be a partially hydrophobic in nature. Therefore, it can be
presumed that the compounds having higher antioxidant activity in the -carotene assay will have higher antimicrobial
capacity.

5. Conclusions
The investigation was carried out to study the effect
on antimicrobial properties of phenolic extracts from
solid-state growth on cranberry using the food grade fungus R. oligosporus. This process resulted in an increase
in the total extractable phenolic content. Antioxidant activity measured by both APF and DRI increased over the
course of growth. Both total phenolics and antioxidant capacity correlated well with the increase in antimicrobial
activity of the extracts against L. monocytogenes and V.
parahaemolyticus, and to a lower degree against E. coli
O157:H7 showing that the process enriched the antimicrobial activity of the pomace. The variation in sensitivities to
the phenolic extracts combined with variable correlations
of the microorganism to different properties of the extracts
may indicate different mechanisms of action of the extracts
against different microorganisms. All the microorganisms
were sensitive to the presence of ellagic acid, which may
indicate a ubiquitous mechanism of action of ellagic acid
either by destabilizing the membrane integrity, blocking ion
channels in the membrane or by inhibiting the electron flow
in the plasma membrane electron transport chain required
for ATP synthesis by virtue of its electron scavenging properties. The results also indicated that partial hydrophobicity
with a good antioxidant capacity are desirable properties
in a phytochemical extract to have good antimicrobial
activity.

1945

Solid-state growth using food grade fungus resulted in


improving the antimicrobial activity of the pomace and the
possible mechanisms of action of phenolic phytochemicals
have been suggested. The fungus, R. oligosporus and other
ingredients used in this solid-state growth process are food
grade and are generally recognized as safe (GRAS). Therefore this process permits an alternative use of the cranberry
pomace as an antimicrobial agent for diverse food and feed
applications.
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