Industrial Crops and Products 51 (2013) 279–288

Contents lists available at ScienceDirect

Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Polyphenol composition, antioxidant capacity, and antimicrobial

activity of the extracts obtained from industrial sour cherry pomace夽
Krzysztof Kołodziejczyk a,∗ , Michał Sójka a , Maribel Abadias b , Inmaculada Viñas c ,
Sylvain Guyot d , Alain Baron d
a
Lodz University of Technology, Institute of Chemical Technology of Food, ul. Stefanowskiego 4/10, 90-924 Łódź, Poland
b
IRTA, XaRTA-Postharvest, 191 Rovira Roure, 25198-Lleida, Catalonia, Spain
c
University of Lleida, XaRTA-Postharvest, 191 Rovira Roure, 25198-Lleida, Catalonia, Spain
d
INRA, UR 1268, Unité BIA, Biopolymères, Interactions Assemblages, équipe PRP, F-35650 Le Rheu, France

a r t i c l e i n f o a b s t r a c t

Article history: Polyphenol extracts from industrial sour cherry pomaces were characterized on polyphenol composi-
Received 26 April 2013 tion, antioxidant capacity and antimicrobial activity. Extracts of pomace were purified and freeze-dried.
Received in revised form Preparations were characterized by high polyphenol contents including anthocyanins, hydroxycinnamic
11 September 2013
acids and flavonoids, selected were characterized by high flavanol content and high antioxidant capacity.
Accepted 14 September 2013
The antimicrobial effect of sour cherry polyphenol extracts was tested against Salmonella, Escherichia coli
O157:H7 and Listeria spp. The bacteriostatic effect was tested by growing the strains in a liquid medium
Keywords:
containing the extracts, the bactericide effect was assayed by putting the strains in direct contact in an
Cherry pomace
Extracts
aqueous suspension of the extract, simulating the disinfection process in the fresh-cut industry. Two of
Polyphenols the sour cherry extracts tested reduced the growth of Salmonella and E. coli O157:H7 at concentrations
LC–MS higher than 2500 ␮g/mL, and inhibited Listeria spp. growth.
Antimicrobial activity © 2013 Elsevier B.V. All rights reserved.

1. Introduction interactions with biomolecules, like enzyme regulators (Lee et al.,

Stone fruit like sour cherries are food products in demand
on the European market due to their taste and nutrition val-
ues. Mean annual crops of sour cherry in Poland reach 200,000 T
(Eurostat, 2010). The fruits are used for the production of juices,
nectars, soft and alcoholic drinks, jams, and as additives in the
dairy and confectionary industry. Sour cherries are a rich source
of polyphenols—phytocomponents characterized by the presence
of more than one phenol group in the molecule. Many research
publications indicate that the increased consumption of fruits and
fruit polyphenols is beneficial for maintaining good human health
(Lee et al., 2007; Rissanen et al., 2003). The effect is attributed
to a number of factors, including polyphenol components which
are characterized by antioxidant properties (Halvorsen et al.,
2002) and contribute to many life processes, resulting from their
夽 Disclaimer: The views and opinions expressed in this publication are purely those
of the writers and may not in any circumstances be regarded as stating an official of
European Commission.
∗ Corresponding author. Tel.: +48 42 631 34 65
E-mail addresses: krzysztof.kolodziejczyk@p.lodz.pl (K. Kołodziejczyk),
michal.sojka@p.lodz.pl (M. Sójka), isabel.abadias@irta.cat (M. Abadias),
ivinas@tecal.udl.es (I. Viñas), sylvain.guyot@rennes.inra.fr (S. Guyot),
alain.baron@rennes.inra.fr (A. Baron).
2007; D’Ischia et al., 2006). The main phenolics present in
sour cherry are anthocyanins, represented by cyanidin glycosides
(Chandra et al., 2009; Simunic et al., 2005). Besides antho-
cyanins, hydroxycinnamic acids, especially caffeoylquinic and
p-coumaroylquinic acids are present in sour cherry. The presence of
quercetin, kaempferol, and isorhamnetin glycosides was detected
in sour cherries as well (Kim et al., 2005). Sour cherry stones are
an interesting material as well. According to Yılmaz and Gökmen
(2013) sour cherry kernel might be utilized as a source of dietary
fibers, proteins and lipids, furthermore oil extracted from seed ker-
nel is rich in polyunsaturated fatty acids, tocopherols, ␤-carotene
and phenolic compounds.
In addition, plant phenolics and terpenoids, have been widely
used because of their strong antimicrobial properties against
foodborne pathogens, and therefore could be applied as novel
preservatives in the food industry (Friedman et al., 2002).
The contamination of foods by foodborne pathogens and
spoilage microorganisms is a problem that is not yet under ade-
quate control despite a wide range of preservation techniques
available. The microbiological safety of food continues to be a major
concern to consumers, regulatory agencies and food industries
throughout the world. Fresh-cut fruits and vegetables are of partic-
ular concern as they do not receive any treatment that guarantees
the complete elimination of pathogens. Currently, chlorine, in the

0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2013.09.030
280 K. Kołodziejczyk et al. / Industrial Crops and Products 51 (2013) 279–288

form of sodium hypochlorite at a concentration of 50–200 ppm 2.2. Plant material

is commonly used to disinfect produce (Beuchat, 1998). Never-
theless, concerns about its limited efficacy and the toxicity of Stoned pomace of sour cherry was a material for the research.
chlorination by-products formed in the presence of organic mat- The pomace came from fruit processing in 2009 at a modern con-
ter, has prompt the search for alternative, safer, more effective ˛
centrated juice production plant Alpex at Łeczeszyce, Poland. Fresh
and environmentally friendly sanitation agents. As far as micro- pomace collected from the production line was sieved on a manual
bial preservatives are concerned, traditional antimicrobials such as 5-mm screen to eliminate stones. The seedless part was extracted
acetic, benzoic, lactic, propionic and sorbic acids and nitrite and promptly, while the seed part was dried and used for other exper-
sulphites have been used for many years to control the growth iments (not described herein).
of microorganisms in food (Sofos et al., 1998). The current con-
sumer demands for more natural and fresh-like foods, with fewer
synthetic additives but increased safety and shelf life, urges food
2.3. Preparation of sour cherry extracts
manufactures to use natural or mild preservation techniques (Negi
et al., 2008). Polyphenols extracted from plants and fruits could
Three kinds of purified extracts, differing in polyphenol contents
be an alternative. Several papers have demonstrated the antimi-
were obtained from the stoneless pomace fraction. Fresh sour
crobial activity of flavonoids extracted from bergamot (Mandalari
cherry pomaces were subjected to water extraction in three steps,
et al., 2007), Garcinia spp. (Negi et al., 2008), pome fruits includ-
at a temperature of 70–75 ◦ C for 30 min. In the first step the weight
ing apple, pears and quinces (Alberto et al., 2006; Fattouch et al.,
ratio of fresh pomace to water was 1:4, while in the second and third
2007, 2008) and grapes (Baydar et al., 2004) among others. How-
extraction step the volume of water was equal to the volume of the
ever, the antimicrobial efficacy of sour cherry extracts has not been
extracts resulting from the preceding extraction step. The first and
evaluated.
second extracts were collected by soaking the pomace on filtra-
The use of sour cherry in the juice industry results in the side
tion cloth, while the third extract, after soaking the pomace was
production of significant amounts of pomaces, which may reach
pressed on own-manufactured laboratory press (Lodz University
from 15% to 28% of transformed raw material, depending on the
of Technology, Poland). The choice of deionized water or option-
process conditions (Toydemir et al., 2013). The main use of such a
ally condensate of technological vapor as a solvent for extraction
by-product is as a fuel, the alternative is its use as an animal food or
resulted from economic factor (inexpensive solvent), safety and
its further transformation, i.e. the production of dietary fibre and
availability of water or vapor as a boiler medium. The excess of
anthocyanin- and other polyphenol-reach extracts.
technological vapor appears in many industrial facilities, in case of
The literature lacks data on the properties of products derived
shortage the installation for deionized water (e.g. reversed osmo-
from sour cherry pomace extracts. Another alternative could be its
sis) can be applied. Unlike technological water, deionized water
use as a source of antimicrobial and antioxidant preservatives for
does not add to extracts any salts, especially calcium and iron
the food industry.
salts, which with polyphenols may result in residues or undesired
The aim of this work was to obtain concentrated polyphe-
color. Disadvantage of deionized water as a solvent is relatively low
nol extracts from industrial sour cherry pomaces by the use of a
yields of some polyphenol groups, in particular flavanols. In pre-
low pressure chromatography method with solvents acceptable
sented research the water was heated to 70 ◦ C in order to increase
in food production. The qualitative and quantitative composition
the yields. Determinations of polyphenols in starting material and
of the preparations obtained was characterized. The antimicro-
post-extraction pomaces showed, that the yields of anthocyanins,
bial capacity of polyphenol preparations on different foodborne
hydroxycinnamic acids and flavonols after triple extraction was
pathogens was determined from two points of view. The bac-
80%, while the yields of flavanols was 30%. All the 3 extracts
tericidal effect was tested with a view to using the extracts as
were put together and filtered on cellulose. The filtered extract
an alternative to chlorine for a washing-disinfection process of
was purified on a 80 × 2.5 cm column (Pharmacia Fine Chemicals,
fresh-cut fruits and vegetables, and the bacteriostatic effect to be
Uppsala, Sweden) filled with Amberlite XAD-7HP (Sigma-Aldrich,
used as a natural preservative to prevent the growth of foodborne
Steinheim, Germany). The extract was applied with a flow rate of
pathogens.
250 mL/h. Next, the column was washed with 300 mL of water and
150 mL of 10% ethanol, with a flow rate of 150 mL/h. The elution
of polyphenols was carried out with the use of 300 mL of 20%, and
2. Materials and methods
750 mL of 60% ethanol, with a flow rate of 150 mL/h. Seven frac-
tions of 150 mL each were collected. The fractions were joined as
2.1. Chemicals
follows: 1 + 2, 3 + 4, and 5 + 6 + 7. Ethanol was removed from each
of the joined fractions on a laboratory vacuum rotary evapora-
Ultrapure water (Millipore System, GmBH, Vienna, Austria) and
tor (type 350P, Unipan-Scientific Instruments, Warsaw, Poland), at
HPLC gradient-grade methanol (J.T. Baker, Deventer, Holland) were
50 ◦ C, and fractions thus prepared were then freeze-dried. That pro-
used to prepare all solutions. HPLC gradient-grade acetonitrile and
cedure yielded 3 concentrated preparations from the pomace. The
formic acid were purchased from J.T. Baker (Deventer, Holland).
sour cherry extracts were labeled as follow: CHPE1 (1 + 2), CHPE2
Cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, peonidin-3-O-
(3 + 4), CHPE3 (5 + 6 + 7).
glucoside, quercetin-3-O-rutinoside, quercetin-3-O-galactoside,
quercetin-3-O-glucoside, kaempferol-3-O-rutinoside, kaempferol-
3-O-glucoside, isorhamnetin-3-O-rutinoside, isorhamnetin-3-O-
glucoside, quercetin, kaempferol, isorhamnetin, ellagic acid from 2.4. Sample preparation for quantification
Extrasynthese (Genay, France) and (+)-catechin, (−)-epicatechin,
chlorogenic acid, phloridzin from Sigma-Aldrich Chemie GmBH Polyphenol extracts from pomaces were diluted in a 50% solu-
(Steinheim, Germany) were used as standards for MS spectral tion of methanol, then centrifuged at 6000 rpm (3600 g) for 5 min.
comparisons. Trolox (6-hydroxy-2,5,7,8-tetramethychroman-2- The samples prepared as above were used to determine the
carboxylic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals total polyphenol content (TCP), the antioxidant activity, and the
to determine anti-oxidant activities were purchased from Sigma- polyphenol, i.e. anthocyanins, hydroxycinnamic acids and flavonols
Aldrich Chemie GmBH (Steinheim, Germany). contents, by the HPLC method.
 K. Kołodziejczyk et al. / Industrial Crops and Products 51 (2013) 279–288 281

Table 1
Method validation parameters for determination of phenolic compounds, and antioxidant activity.

TPC as DPPH (as Cy-Glu Q-Rut Q CLA p-Coum Epi Cat Epi-add
(−)epicatechin Trolox)

Standard linearity
Range (mg/L) 12.5–250 32–260 2–156 3–50 3–60 3–60 3–50 4–200 4–200 5–250
r 0.999 0.999 0.999 0.998 0.999 0.999 0.999 0.999 0.998 0.996

Sensitivity
LOD (mg/L) 6.3 1.5 0.3 0.3 0.8 0.2 0.1 0.1 0.5 0.6
LOQ (mg/L) 12.6 7.5 1.4 1.5 4.2 0.9 0.6 0.6 2.7 3.0
RSD of real samples (%) 1.6 2.1 2.5 2.0 1.8 2.3 0.6 5.4 (2.8* ) 5.9 5.2
n 3 4 3 3 3 3 3 3 3 3

TPC, Total phenolics content; DPPH, radical-scavenging activity; Cy-Glu, cyaniding-glucoside; Q-Rut, quercetin-rutinoside; Q, quercetin; CLA, chlorogenic acid; p-Coum,
p-coumaric acid; Epi, (−)-epicatechin; Cat, (+)catechin; Epi-add, (−)epicatechin-benzylthioeter adduct.
*
RSD for non-thiolysed samples.

2.5. HPLC conditions for identification and quantification 2.7. Total phenolics content (TPC)

High performance liquid chromatograph (HPLC) coupled with a
DAD and an electrospray ion (ESI) trap mass spectrometer was used
for the identification of hydroxycinnamic acids, flavonols, proan-
thocyanidins, and anthocyanins. Separation conditions and mass
detector parameters were described in a previous work (Sójka et al.,
2009).
The quantification of anthocyanins and other phenolics was
determined using a HPLC KNAUER Smartline chromatograph
(Berlin, Germany). The phenolics preparations were separated by
reverse phase according to the method described by Sójka and Król
(2009). Hydroxycinnamic acids were detected at 320 nm, quercetin,
kaempferol and isorhamnetin glycosides as well as their aglycons
were detected at 360 nm, while anthocyanins where detected at
520 nm. Standard curves using external standards of cyanidine-
3-O-glucoside, rutin, quercetin, chlorogenic and p-coumaric acids
were used for quantification. Cyanidin-3-O-glucoside was used to
assay anthocyanins, rutin was used to assay quercetin, kaempferol
and isorhamnetin glycosides, chlorogenic acid was used to assay
chlorogenic acids isomers, whereas p-coumaric acid was used to
assay p-coumaryl derivatives.
The total phenolics were measured by the method described by
Singleton and Rossi (1965), with some modifications. The 0.25 mL
of phenolics extract was placed in a 25 mL volumetric flask, and
0.25 mL of Folin-Ciocalteau reagent. After 5 minutes of the reac-
tion, 2.5 mL of 20% Na2 CO3 were added, filled up with water to
the graduation mark and stirred. The incubation was carried out
at room temperature for 1 h. The absorbance of the solutions was
measured at a wavelength of 720 nm (Metertech SP-880 spec-
trophotometer, Taipei, Taiwan). The results were expressed as
grams of (−)-epicatechin equivalent per 100 g of extract. All the
samples were analyzed in duplicates.
2.8. DPPH radical-scavenging activity
DPPH scavenging activity was determined using the method
described by Kim et al. (2002).
The absorbances were measured on Metertech SP-880 (Taipei,
Taiwan). Results of scavenging activity of sour cherry phenolics
extracts were expressed as ␮M TEAC g−1 of extract or standard
(TEAC—Trolox equivalent anti-oxidative capacity).

2.9. Validation parameters

2.6. HPLC analysis of flavanols
A method applying the acid-catalyzed degradation of poly-
meric proanthocyanidins in the presence of toluene-␣-thiol was
used for the determination of flavanols. Crude methanol solutions
for HPLC determination of free (+)-catechin and (−)-epicatechin)
were prepared by dilution of 5 mg of the extract in 1.2 mL of dry
methanol and sonication at room temperature for 30 min. The thiol-
ysis reaction was performed according to the procedure described
by Guyot et al. (2001). Five milligrams of dry extract were mixed
with 800 ␮L of benzylthiolether (5% (v/v) in anhydrous methanol)
and 400 ␮L of 0.4 M HCl in dry methanol, and the reaction was car-
ried out at 40 ◦ C for 30 min. Thiolysis reactions were performed
in triplicates for each sample. The samples were filtered through
a 0.45 ␮m Teflon membrane (Millipore, Bedford, MA) and then
analyzed by RP-HPLC coupled UV–vis detection according to the
method described by Guyot et al. (2001). The 280 nm HPLC response
factors of (−)-epicatechin, (+)-catechin were obtained from cal-
ibration curves of the respective commercial standards (Sigma
Aldrich) whereas the 280 nm calibration curve of (−)-epicatechin
benzylthioether was pointed out by the use of the standard addi-
tionally purified. The response factors were next used both for the
quantification of total flavanols in the extracts and for the deter-
mination of their average degree of polymerization (Guyot et al.,
1998).
Standard curves used for quantitative assays were characterized
by very good linearity; the correlation coefficient for the majority of
the standards in the ranges applied was between 0.996 and 0.999.
Limits of detection (LOD) and limits of quantification (LOQ) were
determined on the basis of equations of standard curves, where
the coefficient b from the equation y = ax + b stood for LOD. LOQ
was calculated by five-fold multiplication of the limit of detec-
tion (Table 1). Limits of detection (LOD) did not exceeded 0.8 and
6.3 mg/L for chromatographic methods, and spectrophotometry,
respectively. Limit of quantification (LOQ) of method used for
hydroxycinnamic acids and flavonol glycosides determination did
not exceeded 1.5 mg/L. LOQ for quercetin calibration curve was
4.2 mg/L. For spectrophotometric methods LOQ did not exceeded
12.6 mg/L. The relative standard deviation (RSD) of the methods
used did not exceed 3%, except for the determination of flavanols,
where the RSD amounted to 5%, which resulted from low repeat-
ability of the thiolysis process.
2.10. Antimicrobial tests
The strains used for antimicrobial testing were a non-pathogenic
strain of Escherichia coli O157:H7 (NCTC 12900), Listeria mono-
cytogenes serovar 1a (CECT-4031), L. monocytogenes serovar 4d
(CECT-940), L. innocua (CECT-910) and the strains of Salmonella
282 K. Kołodziejczyk et al. / Industrial Crops and Products 51 (2013) 279–288
choleraesuis subsp. choleraesuis (Smith) Weldin serotype Michi-
gan (ATCC BAA-709) and Montevideo (ATCC BAA-710). They were
maintained at −20 ◦ C on nutrient agar slants with glycerol. Prior
to use, they were subcultured on Tryptone Soy Agar (TSA, Oxoid,
UK) or tryptone soy agar plus yeast extract (TYSEA, TSA plus 6 g/L
of yeast extract) and incubated at 37 ◦ C for 20–24 h.
The antimicrobial effect of extracted polyphenols was tested
by two methods. The bacteriostatic effect was tested by growing
the strains in a liquid medium containing the cherry extracts. The
bactericidal effect was assayed by putting in contact the indica-
tor microorganism with an aqueous solution of the polyphenol
extract, simulating the disinfection process of fresh-cut fruits and
vegetables in the industry. In both methods, cell suspensions were
prepared as follows. Salmonella and E. coli strains were grown indi-
vidually in tryptone soy broth (TSB, Oxoid, UK) for 20–24 h at 37 ◦ C.
Listeria spp. strains were grown individually in TSB supplemented
with 6 g/L of yeast extract (tryptone yeast extract soy broth, TYSEB)
for 20–24 h at 37 ◦ C. Bacterial cells were harvested by centrifugation
at 9820 × g, 10 min at 10 ◦ C and then resuspended in saline peptone
(SP; 8.5 g/L NaCl and 1 g/L peptone). For the inoculum preparation,
bacterial concentration was estimated using a spectrophotometer
set at  = 420 nm according to standard curves. A suspension of 108
or 106 cfu/mL of each strain was prepared.
2.10.1. Bacteriostatic test
225 ␮L of TSB (Salmonella and E. coli O157:H7) or TYSEB (Lis-
teria spp.) were dispensed into all wells of a 96-well microtiter
plate. From a 20,000 ␮g/mL stock solution, the required volume
was added to three wells to obtain a final concentration of the
polyphenol extract of 50, 100, 250, 500, 750, 1000, 2000, 2500,
3000, 3500 and 4000 ␮g/mL. A volume of sterile distilled water
was added to each well to obtain a final volume of 250 ␮L in each
well. 25 ␮L of sterile distilled water were placed in control wells. A
5 ␮L volume of the 106 cfu/mL suspension was added to each well.
Three wells of each treatment were left uninoculated and served
as a negative control. Plates were incubated at 37 ◦ C for 16 h. The
absorbance of each well at 600 nm was measured in a spectropho-
tometer (Powerwave, Bio-Tek). In this experiment, the minimal
inhibitory concentration (MIC) was taken as the lowest concen-
tration of polyphenol extract at which there was no perceptible
growth of the organism.
2.10.2. Bactericidal test
In the first experiment sour cherry polyphenol extracts were
tested at 10,000 ␮g/mL and 5 min of contact. If they did not
show any antimicrobial effect at such high concentration, they
were rejected for further experiments. Therefore, 1 mL of cherry
polyphenol extracts at 10,000 ␮g/mL were prepared in duplicate
in sterile 1.5 mL microcentrifuge tubes. Afterwards, 100 ␮L of the
pathogen suspension was added to the tubes and a 107 cfu/mL sus-
pension was obtained. After 5 min, populations in the tube were
determined by 10-fold diluting in SP and plating (20 ␮L) onto Sor-
bitol MacConkey Agar (SMAC, Biokar Diagnostics, Beauvais, France)
supplemented with Cefixime-Tellurite (CT-SMAC, Biokar) for E. coli
O157:H7, Xylose-Lysine-Desoxycholate agar (XLD, Oxoid Ltd., Bas-
ingstoke, England) for Salmonella or onto Palcam agar (Palcam
Agar Base with selective supplement, Biokar Diagnostics, Beau-
vais, France) for Listeria spp. The plates were incubated at 37 ± 1 ◦ C
for 24 ± 2 h (E. coli and Salmonella) and 48 ± 2 h (Listeria spp.).
Deionised water was used as control (CK). In this experiment, detec-
tion limit was 2.5 × 103 cfu/mL (3.40 log cfu/mL).
In the second experiment, different concentrations (50, 100,
250, 500, 750, 1000, 5000 and 10,000 ␮g/mL) of the selected cherry
polyphenol extracts were tested against a cocktail of the 3 strains of
Listeria using the same methodology. In this experiment, detection
limit was 25 cfu/mL (1.40 log cfu/mL).
Reduction and absorbance values were analyzed by a General
Lineal Model (GLM) procedure with SAS software (SAS Institute,
version 9.1, Cary, NC, USA). Statistical significance was judged at
the level P = 0.05. When the analysis was statistically significant
Duncan’ Multiple Range Test was used for the separation of the
means.
3. Results and discussion
3.1. Identification of the sour cherry phenolics
The identification of phenolic compounds was performed by
the comparisons with available standards, basing on the recorded
retention times, UV–vis and MS data and on reference data. In the
case of no standard for a substance, HPLC separated compounds
showing identical UV–vis and MS data as known and described
compounds were quantified as such.
Purified sour cherry extracts were characterized by both qual-
itative and quantitative differences of their polyphenol profile.
The identification of polyphenols is presented in Table 2. In
CHPE1 extract the anthocyanins were identified, and cyanidin-
glucosyl-rutinoside was predominant. Besides the predominant
compound, cyaniding-sophoroside and cyanidin-3-O-glucoside
were identified, and traces of cyanidin-dipentosyl-hexoside were
present as well. The identification of cyanidin-3-O-glucoside was
confirmed on the basis of the available standard. The identifi-
cation of the other three anthocyanins was performed basing
on the recorded MS data and on reference data (Chandra et al.,
2009; Simunic et al., 2005; Kirakosyan et al., 2009; Sanchez-
Rabaneda et al., 2003). Cyanidin-glucosyl-rutinoside, the main
anthocyanin, gave deprotonated molecule at m/z 755 and prod-
uct ions at m/z 593 and 285 (m/z) in the first fragmentation stage
(MS2), which corresponds to glucose and rutinose loss, respec-
tively. This is the main anthocyanin present in sour cherry; its
presence was proved by Chandra et al. (2009), Simunic et al.
(2005), Kirakosyan et al. (2009), Toydemir et al. (2013). Cyanidin-
sophoroside molecule gave 609 (m/z) and fragmented to 285
(m/z), while pseudo-molecule of cyanidin-dipentosyl-hexoside
725 (m/z) fragmented to 285 (m/z) as well. The presence of
these anthocyanins in sour cherries was previously observed by
Chandra et al. (2009), Simunic et al. (2005) and Kirakosyan et al.
(2009). Considering the data of Chandra et al. (2009) on tart
cherry anthocyanins, cyanidin-dipentosil-hexoside can possibly
be identified as cyanidin-arabinosyl-rutinoside. Besides antho-
cyanins two other substances were identified in the researched
extract: dicaffeoylquinic acid and amygdalin. Dicaffeoylquinic acid
gave pseudo-molecule 515 (m/z), which fragmented to 353 (m/z)
through the loss of [M-H-162]− ion (quinic acid). Such fragmen-
tation of dicaffeoylquinic acid was proved by Sanchez-Rabaneda
et al. in artichoke and apple pomaces (Sanchez-Rabaneda et al.,
2003, 2004), besides the maximum of 311 nm was found in UV spec-
trum of the compound. The presence of dicaffeoylquinic acids has
not been detected in sour cherry fruits until now. The presence of
amygdalin was proved on the basis of the available standard and
UV spectrum, characteristic of the compound, i.e. maxima at 257,
263, and 268 nm. The deprotonated molecule of the compound gave
456 (m/z), and fragmented to 323 (m/z). The even mass is an indi-
cation that an uneven number of nitrogen atoms is present in the
molecular formula.
CHPE2 extract was characterized by a higher number of
polyphenols present, compared to CHPE1. The anthocyanins
described above and two additional deprotonated molecules at
m/z 593, giving a product ion at m/z 285, and at m/z 607, giv-
ing a product ion at m/z 299 were identified in this extract. The
first one was identified by the comparison with the standard as
 K. Kołodziejczyk et al. / Industrial Crops and Products 51 (2013) 279–288 283

Table 2
Phenolics identification in sour cherry pomace extract.

RTa (min) max (nm) [M-H]− (m/z) MS/MS product ionsb (m/z) Tentative identificationc CHPE1 CHPE2 CHPE3

Anthocyanins
16.98 280, 515 609 285 Cyanidin-sophorosidec + + +

18.08 280, 518 755 285, 593 Cyanidin-glucosyl-rutinosidec + + +

+ + +
18.08 280, 518 447 285 Cyanidin-3-O-glucoside
19.70 280, 516 725 285 Cyanidin-dipentosyl-hexosidec + + +
+ +
19.70 280, 516 593 285 Cyanidin-3-O-rutinoside
23.07 280, 507 607 299 Peonidin-rutinosidec + +

Hydroxycinnamic acids
10.65 280, 311 515 353, 341, 179 Dicaffeoylquinic acidc + +

12.27 326 353 191, 179 Neochlorogenic acidc + +

14.97 311 337 163, 191 3-p-coumaroylquinic acidc + +

+ +
14.97 326 353 191, 179 Chlorogenic acid
19.98 285, 312 337 191, 173, 163 4-p-coumaroylquinic acidc +

Proanthocyanidins
13.82 280 865 695, 577, 289 Procyanidin trimerc +

14.02 281 1153 865, 577, 289 Procyanidin tetramerc +

15.45 280 577 425, 289 Procyanidin dimerc + +

+
17.83 279 289 245, 205, 178 (−)-epicatechin
18.25 280 865 695, 577, 289 Procyanidin trimerc + +

19.13 280 1153 865, 577, 289 Procyanidin tetramerc + +

20.22 1441 1153 Procyanidin pentamerc +

20.65 280 1153 865, 577, 289 Procyanidin tetramerc +

29.92 279 865 577 Procyanidin dimerc +

Flavonols
18.87 284, 354 771 609, 591, 301, 271, 255 Quercetin-triglycosidec + +

20.22 281, 351 771 609, 301, 271, 255 Quercetin-triglycosidec + +

28.92 282 433 271, 151, 119 Naringenin-hexosidec +

+
31.62 354 609 301, 271, 255 Quercetin-3-O-rutinoside
+
35.88 352 463 301, 300, 271 Quercetin-3-O-glucoside
40.83 332, 347 431 269 Genistinc +

42.60 280, 330 433 271, 151, 119 Naringenin-hexosidec +

+ +
43.12 335 593 285, 255, 227 Kaempferol-3-O-rutinoside
+ +
44.83 355 623 315, 300, 299 Isorhamnetin-3-O-rutinoside
+
45.70 347 447 285, 284 Kampferol-3-O-glucoside
+
47.12 349 477 315, 314, 299 Isorhamnetin-3-O-glucoside

Others
+ +
12.83 281, 332 773 285, 637 Unknown
+
13.02 288 465 – Unknown
+
14.27 281 611 285, 485 Unknown
+
14.92 257, 263, 268 456 323 Amygdalin
+
15.83 252, 257, 263 447 401, 269, 161 Unknow
+
49.13 285 435 273 Phloridzin
a
RT–retention time (min).
b
The m/z values of the predominant ions are given in bold type.
c
tentative identification.
+
Presence of the compound detected by MS.

cyanidin-3-O-rutinoside, the second on the basis of MS and litera-
ture data (Chandra et al., 2009; Mozetič et al., 2006) was tentatively
identified as peonidin-rutinoside. Traces of dicaffeoylquinic acid
and amygdalin were present in the extract. The extract was
characterized by the presence of hydroxycinnamic acids, espe-
cially chlorogenic acid isomers and p-coumaric acid derivatives.
Neochlorogenic acid was identified in the extract, the maximum of
UV spectrum was 325 nm, the pseudomolecule gave 353 (m/z), and
fragmented to 191 and 179 (m/z). Similar values were for chloro-
genic acid, which gave the UV maximum 326 nm, deprotonated
molecule at m/z 353, and the main product ion at m/z 191. The
presence of chlorogenic acid was confirmed by the comparison with
the standard. Conditions applied to chromatography resulted in the
co-elution of chlorogenic acid with 3-p-coumaroylquinic acid, the
latter gave pseudo-molecule 337 (m/z) and 163 (m/z) product ions.
The identification of the above acids by an MS-ESI detector in neg-
ative mode was described by Clifford et al. (2003). Procyanidins
oligomers were detected in the extracts as well: dimer, trimer, and
tetramer were clearly detected on the basis of their mass spectra
showing deprotonated molecules at m/z 577, 865, and 1153, respec-
tively. In all the cases, the deprotonated molecules showed product
ions fragmented to the unit identified as (−)-epicatechin, which
gave pseudo-molecule 289 (m/z). Similar results were obtained in
plums, peaches and nectarines by Tomas-Barberan et al. (2001),
who proved the presence of epicatechin, and procyanidin dimers
and trimers. Flavonols, more precisely, two quercetin glycosides
were the last group of compounds present in CHPE2 extract.
Both compounds were characterized by the UV spectra typical for
flavonols, with the maximum, ca. 350 nm, and gave deprotonated
molecule at m/z 771. The first glycoside gave product ions at m/z
625, 609, and 591, where m/z 591 was predominant, the second gly-
coside fragmented to product ions at m/z 609. Such fragmentation,
in both cases, confirms the presence of triglycoside. As described
by Toydemir et al. (2013), one of above glycosides was tentatively
identified as quercetin-rutinoside-glucoside.
The most differentiated by polyphenols content is CHPE3
extract. That extract contained all polyphenols present on CHPE2.
Besides, the presence of additional procyanidins, quercetin,
284 K. Kołodziejczyk et al. / Industrial Crops and Products 51 (2013) 279–288

Table 3
Phenolics composition (mg/100 g) in cherry pomace extracts.

Compound Cherry pomace extracts

CHPE1 CHPE2 CHPE3

Neochlorogenic acid n.d. 1460.7 ± 6.5 1396.7 ± 3.3

3-p-Coumaroylquinic acid n.d. 853.4 ± 8.1 2143.9 ± 8.2
Chlorogenic acid n.d. 633.1 ± 27.1 1637.5 ± 6.3
Quercetin glucoside and rutinoside n.d. 312.8 ± 0.8 2504.9 ± 1.1
Kaempferol-rutinoside n.d. 48.4 ± 4.3 674.4 ± 1.0
Isorhamnetin-rutinoside n.d. 111.9 ± 2.3 1342.8 ± 21.3
Quercetin n.d. n.d. 22.3 ± 0.1
Kaempferol n.d. n.d. 23.9 ± 1.5
Isorhamnetin n.d. n.d. n.d.
Cyanidin-sophoroside 42.8 ± 1.2 294.6 ± 0.3 88.5 ± 0.9
Cyanidin-glucosyl-rutinoside 1190.9 ± 25.5 4421.6 ± 66.5 949.9 ± 5.6
Cyanidin-glucoside 18.5 ± 0.2 59.9 ± 2.6 78.3 ± 1.7
Cyanidin-dipentosyl-hexoside 17.0 ± 0.8 201.4 ± 7.5 54.7 ± 0.1
Cyanidin-rutinoside n.d. 1657.2 ± 15.5 1184.8 ± 3.0
Peonidin-rutinoside n.d. 88.2 ± 0.5 82.3 ± 2.4
Sum of hydroxycinnamic acids n.d. 2947.2 ± 12.5 5178.1 ± 11.3
Sum of flavonols n.d. 473.1 ± 7.4 4522.1 ± 23.4
Sum of flavonol aglycons n.d. n.d. 46.2 ± 1.3
Sum of anthocyanins 1269.2 ± 23.3 6722.9 ± 92.9 2438.5 ± 10.1
Total phyto-compounds 1269.2 ± 23.3 10143.2 ± 73.0 12184.9 ± 23.6

Values are means ± standard deviations (SD) [mg/100 g] n = 2; n.d., not detected.

kaempferol, isorhamnetin, naringenin (Fig. 2), and phlorizin gly-
cosides was proved.
Additionally, the presence of 4-p-coumaroylquinic acid was
proved in the extract. Pseudo-molecule of the compound gave 337
(m/z) and fragmented to 173 (m/z). The same fragmentation was
shown by Clifford et al. (2003). Procyanidins, i.e. two dimers, three
tetramers and one pentamer were also found in the extract. The
presence of (−)-epicatechin was found as well. Two quercetin gly-
cosides: 3-O-rutinoside and 3-O-glucoside were also present in
the extract. Pseudo-molecules of those compounds gave 609 and
463 (m/z), respectively, and fragmented to 301 (m/z). Similarly to
quercetin glycosides the presence of kaempferol and isorhamnetin
3-O-rutinosides and 3-O-glucosides was proved in the extract,
kaempferol glycosides fragmented to 285 (m/z) ion, while isorham-
netin glycosides fragmented to 315 (m/z) ion. CHPE3 extract was
characterized by phlorizin presence as well. The UV spectrum of
the compound gave the maximum at 280 nm, and pseudo-molecule
435 (m/z) fragmented to 273 (m/z). The presence of above compo-
nents was confirmed by the comparison with the available standard
as well, which gave the same values of Rt , UV–vis maximum
and pseudo-molecules. The presence of two naringenin glycosides
was proved basing on the main pseudo-molecules fragmentations,
which were 433 (m/z) for both compounds, and fragmented to
271 (m/z) ion. In addition, both components were characterized
by UV–vis spectrum characteristic of the compounds, i.e. the maxi-
mum at 280 nm. Naringenin-7-O-glucoside in sour cherry fruit and
press cake was tentatively identified by Toydemir et al. (2013). The
possible presence of genistein, the compound of pseudo-molecule
431 (m/z), fragmenting to 269 (m/z) was found in the extract. Nev-
ertheless, the evidence of the presence of naringenin and genistein
glycosides requires further research, especially the comparison
with available standards.
3.2. Total polyphenol content of sour cherry pomace extracts
The results of polyphenol determination by Folin-Ciocalteau
method proved that there is a high content of polyphenols in sour
cherry extracts (Table 4). The extract CHPE3 was characterized by
the highest content of polyphenols, CHPE2 had a slightly lower con-
tent, while CHPE1 contained the least polyphenols. The contents
of polyphenols were 66 g/100 g, 48 g/100 g and 6.3 g/100 g for
CHPE3, CHPE2 and CHPE1, respectively. This data indicate that the
technology applied for extraction and purification is a good method
for the production of polyphenol extracts from industrial wastes.
As described by Cilek et al. (2012) the content of polyphenols in
the extracts obtained from sour cherry pomace was at the level
of 9.1 g/100 g GAE (gallic acid equivalent), however the extracts
obtained by water: ethanol 1:1 (v/v) extraction were not purified.
In the research presented water was used for the extraction of
polyphenols; thus, water can be used for the extraction of pomace
in every juice-producing plant.
According to Adil et al. (2008), the use of high pressures
(50–200 MPa) and organic solvents, such as ethanol can signifi-
cantly increase the efficiency of the extraction from sour cherry
pomace, yet the use of special equipment, which proves to be
expensive in the industrial practice, is required.
3.3. Flavanol content in sour cherry pomace extract
As the research on flavanol content by the use of thiolysis tech-
nique proved the significant part of polyphenols in researched
extracts are flavanols (proanthocyanidins + catechins) (Table 4).
CHPE3 extract was characterized by the highest flavanols contents
35.1 g/100 g extract, twice as much compared to CHPE2. No proan-
thocyanidins presence was proved in CHPE1. The average degree
of polymerization of flavanols in CHPE2 and CHPE3 was 3.3 and
3.4, respectively. The research proved that polymeric proantho-
cyanidins of sour cherry extracts hydrolyzed to terminal units:
(+)-catechin, (−)-epicatechin, while extender units were in adduct
form: (−)-epicatechin-benzylthioether. It should be noted, that the
predominant part of the terminal unit in the extracts was (−)-
epicatechin (Fig. 1). According to Toydemir et al. (2013) the degree
of polymerization of flavanols in sour cherry pomace is at the
level of 2.6, and the value is higher than in the fruits (2.1). Higher
polymerization degree in pomace results from easier transfer of
proanthocyanidins of lower polymerization degree (White et al.,
2011; Pérez-Jiménez and Torres, 2011). The content of proantho-
cyanidins in sour cherry pomace has been little documented until
present. Previous work showed that the content of procyanidins
in cherry pomace depended on the cultivar and fruit transforma-
tion technology, and varied between 0.5% and 2.9% in dry substance
(unpublished data). The degree of polymerization of the procyani-
dins varied between 4.8 and 14.5, where (−)-epicatechin was the
main terminal and extender unit.
 K. Kołodziejczyk et al. / Industrial Crops and Products 51 (2013) 279–288
Fig. 1. HPLC (280 nm) chromatogram of thiolysed procyanidins from CHPE3 cherry pomace extract.
3.4. Phenolic acids, anthocyanins and flavonols content in sour
cherry pomace extract
The content of polyphenols determined by HPLC in polyphenol
extracts obtained from industrial sour cherry pomaces is presented
in Table 3. Sour cherry extract CHPE1 contained anthocyanins at a
level of 1.2%, where, typically of sour cherry, cyanidin-3-glucosyl-
rutinoside was predominant. CHPE2 extract contained much more
anthocyanins: 6.7%. The main anthocyanins of the extract, similarly
to CHPE1, were cyanidin-3-glucosyl-rutinoside and cyanidin-
3-rutinoside. The significant amounts of cafeoylquinic acids,
p-coumaroylquinic acids and flavonols were found in CHPE2 as
well. The content of neochlorogenic acid in this extract was
at a level of 1460 mg/100 g, chlorogenic acid 633 mg/100 g, and
p-coumaroylquinic acid 853 mg/100 g. Quercetin rutinoside and
glucoside can be considered the main flavonols in the extract—their
total content was 312 mg/100 g. Besides, kaempferol rutinoside
and isorhamnetin rutinoside presence was detected, and the
presence of two quercetin triglycosides was recorded (Table 2).
CHPE3 extract was also characterized by high polyphenol con-
tent, hydroxycinnamic acids (above 5000 mg/100 g), anthocyanins
(above 2000 mg/100 g) and significant amounts of quercetin,
kaempferol and isorhamnetin rutinosides (Fig. 2). The noticeable
amounts of quercetin, kaempferol and isorhamnetin rutinosides in
sour cherry fruits were previously proved by Kim et al. (2005), when
the levels of individual substances depended on cultivar. Besides
the components determined (Table 3), the sour cherry extracts con-
tained lower amounts of other polyphenol components, described
in the “identification” section.
3.5. Antioxidant activity of sour cherry pomace extracts
The antioxidant activity of researched extracts correlated to the
content of polyphenols. Sour cherry extracts CHPE2 and CHPE3
285
revealed antioxidant activity above 1200 ␮M/g TEAC, while CHPE1
gave 192 ␮M/g TEAC (Table 4). The antioxidant activity of the
obtained extracts was ca. 3 times lower, compared to the previ-
ously described extracts obtained from black currant pomace (Sójka
et al., 2009). It should be noticed, that black currant extracts were
purified on more selective octadecyl bed, which made it possible
to obtain the extracts of better purity and higher concentration.
According to the research by Rødtjer et al. (2006), the extracts
obtained from cherry liqueur pomace by means of water extraction
demonstrate stronger antioxidant properties compared to extracts
obtained by means of water-soluble organic extractants, such as
ethanol, methanol or acetone. In this case, despite the increase in
the amount of extracted polyphenols, the free radical scavenging
capability did not increase. The same authors have also shown that
a high concentration of polyphenols in relation to a low concen-
tration of iron in the model/standard solution is a pro-oxidizing
character, which can be important when polyphenols are used as
food additives.
A high antioxidant capacity of cherries and extract obtained
from cherries is shown in many published works, e.g.: Kirakosyan
et al. (2009), Piccolella et al. (2008), Garcia-Alonso et al. (2004),
Halvorsen et al. (2002), Wang et al. (1999). The antioxidant activity
of both (CHPE2 and CHPE3) extracts is also of great interest as their
potential to be used as natural preservatives in the food industry,
e.g. to prevent browning of fresh-cut fruits, but their efficacy should
first be determined.
3.6. Antimicrobial activity of sour cherry polyphenol extracts
3.6.1. Bacteriostatic effect
CHPE1 extract did not completely inhibit Salmonella, E. coli
O157:H7 and Listeria spp. growth at the tested concentrations
(Fig. 3A), it only slightly reduced the growth of Salmonella and Lis-
teria spp. In contrast, CHPE2 inhibited the growth of Listeria spp.
286 K. Kołodziejczyk et al. / Industrial Crops and Products 51 (2013) 279–288

Table 4
Total phenolics content (TPC) determined by FC method, total flavanols (procyanidins + catechins) content determined by thiolysis method and anti-oxidant activity (AA)
determined by DPPH method of cherry pomace phenolic extracts (CHPE).

Extract TPC [g/100 g]a ± SD Total flavanols AA [␮M/g TEAC]c ± SD

[g/100 g]b ± SD DPnd

CHPE1 6.35 ± 0.07 – – 192 ± 2

CHPE2 48.55 ± 0.16 18.3 ± 2.2 3.3 ± 0.0 1207 ± 22
CHPE3 66.09 ± 0.35 35.1 ± 0.6 3.4 ± 0.0 1628 ± 42

Values are means ± standard deviations (SD), n = 2.

a
Total phenolics content determined by the FC method calculated on (−)epicatechin.
b
The content of flavanols determined by the thiolysis method.
c
Anti-oxidant activity determined by the DPPH method, calculated as ␮M TEAC g−1 .
d
Degree of flavanols polymerization.

when tested at 2500 ␮g/mL or above (Fig. 3B). At these concen-
trations it also significantly reduced the growth of Salmonella and
E. coli O157:H7 when compared with the medium without cherry
extracts. CHPE3 was also effective in reducing Salmonella and E. coli
O157:H7 growth but it did not fully prevent it (Fig. 3C). Concerning
L. monocytogenes, MIC concentration for CHPE3 was higher than for
CHPE2.
3.6.2. Bactericidal effect
The results demonstrated that none of the extracts had bac-
tericidal activity against the Gram-negative Salmonella and E. coli
O157:H7 at a high dose (10,000 ␮g/mL, Table 5) when put in direct
contact in an aqueous solution for 5 min. Two of the extracts (CHPE2
and CHPE3) had antilisterial activity at 10,000 ␮g/mL and for that
reason they were selected for further experiments. There was a
positive correlation between the reduction in the population in the
solution with increasing polyphenol concentration (Table 6). Both
extracts at concentrations higher than 1000 ␮g/mL reduced Listeria
spp. below the detection limit (data not shown). At lower concen-
trations, CHPE3 at 250 ␮g/mL or above reduced Listeria spp. more
than 2-log units (Table 6) while concentrations above 500 ␮g/mL
were needed to obtain similar reduction values with CHPE2 extract.
The reduction values obtained for both polyphenol extracts were
not significantly different at concentrations >750 ␮g/mL.
In our study, the efficacy of sour cherry polyphenol extracts
was tested by two methods. Complementary and to some extent
contradictory results were observed. Although cherry extracts
did not have a bactericidal effect against Salmonella and E. coli
O157:H7 at the tested concentrations, CHPE2 and CHPE3 reduced
their growth in a liquid medium, thus demonstrating bacteriostatic
effect but they did not completely inhibit their growth. On the con-
trary, CHPE2 and CHPE3 tested at 1000 ␮g/mL in aqueous solution
for 5 min reduced Listeria spp. population below the detection limit,
meanwhile they only reduced their growth when tested in a liq-
uid growth medium for 16 h. These extracts also had a bactericidal
effect against the Gram-positive Listeria spp. strains tested. Similar
trends were observed in other studies with crude hexane and chlo-
roform extracts from the fruit rinds of Garcinia cowa and Garcinia
pedunculata (Negi et al., 2008), extracts from some Asian edible
plants (Alzoreky and Nakahara, 2002) and pome fruit pulp and peel
aqueous acetone extracts (Fattouch et al., 2008). However, Fattouch
et al. (2008) also found some activity against the Gram-negative
Pseudomonas aeruginosa (tested using the agar and well diffu-
sion method). The reason for a higher sensitivity of Gram-positive
bacteria could be ascribed to their differences in cell membrane
constituents and their arrangement. The Gram-positive bacteria
contain an outer peptidoglycan layer, which is an ineffective per-
meability barrier (Scherrer and Gerhardt, 1971) and the resistance
of Gram-negative bacteria towards antibacterial substances may
be due to the outer phospholipidic membrane carrying the struc-
tural lipopolysaccharide components, which makes it impermeable
to lipophilic solutes whereas porins constitute a selective barrier to

Fig. 2. HPLC chromatogram of flavonols at 360 nm of CHPE3 cherry pomace extract.

 K. Kołodziejczyk et al. / Industrial Crops and Products 51 (2013) 279–288 287

Table 5
Population of the studied foodborne pathogens (log cfu/mL) after 5 min contact in a water solution (CK-Water) or in the studied sour cherry extract polyphenols at
10,000 ␮g/mL. Results in brackets represent the reduction value. Detection limit was 3.4 log cfu/mL.

Extract Salmonella (log cfu/mL) Listeria (log cfu/mL) E. coli (log cfu/mL)

CK-Water 7.2 7.2 7.1

CHPE1 7.2 (0.0) 7.0 (0.2) 7.1 (0.0)
CHPE2 7.2 (0.0) 3.7 (3.5) 7.2 (−0.1)
CHPE3 7.2 (0.0) <3.40 (>3.8) 7.2 (−0.1)

Table 6
Reduction (log cfu/mLinitial − log cfu/mLafter 5 min contact ) of the 3-strain Listeria spp. cocktail after 5 min contact with aqueous solutions of selected polyphenol extracts at different
concentrations.

Concentration (␮g/mL)

Extract 0 50 100 250 500 750 1000

CHPE2 0e 0.41d 0.43d 0.98d* 1.92c* 3.49b 5.73a
CHPE3 0q 0.39p 0.52p 2.14o* 4.60n* 4.20n 5.73m

Initial concentration was 7 log cfu/mL. Values represent means of 4 replications. In the same line, different letters indicate significant differences (P < 0.05) according to a
Duncan Test. Within the same concentration, an asterisk (*) indicates differences between polyphenol fraction. Detection limit was 1.4 log cfu/mL.

the hydrophilic solutes (Nikaido and Vaara, 1985). However, nature

bergamot (Citrus bergamia Risso) peel fractions were inhibitory to
Gram-negative bacteria only (Mandalari et al., 2007) and the Gram-
positive Bacillus subtilis at a high concentration (<1000 ␮g/mL).
Comparing the three extracts, it seems that a higher bac-
tericidal effect was found in those extracts with higher TPC
(CHPE2 and CHPE3). Between both extracts, differences were only
observed when tested at concentrations <750 ␮g/mL. The differ-
ences between them were, in general, a higher content of phenolics
of CHPE3. Fattouch et al. (2007) found that chlorogenic acid was
the strongest inhibitor exhibiting activity against Staphylococcus
aureus, P. aeruginosa, E. coli and Candida albicans and quercitin
and kaempferol activities were lower than other phenolics. Other
authors (Hakkinen et al., 1999) reported that berry extract’s
inhibitory effect was a synergistic effect of various phenolic com-
pounds. In general, the mechanisms thought to be responsible for
phenolic toxicity to microorganisms include adsorption and disrup-
tion of microbial membranes, enzyme inhibition by the oxidized
compounds possibly through reaction with sulfhydryl groups or
through more non-specific interactions with the proteins, and sub-
strate and metal ion deprivation (Cowan, 1999). The activity of
flavones and flavonoids is probably due to their ability to complex
with extracellular and soluble proteins and to complex with bacte-
rial cell walls. More lipophilic flavonoids may also disrupt microbial
membranes (Cowan, 1999).

4. Conclusions

Fresh industrial sour cherry pomace is a good raw material

Fig. 3. Growth of OD (Absorbance at 600 nm) of Salmonella ( ), E. coli () and
Listeria spp. () in liquid medium containing different concentrations of the sour
cherry polyphenol extract: CHPE1 (A), CHPE2 (B) and CHPE3 (C)
for preparation of polyphenol-rich extracts. The application of
purification technology of raw water extracts on polymer bed
type Amberlite XAD makes it possible to easily obtain polyphe-
nol extracts rich in anthocyanins (cyaniding-glucoside-rutinoside
most of all), hydroxycinnamic acids (mainly chlorogenic acid,
neochlorogenic acid and 3-p-coumaroylquinic acid), flavanols
and flavonl glycosides (quercetin, kaempferol and isorhamnetin
glycosides). Researched extracts were characterized by proantho-
cyanidin dimers, trimer, tetramers and pentamers presence. The
quantification proved the flavanols to be predominant group in
the extracts, with the content up to 35 g/100 g. Polyphenol rich
extracts CHPE2 and CHPE3 expressed particular antilisterial activ-
ity in aqueous solutions. For the Gram-negative Salmonella and
E. coli O157:H7 only reduction of growth at relatively high con-
centration of extracts was observed. Considering high antioxidant
and antilisterial capacity and beneficial biological properties of high
concentrated sour cherry extracts, the possibility of use of such
288 K. Kołodziejczyk et al. / Industrial Crops and Products 51 (2013) 279–288
preparations as a potential food product supplement and their use
as natural antioxidant and preservative is an interesting option.
Acknowledgements
The research was financially supported by 6 Framework Pro-
gram EU project “ISAFRUIT”. The ISAFRUIT project is funded by the
European Commission under the Thematic Priority 5-Food Quality
and Safety of the 6th Framework Programme of RTD (Contract no.
FP6-FOOD-CT-2006-016279).
References
Adil, İ.H., Yener, M.E., Bayındırh, A., 2008. Extraction of total phenolics of sour cherry
pomace by high pressure solvent and subcritical fluid and determination of
antioxidant activities of the extracts. Sep. Sci. Technol. 43, 1091–1110.
Alberto, M.R., Canavosio, M.A.R., Manca de Nadra, M.C., 2006. Antimicrobial effect
of polyphenols from apple skins on human bacterial pathogens. Electron. J.
Biotechnol. 9, 205–209.
Alzoreky, N.S., Nakahara, K., 2002. Antibacterial activity of extracts from some edible
plants commonly consumed in Asia. Int. J. Food Microbiol. 80, 223–230.
Baydar, N.G., Özkan, G., Sağdiç, O., 2004. Total phenolic contents and antibacterial
activities of grape (Vitis vinifera L.) extracts. Food Control 15, 335–339.
Beuchat, L.R., 1998. Surface decontamination of fruits and vegetables eaten raw: a
review. In: WHO/FSF/FOS/98.2. Food Safety Unit. World Health Organisation,
Available from http://www.who.int/foodsafety/publications/fs management/
en/surface decon.pdf (accessed 30.01.2010).
Chandra, A., Rana, J., Li, Y., 2009. Separation, identification, quantification, and
method validation of anthocyanins in botanical supplement raw materials by
HPLC and HPLC-MS. J. Agric. Food Chem. 49, 3515–3521.
Cilek, B., Luca, A., Hasirci, V., Sahin, S., Sumnu, G., 2012. Microencapsulation of
phenolic compounds extracted from sour cherry pomace: effect of formula-
tion, ultrasonication time and core to coating ratio. Eur. Food Res. Technol. 235,
587–596.
Clifford, M.N., Johnston, K.L., Knight, S., Kuhnert, N., 2003. Hierarchical scheme for
LC-MSn identification of chlorogenic acids. J. Agric. Food Chem. 51, 2900–2911.
Cowan, M.M., 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12,
564–582.
D’Ischia, M., Panzella, L., Manini, P., Napolitano, A., 2006. The chemical basis of
the antinitrosating action of polyphenolic cancer chemoprotective agents. Curr.
Med. Chem. 13, 3133–3144.
European Comission Eurostat, 2010. Available from http://nui.epp.eurostat.
ec.europa.eu (accessed 15.01.2010).
Fattouch, S., Caboni, P., Coroneo, V., Tuberoso, C., Angioni, A., Dessi, S., Marzouki,
N., Cabras, P., 2008. Comparative analysis of polyphenolic profiles and antioxi-
dant and antimicrobial activities of Tunisian pome fruit pulp and peel aqueous
acetone extracts. J. Agric. Food Chem. 56, 1084–1090.
Fattouch, S., Caboni, P., Coroneo, V., Tuberoso, C.I.G., Angioni, A., Dessi, S., Marzouki,
N., Cabras, P., 2007. Antimicrobial activity of Tunisian quince (Cydonia oblonga
Miller) pulp and peel polyphenolic extracts. J. Agric. Food Chem. 55, 963–969.
Friedman, M., Henika, P.R., Mandrell, R.E., 2002. Bactericidal activities of plant essen-
tial oils and some of their isolated constituents against Campylobacter jejuni,
Escherichia coli, Listeria monocytogenes and Salmonella enterica. J. Food Protect.
65, 1545–1560.
Garcia-Alonso, M., Pascul-Teresa, S., Santos-Buelga, C., Rivas-Gonzalo, J.C., 2004.
Evaluation of the antioxidant properties of fruits. Food Chem. 84, 13–18.
Guyot, S., Marnet, N., Laraba, D., Sanoner, P., Drilleau, J.F., 1998. Reversed-phase
HPLC following thiolysis for quantitative estimation and characterization of the
four main classes of phenolics compounds in different tissue zones of a French
cider apple variety (Malus domestica var. Kermerrien). J. Agric. Food Chem. 46,
1698–1705.
Guyot, S., Marnet, N., Sanoner, P., Drilleau, J-F., 2001. Direct thiolysis on crude
apple materials for high-performance liquid chromatography characterization
and quantification of polyphenols in cider apple tissues and juices. Methods
Enzymol. 335, 57–70.
Hakkinen, S.H., Karenlampi, S.O., Heinonen, I.M., Mykkanen, H.M., Torronen, A.R.,
1999. Content of the flavonols quercetin, myricetin, and kaempherol in 25 edible
berries. J. Agric. Food Chem. 47, 2274–2279.
Halvorsen, B.L., Hotle, K., Myhrstad, M.C.W., Barikmo, I., Hvattum, E., Remberg, S.F.,
Wold, A-B., Haffner, K., Baugerod, H., Andersen, L.F., Moskaug, J.O., Jacobs, D.R.,
Blomhoff, R., 2002. A systematic screening of total antioxidants in dietary plants.
J. Nutr. 132, 461–471.
Kim, D-O., Heo, H.J., Kim, Y.J., Yang, H.S., Lee, C.Y., 2005. Sweet and sour cherry
phenolics and their protective effects on neuronal cells. J. Agric. Food Chem.
53, 9921–9927.
Kim, D-O., Lee, K.W., Lee, H.J., Lee, C.Y., 2002. Vitamin C equivalent antioxi-
dant capacity (VCEAC) of phenolic phytochemicals. J. Agric. Food Chem. 50,
3713–3717.
Kirakosyan, A., Seymour, E.M., Llanes, D.E.U., Kaufman, P.B., Bolling, S.F., 2009. Chem-
ical profile and antioxidant capacities of tart cherry products. Food Chem. 115,
20–25.
Lee, E.-R., Kang, G.-H., Cho, S.-G., 2007. Effect of flavonoids on human health: old
subjects but new challenges. Recent Pat. Biotechnol. 1, 139–150.
Mandalari, G., Bennett, R.N., Bisignano, G., Trombetta, D., Saija, A., Faulds, C.B., Gas-
son, M.J., Narbad, A., 2007. Antimicrobial activity of flavonoids extracted from
bergamot (Citrus bergamia Risso) peel, a byproduct of the essential oil industry.
J. Appl. Microbiol. 103, 2056–2064.
Mozetič, B., Simčič, M., Trebše, P., 2006. Anthocyanins and hydroxycinnamic acids
of Lambert Compact cherries (Prunus avium L.) after cold storage and 1-
methylcyclopropene treatment. Food Chem. 97, 302–309.
Negi, P.S., Jayaprakasha, G.K., Jena, B.S., 2008. Antibacterial activity of the extracts
from the fruit rinds of Garcinia cowa and Garcinia pedunculata against
food borne pathogens and spoilage bacteria. LWT-Food Sci. Technol. 41,
1857–1861.
Nikaido, H., Vaara, M., 1985. Molecular basis of bacterial outer membrane perme-
ability. Microbiol. Rev. 49, 1–42.
Pérez-Jiménez, J., Torres, J.L., 2011. Analysis of nonextractable phenolic compounds
in foods: the current state of the art. J. Agric. Food Chem. 59, 12713–12724.
Piccolella, S., Fiorentino, A., Pacifico, S., D’Abrosca, B., Uzzo, P., Monaco, P., 2008.
Antioxidant properties of sour cherries (Prunus cerasus L.): role of colorless phy-
tochemicals from the methanolic extracts of ripe fruits. J. Agric. Food Chem. 56,
1928–1935.
Rissanen, T.H., Voutilainen, S., Virtanen, J.K., Venho, B., Vanharanta, M., Mursu, J.,
Salonen, J.T., 2003. Low intake of fruits, berries and vegetables is associated with
excess morality in men: the Kuopio ischaemic heart disease risk factor (KIHD)
study. J. Nutr. 133, 199–204.
Rødtjer, A., Skibsted, L.H., Andersen, M.L., 2006. Antioxidative and prooxidative
effects of extracts made from cherry liqueur pomace. Food Chem. 99, 6–14.
Sanchez-Rabaneda, F., Jauregui, O., Lamuela-Raventos, R.M., Bastida, J., Viladomat,
F., Codina, C., 2003. Identification of phenolic compounds in artichoke waste by
high performance liquid chromatography–tandem mass spectrometry. J. Chro-
matogr., A. 1008, 57–72.
Sanchez-Rabaneda, F., Jauregui, O., Lamuela-Raventos, R.M., Viladomat, F., Bastida,
J., Codina, C., 2004. Qualitative analysis of phenolic compounds in apple pomace
using liquid chromatography coupled to mass spectrometry in tandem mode.
Rapid Commun. Mass Spectrom. 18, 553–563.
Scherrer, R., Gerhardt, P., 1971. Molecular sieving by the Bacillus megatrium cell wall
and protoplast. J. Bacteriol. 107, 718–735.
Simunic, V., Kovac, S., Gaso-Sokac, D., Pfannhauser, W., Murkovic, M., 2005. Deter-
mination of anthocyanins in four Croatian cultivars of sour cherries (Prunus
cerasus). Eur. Food Res. Technol 220, 575–578.
Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16,
144–158.
Sofos, J.N., Beuchat, L.R., Davidson, P.M., Johnson, E.A., 1998. Naturally Occurring
Antimicrobials in Food: Task Force Report No. 132. Council for Agricultural Sci-
ence and Technology, Ames, IA.
Sójka, M., Guyot, S., Kołodziejczyk, K., Król, B., Baron, A., 2009. Composition of puri-
fied phenolics preparations obtained from an extract of industrial blackcurrant
(Ribes nigrum L.) pomace. J. Hortic. Sci. Biotechnol. (ISAFRUIT Special Issue),
100–106.
Sójka, M., Król, B., 2009. Composition of industrial seedless black currant pomace.
Eur. Food Res. Technol. 228, 597–605.
Toydemir, G., Capanoglu, E., Gomez Roldan, M.V., C.H. de Vos, R., Boyacioglu, D., Hall,
R.D., Beekwilder, J., 2013. Industrial processing effects on phenolic compounds
in sour cherry (Prunus cerasus L.) fruit. Food Res. Int. 53, 218–225.
Tomas-Barberan, F.A., Gil, M.I., Cremin, P., Waterhouse, A.L., Hess-Pierce, B., Kader,
A.A., 2001. HPLC-DAD-ESIMS analysis of phenolic compounds in nectarines,
peaches, and plums. J. Agric. Food Chem. 49, 4748–4760.
Wang, H., Nair, M.G., Strasburg, G.M., Booren, A.M., Gray, J.I., 1999. Antioxi-
dant polyphenols from tart cherries (Prunus cerasus). J. Agric. Food Chem. 47,
840–844.
White, B.L., Howard, T.R., Prior, R.L., 2011. Impact of different stages of juice
processing on the anthocyanins, flavonol, and procyanidin contents of cranber-
ries. J. Agric. Food Chem. 59, 4692–4698.
Yılmaz, C., Gökmen, V., 2013. Compositional characteristics of sour cherry kernel
and its oil as influenced by different extraction roasting conditions. Ind. Crops
Prod. 49, 130–135.