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Evaluation of the effect of high pressure on

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Innovative Food Science and Emerging Technologies

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

Evaluation of the effect of high pressure on total phenolic content,

antioxidant and antimicrobial activity of citrus peels
Rocío Casquete a,b, Sonia Marilia Castro b,c, Alberto Martín a, Santiago Ruiz-Moyano a,
Jorge A. Saraiva c, María G. Córdoba a, Paula Teixeira b,⁎
a
Nutrición y Bromatología, Escuela de Ingenierías Agrarias, Universidad de Extremadura, Badajoz, Spain
b
CBQF-Centro de Biotecnologia e Química Fina—Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Dr. António Bernardino Almeida,
4200-072 Porto, Portugal
c
QOPNA—Departamento de Química, Universidade de Aveiro, Campus Santiago, Aveiro, Portugal

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

Article history: This study aimed to assess the effect of high pressure at 300 and 500 MPa for 3 and 10 min on the phenolic
Received 12 March 2015 compounds, antioxidant capacity and antimicrobial activity of citrus peel extracts. Total phenolic contents
Received in revised form 9 July 2015 (TPC) and antioxidant properties of extracts were determined as free radical-scavenging ability of DPPH and
Accepted 20 July 2015
using the ABTS radical cation decolorization assay. Additionally, extracts were tested for antimicrobial activity
Available online xxxx
against twenty different strains of bacteria representing both Gram-positive and Gram-negative types. Citrus
Keywords:
peel extracts demonstrated antimicrobial activity against a wide range of bacteria. The maximum level of TPC
Radical cation decolorization as well as antioxidant capacity were observed at 300 MPa for 3 min. Citrus peels extracts demonstrated antimi-
ANTIMICROBIAL activity crobial activity against a wide range of microorganisms. The antimicrobial activity of orange peel extract was the
Citrus by-products highest among the four citrus peels studied. Generally, bacteria Acinetobacter and the strain Listeria innocua were
Purple-coloured solution of 1 more sensitive to the peel extracts.
1-Diphenyl-2-picrylhydrazyl radical © 2015 Elsevier Ltd. All rights reserved.
High pressure
Total phenolic content

1. Introduction Citrus peels contain a high concentration of phenolic compounds

Citrus is one of the most abundant crops in the world. Its worldwide
production is over 88 million tons and one-third of the crop is proc-
essed. Oranges, lemons, grapefruits, limes and mandarins represent
approximately 98% of the entire industrialized crop, oranges being the
most relevant with approximately 82% of the total (Izquierdo &
Sendra, 2003).
Produced in tonnes per day, citrus by-products represent a problem
for management, pollution, and environmental issues, due to microbial
spoilage (Laufenberg, Kunz, & Nystroem, 2003; Montgomery, 2004).
Citrus by-product wastes have been traditionally utilized as molasses
for animal feed (Mirzaei-Aghsaghali & Maheri-Sis, 2008), fiber (pectin)
(Chou & Huang, 2003) and fuel production (Llorach, Espin, Tomas-
Barberan, & Ferreres, 2003). However, researchers are looking for new
uses of these by-products such as production of food additives or sup-
plements with high nutritional value (Delgado-Adámez, Gamero,
Valdés, & González-Gómez, 2012b; Pokorny & Parkanyiova, 2004).
⁎ Corresponding author. Tel.: +351 225580001; fax: +351 22 5090351.
E-mail address: pcteixeira@porto.ucp.pt (P. Teixeira).
and represent a rich source of natural flavonoids (Hayat et al., 2010;
Kamran, Youcef, & Ebrahimzadeh, 2009); these are abundant in the
plant. In addition, there are several compounds such as flavanones,
flavanone glycosides and polymethoxylated flavones unique to
citrus, which are relatively rare in other plants (Manthey &
Grohmann, 2001; Montgomery, 2004). It has been reported that
these compounds have high antioxidant activity (El-Seedi et al.,
2012; Hayat et al., 2010) and exert antimicrobial effects against
foodborne pathogens (Delgado-Adámez, Fernández-León, Velardo–
Micharet, & González-Gómez, 2012b, 2012a; DelgadoAdámez et al.,
2012b; Espina et al., 2011) due to their high contents of terpenoids,
tannins, quinones, phenolic acids and polyphenols (Calvo et al.,
2006; Lee & Lee, 2010). However, these compounds are usually present
in a covalently bound form (Xu, Ye, Chen, & Liu, 2007). Therefore, reliable
and practical methods for liberation of natural antioxidants and antimi-
crobial compounds from plant materials are of considerable interest.
For the analysis and exploitation of bioactive constituents their
extraction from the cellular matrix is needed. The conditions for the
extraction process depend on factors that have a direct influence on
the raw material and they must be fixed to obtain the highest antioxi-
dant and antibacterial activity (Pinelo, Rubilar, Sineiro, & Nunez, 2004).

http://dx.doi.org/10.1016/j.ifset.2015.07.005
1466-8564/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: Casquete, R., et al., Evaluation of the effect of high pressure on total phenolic content, antioxidant and antimicrobial
activity of citrus peels, Innovative Food Science and Emerging Technologies (2015), http://dx.doi.org/10.1016/j.ifset.2015.07.005
2 R. Casquete et al. / Innovative Food Science and Emerging Technologies xxx (2015) xxx–xxx

Several methods are used to extract and activate low-molecular Table 1

weight natural antioxidants and antimicrobials using nonconventional Bacterial strains used as target organisms to assess the antimicrobial activity
of the extracts.
technologies such as microwave and ultrasound-assisted extraction,
supercritical fluid extraction, high temperature/high pressure extrac- Bacterial strains Source
tion and pressurized liquid extraction (Ben Hamissa et al., 2012; Gram-positive
Vilkhu, Mawson, Simons, & Bates, 2008). Several of these methods Bacillus subtilis ESBCC 01
require high temperatures and many organic compounds are heat- Enterococcus faecalis ATCC 29212
Staphylococcus aureus ESBCC 81
sensitive. Therefore they will be degraded, lose biologic activity, or
Listeria innocua PHLS 2030c
change into another compound (Zhang, Junjie, & Changzhen, 2004). Listeria monocytogenes ESBCC 3391
High pressure extraction (HPE) has been initially attempted by Listeria monocytogenes ESBCC 2264
Shouqin, Jun, and Changzheng (2005) to obtain natural plant materials. Listeria monocytogenes ESBCC 1940/1
The inner membrane compounds are extracted due to cell membrane Listeria monocytogenes ESBCC 1853/3
Clostridium sporogenes ESBCC 01
rupture under pressure. High pressure ranging from 100 to 800 MPa Clostridium difficile ESBCC V591359
or even more, up to 1000 MPa, is considered as an alternative extraction Gram-negative
method, which is proven to be fast, effective and heat degradation is Acinetobacter lwoffii ESBCC T7BT5
avoided (Corrales, Garcia, Butz, & Tauscher, 2008; Zhang, Xi, & Wang, Acinetobacter junii UGCC 889
Acinetobacter pittii ESBCC T1BP1
2005; Zhang et al., 2004). Furthermore, this technology has been used
Acinetobacter baumannii ESBCC 05
successfully for the extraction of flavonoids from propolis (bee glue) Klebsiella spp. ESBCC 01
and polyphenols from green tea (Zhang et al., 2004), anthocyanins from Escherichia coli ATCC 25922
grape skin (Corrales, Garcia, Butz, & Tauscher, 2008) and ginsenosides Proteus vulgaris ESBCC 01
from the roots of Panax ginseng (Zhang, Ruizhan, & Changzheng, 2007). Salmonella Typhimurium ESBCC 01
Pseudomonas spp. ESBCC 01
However, limited information is available on the application of high
Cronobacter sakazakii ATCC 51329
pressure to extract phenolic compounds in citrus by-products.
ESBCC: Culture Collection of Escola Superior de Biotecnologia.
The present study established a procedure for the extraction of phe-
ATCC: American Type Culture Collection.
nolic compounds from citrus peels and evaluated their antioxidant and PHLS: Public Health Laboratory Service, Colindale, London.
antimicrobial activity after different high pressure processes. UGCC: University of Gotemburg Culture Collection.

2. Materials and methods

2.1. Plant materials
Samples of lemon (Citrus limon), tahiti lime (Citrus latifolia), mandarin
(Citrus reticulata) and sweet orange (Citrus sinensis) used in this study
were obtained from a local supermarket in Porto, Portugal. Prior to extrac-
tion, fruits were peeled; peels were cut into pieces (ca. 0.25 cm2) and
stored at 4 °C until use.
2.2. Bacterial strains and media
Twenty bacterial strains, ten Gram-positive and ten Gram-negative
bacteria, deposited in the culture collection of Escola Superior de
Biotecnologia as frozen concentrates (− 80 °C) in Tryptone Soy Broth
(TSB; Lab M, UK) containing 30% (v/v) glycerol were used as target
strains to assess the antimicrobial activity of the extracts (Table 1).
2.3. Sample preparation
Each sample of citrus peels (ca. 15 g) was placed in a double-low
permeability polyamide–polyethylene bag and vacuum-sealed. Treat-
ments were conducted in a high pressure system (Unipress Equipment,
Model U33, Institute of High Pressure Physics, Warsaw, Poland). The
equipment has a pressure vessel of “35-mm diameter” and “100-mm
height” “100-mL capacity” surrounded by an external jacket, connected
to a thermostatic bath (Huber Compatible Control CC1, New Jersey,
USA) to control the temperature. As recommended from the manufac-
turer for this HPP equipment, a mixture of propylene glycol and water
(40:60) (Dowcal N fluid, Dow Chemical Company) was used as the
pressurizing fluid and to control the temperature in the external jacket.
Pressure was built up slowly (100 MPa/min) to minimize temperature
increases due to adiabatic heating, and depressurization took place in
less than 60 s. After pressure release, samples were immediately cooled
in an ice-water bath.
Five different batches were prepared: (i) control without pressure
treatments; pressure treatment of (ii) 300 MPa, 3 min; (iii) 300 MPa,
10 min; (iv) 500 MPa, 3 min; (v) 500 MPa, 10 min. All pressure treat-
ments were applied at 10 °C. Each condition was made in triplicate.
2.4. Total phenolic content of peel
Ten grams of each citrus peel was homogenized with 60 mL of
solvent (80% aqueous ethanol, containing 1% conc. HCl). Phenolic com-
pounds were extracted as described by Casquete et al. (2014). Extrac-
tion was performed using a magnetic mixer for 1 h in the absence of
light at room temperature (25 °C) and filtered. This process was
repeated twice. Excess ethanol was removed by heating at 37 °C in a
rotary evaporator under vacuum. The resultant aqueous extracts (crude
extracts) were combined to a final known volume and total phenolic
content (TPC) was measured spectrophotometrically (λ 760 nm)
in a UV-2401PC spectrophotometer (Shimadzu Scientific Instru-
ments, Columbia, MD, USA) using the Folin–Ciocalteu reagent
(Wettasinghe & Shahidi, 1999). Results were expressed as equivalent
of gallic acid (mg GAE/100 g of fresh peel extracts).
2.5. Purification of the crude extract
The crude extract was purified using HyperSep C18 SPE Cartridges
(Thermo Scientific, Cartridge 500 mg, USA). In an initial stage, the
cartridge was preconditioned by sequentially passing 10 mL absolute
methanol and 10 mL deionized water from a Milli-Q water purification
system (Milipore, Bedford, MA, USA) through the cartridge. In a second
stage, the crude extract was loaded into the cartridge. The cartridge was
then washed by adding 10 mL of Milli-Q water. Finally, HPLC-grade
methanol was added to elute the phenolic compounds, and the fraction
was collected. Before antioxidant and antimicrobial activity analysis,
excess methanol was removed by heating at 37 °C in a rotary evaporator
under vacuum. The resultant extracts were combined to a final known
volume with Milli-Q water.
2.6. Total antioxidant activity
2.6.1. Determination of the total antioxidant activity by DPPH assay
The electron donation capacity of aqueous extracts diluted to
0.1 mg/mL with Milli-Q water was measured by bleaching of the
purple-coloured solution of 1,1-diphenyl-2-picrylhydrazyl radical

Please cite this article as: Casquete, R., et al., Evaluation of the effect of high pressure on total phenolic content, antioxidant and antimicrobial
activity of citrus peels, Innovative Food Science and Emerging Technologies (2015), http://dx.doi.org/10.1016/j.ifset.2015.07.005
 R. Casquete et al. / Innovative Food Science and Emerging Technologies xxx (2015) xxx–xxx
(DPPH) according to the method of Teixeira, Canelas, Martins do Canto,
Teixeira, and Dias (2009). The total antioxidant activity (TAA) was
expressed as mg of Trolox/100 g of fresh peel extracts.
2.6.2. Determination of the total antioxidant activity by ABTS assay
The free radical scavenging capacity of aqueous extracts was also
determined using the ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-
sulfonic acid)) radical cation decolorization assay, according to proce-
dure proposed by Cano, Acosta, and Armao (2000) slightly modified
(n = 3). Briefly, 1 mL of the radical cation ABTS (2.20-azinobis (3-
ethylbenz oithiazolone 6-sulphonate)) was placed in a spectrometric
cuvette and 20 μL of peel extract diluted to 0.1 mg/mL with Milli-Q
water were added. The initial absorbance value at λ 730 nm was then
compared with the absorbance obtained after 20 min of reaction. The
results were expressed as mg of Trolox/100 g of peel extracts.
2.7. Antimicrobial activity
Suspensions of target cells (Table 1) were prepared from overnight
cultures at 30 °C on TSA-YE, by transferring colonies into sterile Ringer's
solution (LabM) in order to obtain turbidity equivalent to 0.5 McFarland
standards. Next, 1 mL of each suspension was pipetted into separate ster-
ile petri dishes to which 20 mL of molten TSB-YE with 1% agar (45 °C)
were added. Once set, 10 μL of aqueous extracts with different concentra-
tions (1, 0.6, 0.3, 0.15, 0.08, 0.04 mg/mL) was added. Sterile distilled water
instead of active compounds was used as negative control. The plates
were incubated overnight at 37 °C, and the diameter (mm) of the
resulting zone of inhibition was measured.
2.8. Statistical analysis
Statistical analysis of the data was carried out using SPSS for Windows,
17.0. (SPSS Inc. Chicago, Illinois, USA). Descriptive statistics of the data
was determined, and the differences within and between groups were
studied by one-way analysis of variance (ANOVA) and separated by
Tukey's honest significant differences test (P b 0.01).
3. Results and discussion
3.1. Total phenolic content of peel
The influence of different treatments on TPC of lemon, lime, manda-
rin and orange peel is shown in Table 2. The TPC in control samples of
lemon, lime, mandarin and orange peel was 222.76, 362.98, 530.05
and 284.19 mg GAE/100 g fresh peel extracts, respectively. When ana-
lyzing the effect of high pressure on all citrus samples, it was observed
that whilst some treatments increased, others reduced the content of
TPC in the extracts (P b 0.01). The maximum TPC was observed at
300 MPa/3 min for all samples with values of 266.23, 397.21, 587.28
and 288.16 mg GAE/100 g fresh peel extracts for lemon, lime, mandarin
and orange, respectively. In mandarin peel the TPC also increased
(P b 0.01) when pressure-treated at 500 MPa/10 min (Table 2). High
Table 2
Total phenolic compounds of citrus peel expressed in mg of gallic acid equivalents (GAE)/100 g fresh citrus peels.
Lemon Lime
Mean DTE Mean
Control 222.67 ± 5.213 a1 362.98
300 MPa/3′ 266.23 ± 0.732 b1 397.21
300 MPa/10′ 230.09 ± 1.030a1 367.37
500 MPa/3′ 228.64 ± 3.766a1 347.30
500 MPa/10′ 231.24 ± 0.299 a1 361.19
Data represent means ± SD (n = 3).
a,b,c
Values with different superscripts are significantly different (P b 0.01) between batches.
1,2,3,4Values with different subscripts are significantly different (P b 0.01) between the citrus.
Please cite this article as: Casquete, R., et al., Evaluation of the effect of high pressure on total phenolic content, antioxidant and antimicrobial
activity of citrus peels, Innovative Food Science and Emerging Technologies (2015), http://dx.doi.org/10.1016/j.ifset.2015.07.005
3
pressure treatment resulting in disruption of the cellular walls and
hydrophobic bonds in the cell membrane of citrus peel produces chang-
es in the distribution and aggregation of phenolic compounds, allowing
increased solvent influx and increased contact with the solvent when
the compounds are extracted (Prasad, Yang, Yi, Zhao, & Jiang, 2009).
Therefore, this increase in total phenols may be related to an increased
extractability of some antioxidant components such as anthocyanins,
amino acids and proteins with phenolic hydroxyl groups after high
pressure treatments (Cao et al., 2011). Similar results were obtained
for Cape gooseberry (Vega-Gálvez et al., 2014).
During the process, high pressure is transferred uniformly and
instantly to the whole material. Thus, the equilibration of pressure
between the inside and outside of the cells could occur in a very short
time. Under these circumstances, the extraction of phenolic compounds
can reach the highest value very rapidly (Butz et al., 2003). This can
explain that the TPC for all samples was significantly (P b 0.01) higher
when extraction time was shorter (3 min).
3.2. Total antioxidant activity
Table 3 shows the antioxidant capacity of lemon, lime, mandarin and
orange peel, determined by means of two analyses (DPPH and ABTS).
Regarding DPPH, in control samples of lemon, lime, mandarin and
orange peel was 80.93, 53.11, 69.02 and 102.39 mg Trolox/100 g of
peel extracts, respectively. However, the values of ABTS for control sam-
ples varied between 235.60 to 451.56 mg Trolox/100 g of peel extracts.
The different levels obtained from these assays may indicate a relative
difference in the ability of antioxidant compounds in the extracts to
quench aqueous peroxyl radicals (Thaipong, Boonprakob, Crosby,
Cisneros-Zevallos, & Byrne, 2006). As can be observed from Table 3,
most treatments increased the antioxidant capacity (P b 0.01). Regard-
ing ABTS, all citrus peels (lemon, lime, mandarin and orange) presented
the highest values of antioxidant capacity at 300 MPa/3 min. Similar
results were observed in the DPPH, except for mandarin peel that
presented the highest value of antioxidant capacity at 500 MPa/10 min.
High pressure treatments influence the extraction yield of some bioactive
compounds. However, the effect of pressure on antioxidant activity is not
the same among different matrices. Data in the literature varies consider-
ably with the effect of both time and matrix (Keenan, Rößlea, Gormley,
Butler, & Brunton, 2012; Oey, Lille, Van Loey, & Hendrickx, 2008). Some
authors have reported that high hydrostatic pressure processing either
increases or maintains the antioxidant depending on different levels of
pressure and holding time (Di Scala et al., 2013).
The results reflect that the increase in antioxidant activity with high
pressure processing was promoted by the total phenolic contents. This
mechanism has been corroborated by previous studies (Prasad et al.,
2009, 2010). The reduction of DPPH and ABTS radicals by the peel citrus
extracts has been reported by several authors, by the presence of phenolic
compounds which easily reduce protons. Their capacity varies from one
compound to another and there is a synergy between them and/or
other constituents that might be present in the extracts (Lagha-
Benamrouchea & Madania, 2013).
Mandarin Orange
DTE Mean DTE Mean DTE
± 3.095 a3 530.05 ± 9.635 b4 284.19 ± 3.899 a2
± 12.38 b3 587.28 ± 11.02 c4 288.17 ± 0.965 a2
± 0.952 a3 491.05 ± 1.299 a4 282.34 ± 2.277 a2
± 10.31 a3 544.19 ± 17.42 b4 266.33 ± 1.334 a2
± 6.667 a3 577.58 ± 4.423 c4 280.05 ± 7.934 a2
4 R. Casquete et al. / Innovative Food Science and Emerging Technologies xxx (2015) xxx–xxx

Table 3
Antioxidant activity of citrus peels expressed in mg Trolox/100 g fresh citrus peels.

Lemon Lime Mandarin Orange

Mean SD Mean SD Mean SD Mean SD

DPPH Control 80.93 ± 8.886 a2 53.39 ± 0.698 a,b1 69.03 ± 2.149 a2 102.39 ± 0.659 a3
300 MPa/3′ 189.85 ± 9.516 d3 60.51 ± 0.695 b1 73.70 ± 4.040 a1 114.21 ± 1.477 a2
300 MPa/10′ 134.30 ± 6.626 b3 57.78 ± 0.843 b1 65.11 ± 3.435 a1 112.20 ± 1.177 a2
500 MPa/3′ 155.76 ± 9.716 c4 40.30 ± 0.403 a1 67.72 ± 5.510 a2 106.35 ± 1.509 a3
500 MPa/10′ 142.05 ± 2.148b3 47.47 ± 2.221 a,b1 100.30 ± 1.174 b2 101.82 ± 0.412 a2
ABTS Control 451.56 ± 41.560a2 235.38 ± 9.609 a,b1 301.08 ± 46.352 a1 304.05 ± 1.783 a1
300 MPa/3′ 780.48 ± 25.503c3 334.60 ± 17.579 c1 658.92 ± 32.090 c2 394.80 ± 4.930 b1
300 MPa/10′ 647.45 ± 24.533b3 267.77 ± 5.134a,b,c1 366.35 ± 9.342 a2 348.96 ± 8.464a,b1
500 MPa/3′ 762.57 ± 20.612c4 193.50 ± 8.311 a1 510.96 ± 46.335 b3 287.43 ± 5.509 a2
500 MPa/10′ 673.90 ± 57.476b3 246.10 ± 5.171 a,b1 586.18 ± 28.324b,c2 290.39 ± 5.205a1

Data represent means ± SD (n = 3).

a,b,c
Values with different superscripts are significantly different (P b 0.01) between batches.
1,2,3,4Values with different subscripts are significantly different (P b 0.01) between the citrus.

3.3. Antimicrobial activity
The antimicrobial activity of citrus peel extracts (lemon, lime, manda-
rin and orange) against both Gram-positive and Gram-negative bacteria
(Table 1) was investigated. The inhibition zones (mm) of the citrus peel
extracts (lemon, mandarin and orange) on selected microorganisms are
reported in Tables 4, 5 and 6, respectively. Sterile distilled water, used as
negative control, did not show any inhibition against all tested microor-
ganisms. It is particularly notable that the strain of L. innocua was more
sensitive to the peel extracts than most of the Listeria monocytogenes
strains. L. innocua is frequently used as a surrogate for L. monocytogenes
in food systems (Friedly, 2007). Lime peel extract did not demonstrate
antimicrobial activity against any of the target bacteria investigated.
Previous studies had found that extracts from lime peel at high concentra-
tions were effective against Gram-positive and Gram-negative bacteria
(Palhano et al., 2004; Prabuseenivasan, Jayakumar, & Ignaciumuth,
2006). However, at these high concentrations they would be unaccept-
able for application to food products due to the possible changes in or-
ganoleptic properties, especially aroma. This factor must be considered
when assessing the potential of citrus peel extracts in vitro. The absence

Table 4
Effect of pressure treatments on diameter of the zones of inhibition in mm of lemon peel extract with different concentrations tested against bacteria.

Batches mg/mL Gram-positive bacteria Gram-negative bacteria

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Control 1 11.3a1 10.6a1 10.3a1 11.6a1 – 11.6a1 – 11.6a1 10.6a1 10.6a1 11.3a1 10.6a1 11.6a1 12.3a1 – – 11.6a1 10.6a1 10.6a1 11.6a1
0.6 8.6a2 8.6b2 9.7a1 10.6a1 – – – 9.3b2 8.6a2 8.6a2 11.3a1 8.6b2 11.6a1 8.6 b2 – – 11.3a1 10.6a1 10.6a1 11.3a1
0.3 – – – 6.6b2 – – – 8.3b2 7.6a2 7.6a2 8.7a2 8.6b2 – – – – – – – 11.3a1
0.15 – – – 6.3a2 – – – – – – – 7.6a2 – – – – – – – –
0.08 – – – 6.3a2 – – – – – – – 7.3a2 – – – – – – – –
0.04 – – – 6.3a2 – – – – – – – 7.3a2 – – – – – – – –
300 MPa/3′ 1 10.6a,b1 10.6a1 10.7a1 11.6a1 – 11.6a1 – 8.6b1 10.6a1 10.6a1 11.3a1 11.6a1 12.6a1 12.3a1 – – 12.3a1 11.6a1 11.6a1 11.6a1
0.6 8.6a2 8.6b2 10.3a1 11.6a1 – 11.3a1 – 7.6c1 8.6a2 8.6a2 – 9.6b1,2 11.6a1 12.3a1 – – 10.6a2 11.6a1 11.6a1 11.3a1
0.3 – – – 3.3c2 – – – – 6.6a3 6.6a3 – 7.6b2,3 – – – – – – – 11.3a1
0.15 – – – 3.3b2 – – – – – – – 7.3a2,3 – – – – – – – –
0.08 – – – 3.3b2 – – – – – – – 6.6a3 – – – – – – – –
0.04 – – – 3.3b2 – – – – – – – 6.6a3 – – – – – – – –
300 MPa/10′ 1 11.6a1 10.6a1 10.3a1 11.6a1 – 11.6a1 – 10.6a1 9.6a1 9.6a1 11.3a1 11.6a1 12.6a1 12.3a1 – – 12.3a1 11.6a1 11.6a1 11.6a1
0.6 9.6a2 10.3a1 9.7 a1 11.6a1 – 11.3a1 – 8.6b,c2 8.6a2 8.6a2 – 9.6b1,2 – – – – 11.6a1 11.6a1 11.6a1 11.3a1
0.3 – – – 3.3c2 – – – – – – – 8.6b2,3 – – – – – – – 11.3a1
0.15 – – – 3.3b2 – – – – – – – 7.6a3 – – – – – – – –
0.08 – – – 3.3b2 – – – – – – – 6.6a3 – – – – – – – –
0.04 – – – 3.3b2 – – – – – – – 6.6a3 – – – – – – – –
500 MPa/3′ 1 9.6b1 11.6a1 10.7a1 11.6a1 – 11.6a1 – 11.6a1 9.6a1 9.6a1 12.3a1 11.6a1 12.6a1 12.3a1 – – 12.3a1 11.6a1 11.6a1 11.6a1
0.6 8.6a2 10.3a1 10.7a1 11.6a1 – 11.3a1 – 11.3a1 8.6a2 8.6a2 11.3a1 11.3a1 – – – – 11.6a1 11.6a1 11.6a1 11.3a1
0.3 – – – 11.6a1 – – – 11.3a1 7.6a2 7.6a2 9.3a2 10.3a1 – – – – – – – 11.3a1
0.15 – – – 5.3a,b2 – – – – – – – 7.6a2 – – – – – – – –
0.08 – – – 5.3a,b2 – – – – – – – 7.3a2 – – – – – – – –
0.04 – – – 5.3a,b2 – – – – – – – 7.3a2 – – – – – – – –
500 MPa/10′ 1 11.6a1 10.6a1 10.7a1 11.6a1 – 11.6a1 – 11.6a1 9.6a1 9.6a1 12.3a1 11.6a1 11.6a1 12.3a1 – – 12.3a1 11.6a1 11.6a1 11.6a1
0.6 9.6a2 9.6a,b1 10.7a1 11.6a1 – 11.3a1 – 11.3a1 7.6a2 7.6a1 11.3a1 10.3ª,b1 7.6b2 – – – 11.6a1 11.6a1 11.6a1 11.3a1
0.3 – – – 3.3c2 – – – 11.3a1 7.6a2 7.6a1 9.3a2 10.3a1 – – – – – – – 11.3a1
0.15 – – – 3.3b2 – – – – – – – 6.6a2 – – – – – – – –
0.08 – – – 3.3b2 – – – – – – – 6.3a2 – – – – – – – –
0.04 – – – 3.3b2 – – – – – – – 6.3a2 – – – – – – – –
Pb *** *** *** *** *** *** *** *** *** *** *** ***
Pc *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** ***
Pint *** *** *** *** *** *** *** *** *** *** *** *** ***

1: Bacillus subtilis 01; 2: Enterococcus faecalis 29212; 3: Staphylococcus aureus 81; 4: Listeria innocua 2030c; 5: L. monocytogenes 3391; 6: L. monocytogenes 1940/1; 7: L. monocytogenes 2264;
8: L. monocytogenes 1853/3; 9: Clostridium sporogenes 01; 10: Cl. difficile V591359; 11: Acinetobacter lwoffii T7BT5; 12: A. junii 889; 13: A.pittii T1BP1; 14: A. baumannii 05; 15: Klebsiella spp. 01;
16: Escherichia coli 25922; 17: Proteus vulgaris 01; 18: Salmonella Typhimurium 01; 19: Pseudomonas spp. 01; 20: Cronobacter sakazakii 51329.
– no inhibition; a,b,c: Values with different superscripts are significantly different between batches with equal concentrations 1,2,3: Values with different subscripts are significantly different
between concentrations in one sample; Pb: P-values for batches factor; Pc: P-values for concentrations (mg/mL) factor; Pint: P-values for interaction between batches and concentrations
factor; ∗∗∗: P b 0.01.

Please cite this article as: Casquete, R., et al., Evaluation of the effect of high pressure on total phenolic content, antioxidant and antimicrobial
activity of citrus peels, Innovative Food Science and Emerging Technologies (2015), http://dx.doi.org/10.1016/j.ifset.2015.07.005
 R. Casquete et al. / Innovative Food Science and Emerging Technologies xxx (2015) xxx–xxx 5

Table 5
Effect of pressure treatments on diameter of the zones of inhibition in mm of mandarin peel extract with different concentrations tested against bacteria.

Batches mg/mL Gram-positive bacteria Gram-negative bacteria

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Control 1 8.3a1 9.7a1 7.3 a1 7.3a1 – 6.7a1 – 8.3a1 9.3a1 9.7a1 8.3a1 8.3a1 5.3b1 – 8.9a1 7.3a1 9.3a1 8.3a1 – 6,7a1
0.6 7.7a1 8.3a1 7.3a1 6.7a1 – 6.3a1 – 6.3a1,2 8.7a1 9.3a1 8.7a1 8.3a1 – – 8.5a1 7.3a1 7.7a2 8.3a1 – 6,3a1
0.3 7.3a1 8.3a1 6.7a1 6.3a1 – 5.7a1 – 5.7a2 8.3a1 8.7a1 7.7a1 7.3a1 – – 8.3a1 6.3a1 6.7b2 7.3a1 – 6,3a1
0.15 7.3a1 – – 6.3a1 – 5.7a1 – 5.7a2 5.7a2 6.7a2 7.3a1 – – – – – 6.7b2 – – –
0.08 7.3a1 – – 5.7a1 – 5.7a1 – 5.7a2 5.7a2 6.7a2 5.3b2 – – – – – 6.7b2 – – –
0.04 7.3a1 – – 5.7a1 – 5.3a1 – 5.7a2 5.3a2 6.3a2 5.7a2 – – – – – 6.7b2 – – –
300 MPa/3′ 1 8.7a1 8.7a,b1 7.7a1 6.7a1 – 6.7a1 – 7.7a1 9.7a1 10.3a1 7.7a1 8.7a1 6.7a1 – 8.7a1 7.7a1 8.7a1 8.7a1 – 6,7a1
0.6 8.7a1 8.7a1 7.7a1 6.7a1 – 6.7a1 – 7.7a1 9.3a1 9.7a1,2 7.7a1 8.7a1 – – 8.7a1 7.7a1 8.7a1 8.7a1 – 6,7a1
0.3 8.7a1 7.7a1 7.7a1 6.7a1 – 6.7a1 – 7.7a1 9.3a1 9.3a1,2 7.7a1 8.7a1 – – 8.3a1 7.7a1 8.7a,b1 8.3a1 – 6,7a1
0.15 8.3a1 – – 6.7a1 – 6.3a1 – 6.7a1 6.7a, 2 7.7a2 7.7a1 – – – – – 7.7a,b1 – – –
0.08 8.3a1 – – 6.7a1 – 6.3a1 – 6.3a1 6.3a, 2 6.7a2 6.3a,b1 – – – – – 7.7a,b1 – – –
0.04 8.3a1 – – 6.3a1 – 6.3a1 – 6.3a1 6.3a, 2 6.7a2 6.3a1 – – – – – 7.3a,b1 – – –
300 MPa/10′ 1 8.7a1 7.7b1 7.7a1 6.7a1 – 6.7a1 – 7.7a1 9.7a1 10.3a1 7.7a1 7.7a1 6.7a1 – 8.7a1 7.7a1 9.7a1 8.7a1 – 6,7a1
0.6 8.7a1 7.7a1 7.7a1 6.7a1 – 6.7a1 – 7.7a1 9.7a1 9.3a1,2 7.7a1 7.7a1 6.3a1 – 8.7a1 7.7a1 9.7a1 8.7a1 – 6,7a1
0.3 8.7a1 7.7a1 7.7a1 6.7a1 – 6.7a1 – 7.7a1 9.7a1 9.3a1,2 7.7a1 7.3a1 – – 8.3a1 7.7a1 9.7a1 8.7a1 – 6,7a1
0.15 8.7a1 – – 6.7a1 – 6.3a1 – 6.7a1 7.7a2 7.7a2 7.7a1 – – – – – 9.3a1 – – –
0.08 8.7a1 – – 6.7a1 – 6.3a1 – 6.3a1 7.3a2 7.7a2 6.3a,b1,2 – – – – – 8.7a,b1 – – –
0.04 8.3a1 – – 6.3a1 – 6.3a1 – 6.3a1 7.3a2 7.3a2 5.3a2 – – – – – 8.7a,b1 – – –
500 MPa/3′ 1 8.7a1 7.7b1 7.7a1 5.7a1 – 6.7a1 – 7.7a1 9.7a1 10.7a1 7.7a1 7.7a1 6.7a1 – 8.7a1 5.7b1 8.7a1 8.7a1 – 6,7a1
0.6 8.7a1 7.7a1 7.7a1 5.7a1 – 6.7a1 – 7.7a1 9.7a1 10.3a1 7.7a1 7.7a1 – – 8.7a1 5.7b1 8.7a1 8.7a1 – 6,7a1
0.3 8.7a1 7.7a1 7.3a1 5.7a1 – 6.7a1 – 7.7a1 9.3a1 9.7a1 7.7a1 7.7a1 – – 7.7a1 5.7b1 8.7a,b1 8.7a1 – 6,7a1
0.15 8.3a1 – – 5.7a1 – 6.3a1 – 7.3a1 6.7a, 2 6.7a2 7.7a1 – – – – – 8.7a,b1 – – –
0.08 8.3a1 – – 5.7a1 – 6.3a1 – 6.7a1 6.3a, 2 6.7a2 6.7a,b1,2 – – – – – 8.7a,b1 – – –
0.04 7.7a1 – – 5.7a1 – 6.3a1 – 6.3a1 6.3a, 2 6.7a2 5.7a2 – – – – – 8.3a,b1 – – –
500 MPa/10′ 1 8.7a1 8.7a,b1 7.7a1 6.7a1 – 6.7a1 – 7.7a1 9.7a1 10.3a1 8.7a1 8.7a1 6.7a1 – 9.7a1 6.7a,b1 9.7a1 8.7a1 – 6,7a1
0.6 8.7a1 8.7a1 7.7a1 6.7a1 – 6.7a1 – 7.7a1 9.3a1 9.3a1,2 8.7a1 8.7a1 – – 8.3a1 6.7a,b1 9.3a1 8.7a1 – 6,7a1
0.3 8.7a1 8.3a1 7.3a1 6.7a1 – 6.7a1 – 7.3a1 9.3a1 9.3a1,2 8.3a1 8.7a1 – – 8.3a1 6.7a,b1 9.3a1 7.7a1 – 6,7a1
0.15 8.7a1 – – 6.7a1 – 6.3a1 – 6.7a1 6.7a, 2 7.7a2,3 8.3a1 – – – – – 9.3a1 – – –
0.08 8.3a1 – – 6.3a1 – 6.3a1 – 6.3a1 6.3a, 2 6.7a3 7.7b1,2 – – – – – 9.3a1 – – –
0.04 8.0a1 – – 6.3a1 – 6.3a1 – 6.3a1 5.7 a2 6.7a3 6.3a2 – – – – – 9.3a1 – – –
Pb *** *** *** *** ***
Pc *** *** *** *** *** *** *** *** *** *** *** *** ***
Pint *** *** *** ***

1: Bacillus subtilis 01; 2: Enterococcus faecalis 29212; 3: Staphylococcus aureus 81; 4: Listeria innocua 2030c; 5: L. monocytogenes 3391; 6: L. monocytogenes 1940/1; 7: L. monocytogenes 2264;
8: L. monocytogenes 1853/3; 9: Clostridium sporogenes 01; 10: Cl. difficile V591359; 11: Acinetobacter lwoffii T7BT5; 12: A. junii 889; 13: A.pittii T1BP1; 14: A. baumannii 05; 15: Klebsiella spp. 01;
16: Escherichia coli 25922; 17: Proteus vulgaris 01; 18: Salmonella Typhimurium 01; 19: Pseudomonas spp 01; 20: Cronobacter sakazakii 51329.
–: no inhibition; a,b,c: Values with different superscripts are significantly different between batches with equal concentrations 1,2,3: Values with different subscripts are significantly differ-
ent between concentrations in one sample; Pb: P-values for batches factor; Pc: P-values for concentrations (mg/mL) factor; Pint: P-values for interaction between batches and concentra-
tions factor; ∗∗∗: P b 0.01;

of antimicrobial activity in lime peel extract might be due to a number of
factors such as collection time of plant material, its storage, climate,
which might, in turn, affect the amount of the active principal constitu-
ents (tannins, terpenoids, alkaloids, flavonoids and others) in the plant
material (Mahmud et al., 2009).
The lemon peel extract at 0.6 and 0.3 mg/mL concentrations demon-
strated antibacterial activity against all the tested microorganisms
except for L. monocytogenes 3391 (5), L. monocytogenes 2264 (7),
Klebsiella spp. 01 (15) and Escherichia coli 25922 (16) that were resis-
tant to all tested samples even at 1 mg/mL. L. innocua 2030c (4) and
Acinetobacter junii 889 (12) were sensitive towards all of the tested con-
centrations (Table 4). Iturriaga, Olabarrieta, and Martínez de Marañón
(2012) reported that L. innocua was one of the most sensitive microorgan-
isms towards all of the tested natural extracts. For all pressure-treated
batches of lemon peel at 0.3 mg/mL concentration, inhibition zones
were absent or significantly (P b 0.01) lower than control (Table 4).
The higher or lower antimicrobial activity of these samples may be
related to the concentrations of rutine, quercetin, naringenin, even
though the mechanisms of action are not fully understood. These
compounds increase the permeability of the inner bacterial membrane,
nullifying its potential, decreasing ATP production, membrane transport
and its mobility (Tsuchiya & Iinuma, 2000). In addition, they inhibit DNA
gyrase which is involved in the mechanism of DNA and RNA synthesis in
bacteria (Mirzoeva, Grishanin, & Calder, 1997). The diameter of the
inhibition zone produced by the mandarin peel extracts is reported in
Table 5. Though larger inhibitory zones were observed when using
lemon peel extracts, for most of the target organisms investigated
mandarin peel extracts demonstrated antimicrobial activity at lower
concentrations. Espina et al. (2011) previously demonstrated that man-
darin peel had greater antimicrobial activity than lemon peel. The man-
darin peel extract manifested antibacterial activity against all the tested
microorganisms except for L. monocytogenes 3391 (5), L. monocytogenes
2264 (7), Acinetobacter baumannii 05 (14) and Cronobacter sakazakii
51329 (19) without encountering major differences between batches
studied (Table 5). Globally, Gram-positive bacteria were more sensitive
than Gram-negative bacteria to the action of the mandarin peel extracts.
All Gram-positive bacteria except Enterococcus faecalis 29212 (2) and
Staphylococcus aureus 81 (3) were sensitive to all concentrations tested.
However, most of the Gram-negative bacteria were resistant to concen-
trations lower than 0.3 mg/mL of mandarin peel extract. These results
agree with the literature (Gutierrez, Barry-Ryan, & Bourke, 2009; Holley
& Patel, 2005; Kalemba & Kunicka, 2003), as it is generally recognized
that Gram-negative bacteria are, in general, less sensitive than Gram-
positive bacteria to natural extracts. Nevertheless there are exceptions
in which Gram-negative bacteria are more susceptible than Gram-
positives towards some natural extracts (Kalemba & Kunicka, 2003).
The effect of orange peel extract on the inhibition of the microorganism
tested is shown in Table 6. In general, both Gram-positive and Gram-
negative bacteria were inhibited by orange peel extract at 0.15 mg/mL
concentrations. L. monocytogenes 3391 (5), L. monocytogenes 2264
(7) and Klebsiella spp. 01 (15) showed resistance to all test samples
even at 1 mg/mL. Compared to the control, there was no increase in
antimicrobial activity when samples were subjected to pressure treat-
ments. However, a significant decrease in the diameter of the inhibition

Please cite this article as: Casquete, R., et al., Evaluation of the effect of high pressure on total phenolic content, antioxidant and antimicrobial
activity of citrus peels, Innovative Food Science and Emerging Technologies (2015), http://dx.doi.org/10.1016/j.ifset.2015.07.005
 6
activity of citrus peels, Innovative Food Science and Emerging Technologies (2015), http://dx.doi.org/10.1016/j.ifset.2015.07.005
Please cite this article as: Casquete, R., et al., Evaluation of the effect of high pressure on total phenolic content, antioxidant and antimicrobial

Table 6
Effect of pressure treatments on diameter of the zones of inhibition in mm of orange peel extract with different concentrations tested against bacteria.

Batches mg/mL Gram-positive bacteria Gram-negative bacteria

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Control 1 11.7a1 7.7a1 11a1 10.3a1 – 11.7a1 – 11.3a1 9.3a1 14.3a1 9.7ª,b1 10.0a1 9.3a1 10.7a,b1 – 9.3a1 9.3a1 10.3a1 10.3a1 10.3a1
0.6 11.3a1 7.7a1 9.7 a1,2 10.3a1 – 11.7a1 – 9.3a2 9.3a1 12.3b,c2 9.7a1 9.7a1 7.7a2 10.3a1 – 9.3a1 9.3a1 9.3a1.2 10.3a1 10.3a1

R. Casquete et al. / Innovative Food Science and Emerging Technologies xxx (2015) xxx–xxx
0.3 10.7a1 7.7a1 8.7a2 9.7a1 – 11.7a1 – 8.3a2 8.7a1 12.3a2 9.7a1 9.3a1 7.7a.b2 9.7a1 – 9.3a1 8.7a1 9.3a1.2 9.7 a1 9.7a1
0.15 10.7a1 – 8.7a2 9.7a1 – 11.7a1 – 8.3a2 8.7a1 12.3a2 6.7b2 9.3a1 7.7a.b2 9.3a1 – 8.3a1 8.7a1 8.7a2 9.7a1 9.7a1
0.08 – – – – – – – – – – – – – – – – – – – –
0.04 – – – – – – – – – – – – – – – – – – – –
300 MPa/3′ 1 11.7a1 8.7a1 10.3a,b1 10.7a1 – 11.3a1 – 11.3a1 8.7a1 13.7a1 11.3a1 10.3a1 8.3a1 12.3a1 – 9.7a1 9.3a1 10.3a1 10.3a1 11.7a1
0.6 10.7a1 8.5a1 9.0a2 10.3a1 – 10.3a.b1,2 – 10.3a1 8.7a1 12.3b,c12 9.7a1,2 9.7a1.2 7.7a1.2 8.7b2 – 9.3a1 8.7a1 9.7a1 6.7b2 8.7a2
0.3 10.7a1 8.3a1 8.7a2 10.3a1 – 9.5b2 – 8.0a2 8.0a1 11.5a2 9.5a2 9.5a1.2 6.7b.c2 8.7a.b2 – 6.7b2 – 5.5c2 – 6.7c3
0.15 10.5a1 – 8.7a2 9.5a1 – 5.7d3 – 7.7a2 – 10.7a2 9.3a2,3 9.3a1.2 6.5b.c2 8.5a.b2 – 6.5a2 – – – 6.5b3
0.08 7.3a2 – – – – – – – – – 7.3a,b3 8.3a2 5.7a3 7.3a2 – – – – – –
0.04 – – – – – – – – – – – – – – – – – – – –
300 MPa/10′ 1 11.7a1 8.7a1 8.7b1 10.3a1 – 10.7a1 – 11.3a1 9.3a1 14.3a1 9.5b1 10.3a1 9.7a1 10.3b,c1 – 9.7a1 9.3a1 9.7a.b1 9.3 a1 10.3a1
0.6 11.3a1 8.7a1 8.7a1 9.7a1 – 10.7a.b1 – 10.3a12 8.7a1 14.3a1 9.3a1,2 9.7a1 8.3a1 9.7a1 – 9.3a1 6.7b2 8.7a1 6.7b2 9.7a1
0.3 9.7 a2,3 7.7a1 8.7a1 9.7a1 – 10.3a.b1 – 9.3a2.3 8.7a1 12.3a2 7.7b2,3 9.7a1 8.3a1 9.3a1 – 7.3a,b2 6.7b2 6.3b.c2 – 8.7a.b1,2
0.15 9.3a3 – 8.3a1 9.3a1 – 7.7c2 – 8.7a3 8.7a1 12.3a2 6.7b3 9.3a1 8.3a1 8.7a.b1 – – 5.7b2 6.3b2 – 7.7b2
0.08 – – – – – – – – – – – – – – – – – – – –
0.04 – – – – – – – – – – – – – – – – – – – –
500 MPa/3′ 1 11.7a1 7.7a1 10.3a,b1 10.7a1 – 11.7a1 – 11.3a1 9.3a1 14.3a1 9.7ª,b1 10.3a1 8.7a1 9.3b.c1 – 9.7a1 7.7b1 9.7a.b1 9.3 a1 11.7a1
0.6 11.3a1 7.7a1 9.7a1.2 10.7a1 – 10.7a.b1,2 – 10.3a12 8.7a1 11.7c2 9.7a1 9.3a1.2 8.3a1 8.7b1 – 9.3a1 7.7a.b1 8.7a1.2 9.3 a1 9.7a2
0.3 9.7a1,2 7.7a1 8.7a2 10.3a1 – 10.3a.b1,2 – 9.3a2.3 7.7a1.2 11.3a2 9.3a1 8.7a2 5.7c2 8.7a.b1 – 7.7a,b2 5.7b2 7.3b,c2 7.3 b2 7.7b.c3
0.15 9.3a2 – 8.7a2 10.3a1 – 9.3b2 – 8.7a3 5.7b3 10.7a2 9.3a1 – 5.7c2 8.7a.b1 – 7.7ab2 5.7b2 5.3b3 7.3 b2 6.7b3
0.08 – – – 8.3a2 – – – – – – – – – – – – – – – –
0.04 – – – – – – – – – – – – – – – – – – – –
500 MPa/10′ 1 11.7a1 8.7a1 10.7a1 10.7a1 – 10.3a1 – 10.3a1 9.3a1 14.3a1 10ab1 10.3a1 9.3a1 8.7c1 – 9.7a1 8.3a.b1 8.3b1 9.3 a1 11.7a1
0.6 11.3a1 8.7a1 9.7a1.2 10.7a1 – 9.7b1 – 9.7a1 8.7a1 11.7c2 9.7a1 9.3a1.2 7.7a2 8.3b1.2 – 9.3a1 7.7a.b12 6.7b2 9.3 a1 9.3 a2
0.3 10.7a1 8.3a1 8.7a2 10.3a1 – 9.7b1 – 9.3a1.2 8.3a1 11.7a2 9.7a1 9.3a1.2 7.7a2 7.7b2 – 7.7a,b2 6.7b2,3 6.3b.c2 7.3 b2 8.7 a.b2
0.15 9.7a1 – 8.7a2 10.3a1 – 9.3b1 – 8.3a2 5.7b2 11.3a2 9.3a1 8.3a2 5.7b3 7.7a2 – 7.3a.b2 5.7b3 6.3a2 – 7.7b2
0.08 – – – 9.3a1 – – – – – – – – – – – – – – – –
0.04 – – – – – – – – – – – – – – – – – – – –
Pb *** *** *** *** *** *** *** *** *** *** *** ***
Pc *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** ***
Pint *** *** *** *** *** *** *** *** *** *** *** *** ***

1: Bacillus subtilis 01; 2: Enterococcus faecalis 29212; 3: Staphylococcus aureus 81; 4: Listeria innocua 2030c; 5: L. monocytogenes 3391; 6: L. monocytogenes 1940/1; 7: L. monocytogenes 2264; 8: L. monocytogenes 1853/3; 9: Clostridium sporogenes 01; 10: Cl.
difficile V591359; 11: Acinetobacter lwoffii T7BT5; 12: A. junii 889; 13: A. pittii T1BP1; 14: A. baumannii 05; 15: Klebsiella spp. 01; 16: Escherichia coli 25922; 17: Proteus vulgaris 01; 18: Salmonella Typhimurium 01; 19: Pseudomonas spp. 01; 20: Cronobacter
sakazakii 51329.
–: no inhibition; a.b.c: Values with different superscripts are significantly different between batches with equal concentrations 1.2.3: Values with different subscripts are significantly different between concentrations in one sample; Pb: P-values for
batches factor; Pc: P-values for concentrations (mg/mL) factor; Pint: P-values for interaction between batches and concentrations factor; ∗∗∗: P b 0.01;
 R. Casquete et al. / Innovative Food Science and Emerging Technologies xxx (2015) xxx–xxx
zone to lower concentrations for Clostridium sporogenes (9), Proteus
vulgaris 01 (17), Salmonella Typhimurium 01 (18), Pseudomonas spp.
01 (19) and Cr. sakazakii 51329 (20) was noticed for pressure-treated
samples (300 MPa, 3 min) (Table 6). As in the samples of lemon peel ex-
tract, low or no antimicrobial activity of these samples may be related to
the concentration of rutine, quercetin and naringenin. The highest resis-
tance of L. monocytogenes 3391 (5) and L. monocytogenes 2264 (7) to the
three citrus peel extracts might be attributed to the fact that these
strains are clinical isolates resistant to one or more antibiotics of differ-
ent classes (data not shown). According to Silva, Rodrigues, Feás, and
Estevinho (2012) the drug-resistant strains are more resistant to the
actions of hydro-alcoholic extracts from natural sources. A more detailed
biological/physiological explanation for sensitivity or resistance to citrus
peel extracts, cannot be advanced at this time due to lack of composi-
tional analyses of the various extracts and of information on the interac-
tions of cell membranes of the different bacteria with compounds in the
citrus peel extracts.
4. Conclusions
Results of this study indicated that the applied pressure treatments
on citrus peels affect the total phenolic contents and antioxidant activity
of subsequently prepared ethanolic extracts. The maximum TPC value
was achieved when samples were subjected to 300 MPa/3 min, which
also demonstrated the highest values for antioxidant capacity. With
the exception of lime extracts, all the other extracts demonstrated anti-
microbial activity against a wide range of microorganisms. Orange peel
extracts demonstrated the highest activity. However, no significant dif-
ferences were found between un-treated or pressure-treated samples.
Results indicated that citrus peels subjected to pressure treatments
(300 MPa/3 min) could be used as a source of high added-value bioac-
tive compounds for antioxidant applications.
Acknowledgements
R. Casquete is the beneficiary of a post-doctoral grant (PO12018)
funded by the Junta de Extremadura, Consejería de Empleo, Empresa e
Innovación and co-funded with Fondo Social Europea (FSE). Sónia
Marília A. Castro acknowledged financial support from Fundação para
a Ciência e Tecnologia (Portugal), via a post-doctoral fellowship
(SFRH/BPD/71723/2010). This work was supported by funding from
the National Funds from the Fundação para a Ciência e a Tecnologia
(FCT) through project Pest-OE/EQB/LA0016/2013. Fundação para a
Ciência e Tecnologia, European Union, QREN, FEDER, and COMPETE
are also acknowledged for funding the Organic Chemistry Research
Unit (QOPNA) (project PEst-C/QUI/UI0062/2013; FCOMP-01-0124-
FEDER-037296). Editing of this paper by Dr. P. A. Gibbs is gratefully
acknowledged.
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Please cite this article as: Casquete, R., et al., Evaluation of the effect of high pressure on total phenolic content, antioxidant and antimicrobial
activity of citrus peels, Innovative Food Science and Emerging Technologies (2015), http://dx.doi.org/10.1016/j.ifset.2015.07.005