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  • Institute of Modeling and Innovation on Technology - IMIT CONICET/UNNE
  • Instituto de Modelado e Innovación Tecnológica (CONICET - UNNE) Universidad Tecnológica Nacional (UTN)
  • Hospital Dr. Julio C. Perrando, Resistencia, Chaco. Argentina


The aim of this work was to determine the effectiveness of Grapefruit (Citrus paradisi) essential oil to inhibit the growth of wild food-borne spoilage and pathogenic bacterial strains. Additionally, the chemical composition and physical properties of this essential oil was evaluated. Essential oil was obtained as a byproduct from agro-processing industry in the province of Corrientes, Argentina. Monoterpene hydrocarbon limonene representing 93% (v/v), quantified by gas chromatography, was the major component of essential oil. Citrus paradisi essential oil inhibited growth of Escherichia coli, Staphylococcus aureus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. diacetylactis, Leuconostoc mesenteroides subsp. dextranicum and Lactobacillus plantarum. The lowest concentration of essential oil (4.29ppm) was required to inhibit Lactococcus lactis subsp. lactis 207c VCOR (8.27±0.13 log10 CFU/ml) according to the Minimum Inhibitory Concentration. The effect of this oil on growth of wild strain 207c VCOR (9.17±0.024 log10 CFU/ml) was determined at different times by total count and spectrophotometric absorption to 560 nm. At the moment of essential oil injection, the number of microorganisms was 1.29±0.17x109 CFU/ml and, at 24 hours of contact, only 6.3±0.5x106 CFU/ml was detected. These results show that oil has a bactericidal effect if counts with and without addition of essential oil were compared. The loss of viability was 1.82x109 CFU/ml under these experimental conditions. This essential oil has very strong potential applicability as a natural antibacterial agent for food industry, particularly for pasta manufacture which is facing serious spoilage problems due to lactic bacteria activity.
O. M. Vasek, et al. /Journal of Natural Products, Vol. 8(2015): 16-26
Copyright © 2015, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
ISSN 0974 – 5211
Antibacterial activity of Citrus paradisi essential oil
O.M. Vasek
*, L.M. Cáceres
, E.R. Chamorro
, G.A. Velasco
Biotecnología Microbiana para la Innovación Alimentaria (BiMIA, Microbial Biotechnology
for Food Innovation), Institute of Modeling and Innovation on Technology, CONICET and
National Northeastern University, Corrientes, Argentina
Centro de Investigación en Química Orgánica-Biológica (QUIMOBI, Research Center on
Biological Organic Chemistry), National Technological University and Institute of Modeling
and Innovation on Technology, CONICET, Resistencia, Chaco, Argentina
*Corresponding Author
(Received 08 October 2014; Revised 25 December 2014-31 March 2015; Accepted 12 April 2015)
The aim of this work was to determine the effectiveness of Grapefruit (Citrus
paradisi) essential oil to inhibit the growth of wild food-borne spoilage and
pathogenic bacterial strains. Additionally, the chemical composition and physical
properties of this essential oil was evaluated. Essential oil was obtained as a by-
product from agro-processing industry in the province of Corrientes, Argentina.
Monoterpene hydrocarbon limonene representing 93% (v/v), quantified by gas
chromatography, was the major component of essential oil. Citrus paradisi essential
oil inhibited growth of Escherichia coli, Staphylococcus aureus, Lactococcus lactis
subsp. lactis, Lactococcus lactis subsp. diacetylactis, Leuconostoc mesenteroides
subsp. dextranicum and Lactobacillus plantarum. The lowest concentration of
essential oil (4.29ppm) was required to inhibit Lactococcus lactis subsp. lactis 207c
VCOR (8.27±0.13 log
CFU/ml) according to the Minimum Inhibitory
Concentration. The effect of this oil on growth of wild strain 207c VCOR (9.17±0.024
CFU/ml) was determined at different times by total count and
spectrophotometric absorption to 560 nm. At the moment of essential oil injection, the
number of microorganisms was 1.29±0.17x10
CFU/ml and, at 24 hours of contact,
only 6.3±0.5x10
CFU/ml was detected. These results show that oil has a bactericidal
effect if counts with and without addition of essential oil were compared. The loss of
viability was 1.82x10
CFU/ml under these experimental conditions. This essential oil
has very strong potential applicability as a natural antibacterial agent for food
industry, particularly for pasta manufacture which is facing serious spoilage problems
due to lactic bacteria activity.
Keywords: Natural preservative; Spoilage control; Essential oil; Lactic acid bacteria.
Presence of undesirable microorganism in food and their multiplication is a problem
for whole world. They can lead to spoilage and deteriorate the quality of food or
cause illness. In 2011, 1.8 million child deaths were registered (WHO, 2013), most
Journal of Natural Products
Volume 8 (2015)
Research Paper
O. M. Vasek, et al. /Journal of Natural Products, Vol. 8(2015): 16-26
Copyright © 2015, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
of them caused by contaminated water and food. In recent years, emergence of
bacterial resistance against multiple antibiotics has accelerated dramatically.
Community- and hospital-acquired pathogens and larger part of them are multi-drug-
resistant bacteria (Lai et al., 2011; Solórzano-Santos and Miranda-Novales, 2012).
So, food safety is a past, present and future public health concern all over the world.
Growing interest in substitution of synthetic antimicrobial agents by natural
ones has fostered research on vegetable sources and screening of plant materials in
order to identify new compounds or test natural chemicals already known for
important activities that have not been discovered so far (Ait-Ouazzou et al., 2011;
Lv et al., 2011; Badawy and Abdelgaleil, 2014).
Essential oils (EOs) extracted from different plant genera are, in many cases,
biologically active. These aromatic compounds are relatively inexpensive and there
is abundant raw material with practical applications for different industries.
Citrus is considered an important fruit in world production because its great
value for human diet. Citrus is member of Rutaceae family from sub-tropical origin
and is known for its semi-sweet taste.
Due to nutraceutical and economic importance, numerous investigations have
been performed aiming at identifying chemical composition, antimicrobial activities
of EOs from peel of different citrus species.
Citrus EOs has been recognized as safe due to their wide spectrum of
biological activities, such as antimicrobial, antioxidant, anti-inflammatory and
anxiolytic (Rehman, 2006; Chutia et al., 2009). These antimicrobial properties have
shown to create a particularly interesting scope for application within food industry
(Imran et al., 2013), for veterinary use (Fuselli et al., 2008; Roussenova, 2011),
human medicine (Yin et al., 2012; Oliveira, et al., 2014) and plants for agricultural
production (Badawy and Abdelgaleil, 2014).
Argentina is the world's eighth largest citrus grower and the first largest
lemon grower. It has exported dry fruit, juices and EOs since 1970. Citrus production
area covers 147.000ha, and has a total annual production of, almost, 3.000.000t.
Main citrus fruit include lemon (47%), followed by orange (29%), mandarin orange
(16%) and grapefruit (8%) (UIA, 2008).
Here we are try to determine physical characteristics and chemical
composition of Citrus paradisi essential oil; to screen antibacterial properties of EO
against wild food-borne spoilage and pathogenic bacterial strains and; to evaluate the
effect of adding EOs to growth of sensitive microorganisms in dose according to the
Essential oil: Citrus paradisi essential oil (EO) was obtained as a by-product in a
citrus juice extraction plant. The method used for juice extraction was extruding or
cold pressing in which follicular glands of citrus peel is mechanically ground to
release its content. For this purpose, a Food Machinery Corporation (FMC) extractor
was used. The objective of this process was to separate oil from fruit juice.
Essential oil characterization: Grapefruit oil characterization was performed by
determination of the following physical properties: refractive index, relative density
and optical rotation using standardized methods.
Relative density method was performed according to recommendations of Argentine
Institute of Standardization and Certification, IRAM-SAIPA Standard 18504
(2002), by pycnometer method. For this purpose, a 0.1 milligram-precision scale
O. M. Vasek, et al. /Journal of Natural Products, Vol. 8(2015): 16-26
Copyright © 2015, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
METTLER AJ150 (Germany) was used. Procedure consisted of determination of
body of water (m
) contained in the pycnometer, by weight difference of
pycnometer with or without distilled water, and body of water using the same
technique but with EO and using the same pycnometer. Both at reference temperature
= 20±0.2°C.
Relative density of oil was calculated applying the following equation:
= m
/ m
20°C/4°C: oil
density at 20°C, relative to water to 4°C,
: body of oil at full pycnometer at 20°C (g),
: body of water calculated at pycnometer calibration (g).
Additionally, refractive index was determined through IRAM-SAIPA Standard
18505 (2002) with an ABBE DR-M2 refractometer from ATAGO USA, Inc., with a
temperature thermostatic control through water circulation sleeve among prisms and
white light lamp. Tool precision is 0.0001, for a dynamic interval of n
= 1.300-
1.700. The method is based on determination of critical angle of total reflection
between oil and prism Flint glass, looking critical angle in observation field,
visualizing as a clear separation between two fields (pale and dark) focused on two
hair cruces. Using a 20ºC thermostatized oil sample (1 or 2 drops) reading in
refractometer was performed. Determination of optical rotation [α]
was done under
IRAM-SAIPA Standard N° 18507 (2002), using a A&E type WXG-4 disc polarimeter
from Shanghai, China, measuring range ± 180° and precision of 0.05°. Procedure was
based on observation of angle of deviation for polarized plane light 589-nm
wavelength (Sodium D line) caused by essential oil placed on a 10-dm-long sample
holder at 20±0.2°C. For this purpose, oil sample was stabilized on a water bath at a
temperature of 20±0.2°C. Angle observation was visualized in the observation field as
the intermediate of two fields (a dark circle with a pale line in the middle and another
pale circle with a dark line in the middle). Reading was done at external scale
Optical rotation of oil was calculated applying the following equation:
= α
: optical rotation to 20°C for Sodium D line (º), α
: optical rotation angle observed (º), l: leng
of sa
mple-holder flask (mm).
Chemical constitution of essential oil: identification of Citrus paradisi EO
components was conducted by gas chromatography-mass spectrometry (GC/MS). A
two-capillary column QP 5050 SHIMADZU equipment was used: one SE 52
(MEGA, Legnano, Italia) chemically bonded (25m x 0.25mm internal diameter;
0.25µm-thick stationary phase) column, covered by 5% phenil-polydimethylsiloxane
(0.25µm-thick stationary phase) at a column temperature of 60ºC (8min), increasing
up to 180ºC at a 3ºC/min
speed, then up to 230ºC at a 20ºC/min speed. 250ºC injector
temperature, split injection mode; 1:40 split ratio; 0.2µl oil injection volume. Mobile
phase: Helium, 122.2kPa (51.6cm/sec), 250ºC interface temperature and 40-400m/z
mass range acquisition. Another BP-20 (SGE, Australia) 25m x 0.25mm internal
diameter fused silica capillary column covered by polyethyleneglycol 20.000Da (0.25
µm thick stationary phase). Column temperature at 40ºC (8min), increasing up to
180ºC (3 ºC/min), and up to 230ºC (20ºC/min). 250ºC, injector temperature, split
injection mode; 1:40 split ratio; 0.2µl oil injection volume. Mobile phase: Helium,
92.6kPa (55.9cm/s), 250ºC interface temperature and mass range acquisition: 40-
O. M. Vasek, et al. /Journal of Natural Products, Vol. 8(2015): 16-26
Copyright © 2015, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
Fragmentation patterns in each component were compared to those stored in the
software library spectra (Mc Lafferty and Stauffer, 1991; Adams, 2001).
Microorganisms and growth conditions: forty autochthonous bacterial strains were
obtained from Institutional Collection of Wild Microorganisms” - Facultad de
Ciencias Exactas y Naturales (School of Natural and Exact Sciences, Northeastern
University of Argentina (Acronym: VCOR). In this study, the following were used as
food spoilage bacteria: Lactobacillus (Lb.) plantarum (11 strains), Leuconostoc
(Leuc.) mesenteroides subsp. dextranicum (1 strain), Lactococcus (L.) lactis subsp.
diacetylactis (1 strain) and L. lactis subsp. lactis (11 strains). Additionally,
Staphylococcus aureus (3 strains) and Escherichia (E.) coli (13 strains) were tested as
pathogenic bacteria of food.
Bacterial strains were preserved on Milk-Yeast extract with glycerol (15%, v/v) at -
20ºC. Lactobacillus strains were cultivated in MRS medium (Merck) at 30ºC;
Lactococcus lactis strains were cultivated in Elliker medium (Biokar Diagnostic) at
35ºC; Staphylococcus aureus were cultivated in Brain Heart Infusion (Merck) at 37ºC
and Nutrient medium (Britania) was used for proliferation of E. coli strains at 37ºC.
Working bacterial cultures were transferred (2%, v/v) to a fresh broth three times
prior to experiences.
Lactic bacteria count: appropriate further decimal dilutions of bacterial suspensions
were made in a peptone-saline solution (0.1-0.85%, w/v) for enumeration of lactic
bacteria (LAB) according to Aerobic Plate Count (Maturin and Peeler, 2001). For
growth, Elliker agar (Biokar Diagnostic) was used with incubation at 30°C for 48h.
Screening of antibacterial effect: antibacterial activity in vitro of EOs was assayed
using Disk diffusion method (Ortez, 2005). Briefly, 50µl of EO were placed on sterile
0.55cm diameter filter paper discs (Whatman 1) located on the surface of adequate
media in plates previously spread with 100µl of 10
CFU/ml overnight cultures. Plates
have been allowed to dry for 15min in a sterile environment, inverted and incubated
for 24h at optimal temperature of growth. Diameters of zones of inhibition (ZOI) were
measured using Vernier caliper. Controls were bacterial cultures without EO exposure.
Minimum inhibitory concentration (MIC): according to Rankin (2005), an aliquot
(5µl) of 10
CFU/ml overnight cultures was added to wells of sterile 96-well micro-
titerplate, containing (135µl) adequate medium added of Bromocresol Purple to
provide a final medium concentration of 0.16% (w/v). EO was diluted in sterile
solution (0.5%, v/v) of Tween 80 and added (50µl) to wells to give final EO
concentrations between 0.50 and 95.00% (v/v). Positive control wells contained
adequate broth and cells without EO while negative controls wells contained,
individually, medium, EO and Tween 80. Plates were incubated under normal
atmospheric conditions at respective temperatures according to strain for 24h.
Inhibition of growth was made visible from change in color in pH indicator observed
in a stereomicroscope (OLYMPUS C011-092879, Japan). The Minimum Inhibitory
Concentration (MIC) was defined as the lowest concentration of essential oil at which
microorganism does not demonstrate any visible growth (Tao, et al., 2009).
Characterization of EO antagonistic effect on cell growth: considering MIC results,
the effect of this essential oil on the growth of wild strain was determined at different
times by total count and spectrophotometric absorption (JASCO spectrophotometer V-
630, Japan) to 560nm (Abs
). Active strains were transferred (2%, v/v) to two
series of flasks containing Elliker broth, in sufficient number (30 tubes) for total
monitoring of growth; third series of flasks without inoculum were used for blank test.
Three series were incubated in water bath with low speed agitation (VICKING
O. M. Vasek, et al. /Journal of Natural Products, Vol. 8(2015): 16-26
Copyright © 2015, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
Thermostatic bath, Dubnoff model, Argentina) to 30°C during 120h. When cells
reached exponential phase of growth, corresponding to 0.7-0.8 of spectrophotometric
absorption, an aliquot (100µl) of EO dilution (5:95, EO: Tween 80) was added to each
flask of one series and the other was used as positive control to normal growth. At
different times, one flask from each series was used for determination of Abs
count of viable cells.
Statistical analysis: experiments were replicated three times. All results reported were
expressed as Mean±SD. Data were analyzed using INFOSTAT Software by one-way
ANOVA procedure (Di Renzo et al., 2008). Differences among means were detected
by the Hotelling Test. Significance of all tests was set at P<0.05.
Essential oil characterization: the relative density value obtained (0.8573mg/ml) is
within specifications of the given quality in IRAM-SAIPA Standard for Citrus
paradisi oil (0.8520-0.8600mg/ml). Although it was greater than the value reported by
other authors: 0.8433 (Pino, et al., 1999), 0.8500 (Viuda, et al., 2008) and a 0.8532-
0.8508 mg/ml (Kesterson and MacDuff, 1948) range for cold-pressed EO of this same
fruit; besides, it is within the range reported by (Güenther, 1961) from 0.8550 to
0.8600 mg/ml obtained under same conditions.
Refractive index measured (1.4723) was slightly under IRAM-SAIPA
(1.4740-1.4790) and Güenther (1961) specifications (1.4745-1.4778), but resulted
similar to those reported by Kesterson and MacDuff (1948) of 1.4714-1.4726.
However, it was greater than those reported by other authors, 1.4692 (Pino, et al.,
1999) and 1.4700 (Viuda, et al., 2008). Density values lower than 0.9000 mg/ml and
refractive indexes close to 1.4700 indicate a high content of terpenes (Kesterson and
MacDuff, 1948), consistently with ours chromatographic results.
Optical activity of grapefruit EO was +90.25° being in the range +91.45-
+94.36° reported by Güenther (1961) as well as in the range from +91.50° to +96.50°
reported by Kesterson and MacDuff (1948). Instead, other reference (Pino, et al.,
1999) reported a value greater than +94°. Values obtained from activity can be due to
content in terpenes, mainly from limonene which is the greatest, as they have more
optical and dextrorotatory activity compared to oxygenated components where optical
activity is null and to those of sesquiterpenes where it tends to be levorotatory (Weast,
Chemical constitution of essential oil: 21 components were identified in the test by
GC/MS: 9 terpenes, 3 sesquiterpenes, 2 aldehydes, 7 alcohols, and 1 ester
(representing 99.10% of components).
In Table 1, identified components together with retention times (Tr), and
composition average with its respective percentage values are shown. Limonene was
the monoterpene present in greater proportion, for this compound 92.60% was
obtained being in the upper limit of reported range (76.00-96.00%) for citrus EOs
(Kirbaslar, et al., 2006; Espina, et al., 2011). Monoterpene in greater proportion
following limonene was β-myrcene (1.20%), slightly under the percentage reported in
literature for citrus EOs (Kirbaslar, et al., 2006). Other components are found in lower
proportions (below 1.00%).
Screening of antibacterial effect: Assay results for inhibition tested of
microorganism growth, indicated that EO exhibited varying levels of antibacterial
activity against studied bacteria. Although a greater number of Gram (+) strains than
O. M. Vasek, et al. /Journal of Natural Products, Vol. 8(2015): 16-26
Copyright © 2015, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
Gram (-) strains was used for experiments (27 and 13, respectively), under identical
assay conditions, 40.74% of Gram (+) and 100.00% of Gram (-) were sensitive to EO.
All tested E. coli (13 strains) showed sensibility to this EO.
Of tested strains of Gram (+) lactobacillus, only 3 of them showed a minimum
inhibition in the assay conditions (ZOI: 5.0-6.5mm). Among Gram (+) cocci tested
strains, Leuc. mesenteroides subsp. dextranicum 14c isolated from caseario
environment in the province of Corrientes (Argentina) presented a significant
sensitivity (ZOI=11.0mm); L. lactis subsp. diacetylactis 166c showed less sensitivity
to grapefruit EO (ZOI=9.1mm), and L. lactis subsp. lactis strains showed a variable
response. Four strains can be highlighted (207c, 138c, 199c, and 140c, in descending
order of importance) to have shown an interesting inhibitory response against
essential oil. These results are shown in Fig. 1, L. lactis subsp. lactis 35c VCOR was
used as negative control.
Table-1: Chemical composition of Citrus paradisi oil by GC/MS.
Peak Nº TR (min) Identified Components %
1 12.62
2 15.38 Sabinene 0.60
3 16.71
4 17.38 n-octanal 0.40
5 21.21 Limonene 92.60
6 2.46 Linalool oxide cis 0.10
7 23.72 Linalool oxide trans 0.10
8 24.19 Linalool 0.20
9 25.49 Mentha-2,8-dien-1-ol trans p 0.30
10 26.27
Limonene oxide (Z)+mentha 2,8-
dien-1-ol cis p- +limonene oxide (E)
11 27.5
12 30.17 Decanal 0.10
13 32.03 Carveol trans 0.30
14 32.87 Carveol cis 0.30
15 33.63 Carvone 0.30
16 35.33 Geranial + E-ocimenone 0.10
17 40.76
18 41.23 Neryl acetate 0.10
19 43.77
20 48.58
21 50.83 Spathulenol+caryophyllene oxide 0.20
Total 99.10
: Retention time.
: Trace amounts.
Figure-1: Inhibition in growth of L. lactis subsp. lactis 207c y 138c VCOR generated by C.
paradisi EO.
O. M. Vasek, et al. /Journal of Natural Products, Vol. 8(2015): 16-26
Copyright © 2015, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
Minimum inhibitory concentrations: Minimum concentration of EO required
inhibiting the growth was determined in 2 strains of L. lactis subsp. lactis: 138c and
207c. Cell reproduction of first strain, inoculated at 9.27±0.90 log
CFU/ml density,
was stopped by a 15% (v/v) concentration of EO:Tween 80. While second strain
growth, 207c, at a cell density of 8.27±0.13 log
CFU/ml, was inhibited with just 5%
(v/v) of EO:Tween 80.
Considering the relation between volume and mass (mass = density/volumen), the
experimental density (0.853mg/ml) and the EO volume used in the dilution that
resulted inhibitory at the experiences (5 %, v/v), our results expressed that the lowest
concentration of Citrus paradisi EO needed to inhibit L. lactis subsp. lactis 207c
VCOR with a cellular density of 8.27±0.13 log
CFU/ml, was 4.29ppm, according to
the MIC using the Broth Microdilution testing.
Characterization of antagonistic effect of EO on cell growth: growth kinetics of
207c VCOR wild-type strain with and without EO is presented in Table 2. Test strain,
at 9.17±0.024 log
CFU/ml cellular density, was inoculated in two series of flasks.
Once growth exponential phase was fully reached, 3 to 4 growing hours, showing an
increasing spectrophotometric turbidity speed of 0.398 units Abs
/h and a number
of cells of 1.29± 0.17x10
CFU/ml, an aliquot of EO dilution was injected into flasks.
Table-2: Growth kinetics of L. lactis subsp. lactis.
Time (h) Strain 207c Strain 207c+EO
Viable count cell
Viable count cell
0.05 9.17
0.24 0.090
0.05 9.17
0.02 9.11
0.09 0.436
0.03 9.11
0.00 9.11
0.16 0.859
0.02 9.11
0.08 9.11
0.17 1.405
0.08 9.11
0.07 9.43
0.09 1.283
0.05 7.11
0.02 9.26
0.01 1.270
0.01 6.80
0.02 7.95
0.05 1.200
0.03 4.48
: no determined.
Adding EO has no effects (P>0.05) on incremental speed of Abs
by 207c VCOR strain with and without addition of EO (0.407/h, 0.492/h,
respectively). However, smaller Abs
was produced when reaching cryptic
growing phase, showing a statistically significant difference (P<0.05) in the population
measured by Abs
of 0.280. Considering the methodology used for its
determination, this difference might suggest a partial cell lysis.
Although kinetics has been tested for 120h, only the results of the first 54h are shown.
No significant differences (P>0.05) were observed in absorbance measurements in
each series over time, or major differences were seen in both series than those detected
at 54h incubation at subsequent incubation periods.
O. M. Vasek, et al. /Journal of Natural Products, Vol. 8(2015): 16-26
Copyright © 2015, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
Chemical composition and values of grapefruit EO physical properties obtained were
found near or within values reported in literature for this type of oil, and are consistent
with extraction method used and its composition-high monoterpenic hydrocarbon
content, especially limonene.
Reviewing opposing results about antibacterial effect, regarding differential
sensitivity of bacteria (Gram + and Gram -) against EOs, some (Smith-Palmer, et al.,
2001; Burt, 2004) support a greater Gram (+) sensitivity due to relative external
membrane impermeability in Gram (-), and others (Tassou, et al., 2000; Fisher and
Phillips (2006), reported similar sensitivity results both in Gram (+) and in Gram (-).
Researchers postulated that differential sensitivity in both bacteria groups (Gram +
and Gram -) does not depend exclusively from chemical or structural characteristics
of cell wall, but there are other factors that influence response (Fisher and Phillips
2008; Bajpai et al., 2012). This postulate is consistent with our results.
Grapefruit EO had an important spectrum of antibacterial activities against E.
coli (Fig. 1) with zones of inhibition ranging from 8.3 to 17.2mm. These results are
consistent with those reported by other authors (Akroum, et al., 2009; Uysal, et al.,
2011; Imran, et al., 2013) for grapefruit EO antibacterial effect and other related
species such as C. union (Sarmah and Kumari, 2013), C. limettioides (Vasudeva and
Sharma, 2012) and Citrus spp. (Chanthaphon, et al., 2008; Fuselli, et al., 2008).
Lactic acid bacteria, particularly cocci, showed an interesting inhibition from
this EO. In northeastern provinces of Argentina, there a great amount of fresh
handmade pasta factories corresponding to classical Italian pasta in addition to those
factories of traditional "chipa"-a tapioca-flour roll with cheese, eggs, and water,
inherited from the Republic of Paraguay which, given its geographical position,
borders Corrientes, Chaco, Formosa, and Misiones provinces. In these places,
production and selling of "chipa" was so popular that nowadays it is sold baked for its
direct consumption and as undercooked hard rolls or dough for preparation,
refrigerated or frozen with different shelf life periods according to the conservation
methodology. Considering that during spring and summer seasons high ambient
temperatures are reached (42-46ºC), together with high humidity, commercial low
temperature preservation systems do not bear this climate conditions minimizing its
performance. As a consequence, this sector manufacturers face severe microbial
reproduction problems, mainly because of lactic bacteria naturally present in raw
material that do not involve a risk for consumer health since they are Generally
Recognized As Safe (GRAS) according to American Food and Drug Administration
(FDA) (FDA, 2013). However, their growth and proliferation can reach the order of
CFU/g, causing acidification (50-60°Dornic), typical glycolytic metabolism
of this microbial group (data not shown), obviously, with changes in the organoleptic
characteristics of the product. This is why this modifying microorganism group gains
importance in this food sector, especially for future transference of results to regional
Although antagonistic effect to growth of this EO, with GRAS status (FDA,
2013), was determined against spoilage bacteria which are of interest for this study, we
must consider that, for its potential application to food preservation, effective activity
concentration shall not produce changes in its sensorial properties. Determined MIC
for this EO (4.29ppm) against L. lactis subsp. lactis (1.86x10
CFU/ml) is low enough
to be used in prevention of food spoilage with no cytotoxic effects for human health, or
sensory changes in product. However, specific food system must be experimentally
tested as factors like fat content, and pH contained, can reduce this beneficial effect
O. M. Vasek, et al. /Journal of Natural Products, Vol. 8(2015): 16-26
Copyright © 2015, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
determined in vitro.
There was a decrease (P<0.05) in the number of viable cells originally present
when adding EO (1.29±0.17x10
CFU/ml). After 24h of contact, 6.3±0.50x10
CFU/ml was detected, while cell density of control microorganism series remained in
the order of 10
CFU/ml (1.83±0.07x10
CFU/ml). The antagonistic effect of EO
lasted during all experimental incubation period, making only 4.48±0.07x10
microorganisms keep their viability at 54h of contact and subsequently. These results
verified EO bacterial effect on 207cVCOR strain under these assay conditions.
Grapefruit EO has undergone less studies than sweet orange, lemon and
bergamot EOs and their main components respect to antimicrobial effect. Particularly,
Fisher and Phillips (2006), reported absence of limonene antimicrobial effect against
some pathogenic bacteria related to food-borne diseases. These results are in
opposition to our experience.
However, it is necessary to consider the possibility of chemical interactions
(positive or negative) between principal components and other components present at
level of traces that could generate synergy.
Comparing series cell counts with and without addition of EO to this time,
there was 1.82x10
CFU/ml (99.29%) viability loss at 24h of contact and 8.95x10
CFU/ml (99.97%) viability loss at 54h under these experimental conditions.
EO of Citrus paradisi can be used as safer and alternative means of food
preservation to minimize microbial contamination and to improve quality of foods as a
shift from synthetic chemicals to botanical antimicrobials is gaining popularity because
of their environment safety and bio-rational mode of action. This essential oil has very
strong potential applicability as natural antibacterial agent for food industry, specially,
for pasta manufacture which is facing serious problems of spoilage due to lactic
bacteria activity. EOs could have important implications for the development of
antimicrobial strategies. It is likely that it will be more difficult for bacteria to develop
resistance to the multi-component EOs than to common antibiotics or chemical
preservers, generally constituted by a single molecular entity.
In our knowledge, this is the first time the Argentinean grapefuit essential oil
is evaluated as control agent, for the growth of undesirable bacteria in food, from the
viewpoint of their potential application in bio-preservation.
Essential oil of Citrus paradisi from Bella Vista, Corrientes, Argentina has limonene
as main constituent (92.6%), inhibits spoilage and pathogenic bacterial growth, both
Gram (+) and Gram (-), and has bactericidal effect on Lactococcus lactis subsp. lactis
with a dose low enough to be used as bio-preservative in food where this
microorganism causes spoilage.
Acknowledgments: the authors would like to thank General Secretariat of Science and Technology of
Northeastern University and National Technological University of Argentina for their financial support
for this study and scholarships (PI:F011-11. Res. 976/11-CS-UNNE, 2012-2015 and PI:25/L054-
UTI1672-R-UTN, 2012-2014, respectively).
Adams, R.P., (2001): Identification of Essential Oil Components by Gas Chromatography,
Quadrupole Mass Spectroscopy, Allured: C. Stream, IL.
Ait-Ouazzou, A., Cherrat, L., Espina, L., Lorán, S., Rota, C., Pagán, R., (2011): The
antimicrobial activity of hydrophobic essential oil constituents acting alone or in
combined processes of food preservation. Innov. Food Sci. Emerg., 12:320-329.
O. M. Vasek, et al. /Journal of Natural Products, Vol. 8(2015): 16-26
Copyright © 2015, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
Akroum, S., Satta, D., Lalaoui, K., (2009): Antimicrobial, Antioxidant, Cytotoxic activities &
phytochemical screening of some Algerian plants. Eur. J. Sci. Res., 31(2):289-295.
Badawy, M.E.I., Abdelgaleil, S.A.M., (2014): Composition and antimicrobial activity of
essential oils isolated from Egyptian plants against plant pathogenic bacteria and
fungi, Ind. Crops Prod., 52:776-782.
Bajpai, V.K., Baek, K.H., Kang, S.C., (2012): Control of Salmonella in foods by using
essential oils: A review, Food Res. Int., 45:722-734.
Burt, S., (2004): Essential oils: their antibacterial properties and potential applications in
foods, A review, Int. J. Food Microbiol., 94(3):223-253.
Chanthaphon, S., Chanthachum, S., Hongpattarakere, T., (2008): Antimicrobial activities of
essential oils and crude extracts from tropical Citrus spp. against food-related
microorganisms, Songklanakarin J. Sci. Technol., 30(1):125-131.
Chutia, M., Bhuyan, P.D., Pathak, M.G., Sharma, T.C., Boruah, P., (2009): Antifungal
activity and chemical composition of Citrus reticulata Blanco essential oil against
phytopathogens from North East India, Food Sci. Technol., 42:777-780.
Di Renzo, J.A., Casanoves, F., Balzarini, M.G., Gonzalez, L., Tabalda, M., Robledo, C.W.,
(2008): Statistical Software Infostat, Groip Infostat, Córdoba, Argentina.
Espina, L., Somolinos, M., Lorán, S., Conchello, P., García, D., Pagán, R., (2011):Chemical
composition of commercial citrus fruit essential oil & evaluation of their
antimicrobial activity acting alone/ in combined processes. Food Cont., 22:896-902.
FDA, (2013): Food and Drug Administration. Department of Health and Human Service.
Code of Federal Regulations, 21(3). Available online at:
Fisher, K., Phillips, C., (2006): The effect of lemon, orange and bergamot essential oils and
their components on the survival of Campylobacter jejuni, Escherichia coli O157,
Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus in vitro and in
food systems, J. Appl. Microbiol., 101(6):1232-1240.
Fisher, K., Phillips, C., (2008): Potential antimicrobial uses of essential oils in food: is citrus
the answer? Review, Trends Food Sci. Tech., 19:156-164.
Fuselli, S.R., García, S.B., Eguaras, M.J., Fritz, R., (2008): Chemical composition and
antimicrobial activity of Citrus essences on honeybee bacterial pathogen
Paenibacillus larvae, the causal agent of American foulbrood, World J. Microbiol.
Biotechnol., 24:2067-2072.
Güenther, E., (1961): The Essential Oils, Individual essential oils of the plant families
Rutaceae and Labiatae, D. Van Nostrand Comp., Toronto, Canada, Vol 3:349-357.
Imran, K., Saeed, M., Randhawa, M.A., Rizwan Sharif, H., (2013): Extraction and
applications of Grapefruit (Citrus paradise) peel oil against E. coli, Pak. J. Nut.,
IRAM-SAIPA 18504, (2002): Products Flavorings. Determination of relative density at 20ºC,
IRAM-SAIPA 18505, (2002): Products Flavorings. Determination of refractive index,
IRAM-SAIPA 18507, (2002): Products Flavorings. Determination of optical rotation,
Kesterson, J.W., MacDuff, O.R., (1948): Florida Citrus Oils. Commercial production methods
and properties of essential oils (1947-1948 season). University of Florida,
Gainesville, FL. Agr. Exp. Sta. Tech. Bull., 452:1-44.
Kirbaslar, S., Boz, I., Kirbaslar, F., (2006): Composition of Turkish lemon and grapefruit peel
oils, J. Essent. Oil Res., 18:525-543.
Lai, C.C., Wang, C.Y., Chu, C.C., Tan, C.K., Lu, C.L., Lee, Y.C., (2011): Correlation
between antibiotic consumption and resistance of Gram-negative bacteria causing
healthcare-associated infections at a university hospital in Taiwan from 2000 to 2009,
J. Antimicrob. Chemoter., 66:1374-1382.
Lv, F., Liang, H., Yuan, Q., Li, Ch., (2011): In vitro antimicrobial effects and mechanism of
action of selected plant essential oil combinations against four food-related
O. M. Vasek, et al. /Journal of Natural Products, Vol. 8(2015): 16-26
Copyright © 2015, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
microorganisms. Food Res. Int., 44:3057-3064.
Mc Lafferty, F.W., Stauffer, D.B., (1991): The Wiley/NBS Registry of Mass Spectral Data,
Wiley, New York.
Maturin, L., Peeler, J.T., (2001): Aerobic Plate Count, Bacteriological Analytical Manual
Food and Drug Administration, U.S. Available online at: [
Oliveira, A.C.S., Rabelo M.Z.J., Bispo Reis Di Iorio, F., Pereira, C.A., Cardoso, A.O., (2014):
The antimicrobial effects of Citrus limonum and Citrus aurantium essential oils on
multi-species biofilms, Braz. Oral Res., 28(1):22-27.
Ortez, J.H., (2005): Test methods, Disk Diffusion Testing, In: Manual of Antimicrobial
Susceptibility Testing, M.B. Coyle Coordinating, American Society for
Microbiology, Panamerican Health Organization, pp. 39-52.
Pino, J., Acevedo, A., Rabelo, J., González, C., Escandón, J., (1999): Chemical Composition
of Distilled Grapefruit Oil, J. Essent. Oil Res., 11(1):75-76.
Rankin, I.D., (2005): Test methods. MIC Testing. In: Manual of Antimicrobial Susceptibility
Testing, M.B. Coyle Coordinating (Ed). American Society for Microbiology,
Panamerican Health Organization, pp. 53-62.
Rehman, Z., (2006): Citrus peel extract: A natural source of antioxidant. Food Chem., 99:450-
Roussenova, N., (2011): Antibacterial activity of essential oils against the etiological agent of
American foulbrood disease (Paenibacillus larvae), Bulg. J. Vet. Med., 14(1):17-24.
Sarmah, N., Kumari, S., (2013): Comparative study of antibacterial activity of ripen &unripen
Indigenous Citrus union of Assam India. Int. J. Adv. Res. Technol., 2(9):25-31.
Smith-Palmer, A., Stewart, J., Fyfe, L., (2001): The potential application of plant essential
oils as natural food preservatives in soft cheese, Food Microbiol., 18(4):463-470.
Solórzano-Santos, F., Miranda-Novales, M.G., (2012): Essential oils from aromatic herbs as
antimicrobial agents, Curr. Opinion Biotechnol., 23:136-141.
Tao, N., Liu, Y., Zang, M., (2009): Chemical composition and antimicrobial activities of
essential oil from the peel of bingtant sweet orange (Citrus sinensis Osbeck), Int. J.
Food Sci. Technol., 44:1281-1285.
Tassou, C., Koutsoumanis, K., Nychas, G.J.E., (2000): Inhibition of Salmonella enteritidis
and Staphylococcus aureus in nutrient broth by mint essential oil, Food Res. Int.,
UIA, (2008): National Agency for Science and Technology-Profecyt-Mincyt, Weaknesses
and technological challenges of the productive sector. Citrus fruits (lemon, tangerine
Available on line at:
Uysal, B., Sozmen, F., Aktas, O., Oksal, B.S., Kose, E.O., (2011): Essential oil composition
and antibacterial activity of the grapefruit (Citrus paradisi L.) peel essential oils
obtained by solvent-free microwave extraction: comparison with hydrodistillation,
Int. J. Food Sci. Technol.,46(7):1455-1461.
Vasudeva, N., Sharma, T., (2012): Chemical composition and antimicrobial activity of
essential oil of Citrus limettioides Tanaka, J. Pharm. Technol. Drug Res. Available
online at: [, doi:
Viuda, M., Navajas, R., Fernández, J., Pérez, J., (2008): Antifungal activity of lemon (Citrus
lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.) and orange
(Citrus sinensis L.) essential oils, Food Control, 19:1130-1138.
Weast, R., (2010): Handbook of Chemistry and Physics, Ed. 90
. CRC Press, USA, pp. 318-
WHO, (2013): World Health Organization. Statistic. Available online at:
Yin, X., Gyles, C.L., Gong, J., (2012): Grapefruit juice and its constituents augment the effect
of low pH on inhibition of survival and adherence to intestinal epithelial cells of S.
enterica serovar typhimurium PT193. Int. J. Food Microbiol., 158:232-238.
... Its content ranges from 84.8 to 93.45%. The remaining ingredients are present in small quantities: myrcene (6.9%) and alpha-pinene (1.7%), belonging to monoterpenes; and beta-caryophyllene (1.1%), belonging to sesquiterpenes [40, 59,73]. C. paradisi EO inhibited the growth of E. coli, S. aureus, E. faecalis, S. Typhimurium, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus plantarum, Staphylococcus epidermidis, Serratia marcescens, and Proteus vulgaris [73][74][75]. ...
... The remaining ingredients are present in small quantities: myrcene (6.9%) and alpha-pinene (1.7%), belonging to monoterpenes; and beta-caryophyllene (1.1%), belonging to sesquiterpenes [40, 59,73]. C. paradisi EO inhibited the growth of E. coli, S. aureus, E. faecalis, S. Typhimurium, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus plantarum, Staphylococcus epidermidis, Serratia marcescens, and Proteus vulgaris [73][74][75]. The effectiveness of C. paradisi EO against fungi: A. niger, A. flavus, C. albicans, Penicillium chrysogenum, Fusarium moniliforme and Saccharomyces cerevisiae has been confirmed [76]. ...
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The role of purified natural products in the prevention and treatment of countless diseases of bacterial, fungal, and viral origin cannot be overestimated. New antiviral drugs have been obtained from natural sources and transformed into preparations for prophylactic and therapeutic purposes. Flavonoids, polyphenols, saponins, proanthocyanins, polysaccharides, organic acids, proteins, polypeptides, and essential oils derived from plants, animals, or microorganisms can control and combat foodborne viral infections, including hepatitis A. The components of essential oils are characterized by numerous therapeutic and antioxidant properties and exhibit a broad spectrum of antimicrobial and antiviral activity. Due to these properties, they can be used to preserve meat, fruit, vegetables, and their products. Over the past two decades, much effort has been made to identify natural products, mostly of plant origin, to combat foodborne viruses. Natural plant extracts have several potential uses, not limited to increasing the safety of food products and improving their quality, but also as natural antiviral agents.
... Limonene is the major volatile component identified in the peel of different Citrus species. Citrus peel essential oils have several biological activities as antioxidant, anti-inflammatory, analgesic, antimicrobial and anticancer activities (Singh et al., 2021 (Chanthaphon et al., 2008;Jafari et al., 2011;Bourgou et al., 2012;Jing and al., 2014;Ngele et al., 2014;Vasek et al., 2015;Sajid et al., 2016;Kademi and Garba, 2017). The antifungal activity of from different Citrus peel essential oils species against Alternaria alternata has been also studied (Sharma and Tripathi, 2006;Phillips et al., 2012;Bozkurt et al., 2017;Ajayi-Moses et al., 2019;Sedeek et al., 2021). ...
The essential oil extraction from Citrus peels constitutes an effectual approach for the valorization of Citrus fruit waste. Citrus peel essential oils can have an antifungal activity. The peel essential oils of seven species of Citrus, namely clementine, pummelo, tangelo, sweet orange, sour orange, tangerine and lemon, were extracted by steam distillation and subjected to chemical analysis to identify and evaluate the chemical constituents. Tangerine exhibited the highest yield (1.23%) followed by lemon (0.66%), clementine (0.56%), sweet orange (0.55%), tangelo (0.50%) and sour orange (0.47%). The lowest yield was obtained in pummelo (0.16%). The main compound in all Citrus essential oils was limonene with 62.1% for tangerine, 69.7% for lemon, 72.2% for tangelo, 76.4% for clementine, 89.8% for pummelo, 92.8% for sour orange and 94.1% for sweet orange. All these Citrus essential oils had a weak antifungal activity (MIC = 8000-12000 µg/ml) against Alternaria alternata strain.
... Therefore, fortification of essential herb oil in dairy products can deliver value-added and functional properties to different dairy products (El-Sayed and Ibrahim, 2021;El-Sayed and Youssef, 2019). Moreover, the use of some essential herb oils will help to extend shelf life and protect final products from bacterial infection and spoilage caused by storing the product (Vasek et al., 2015). The favorable essential oil can be used from cumin. ...
White soft cheese is common to become contaminated with pathogenic and spoilage microorganisms during storage. Thus, the influence of cumin essential oil (CEO) nanoemulsion and used as a brined solution on the quality of white soft cheese was reported in this study. CEO nanoemulsion was exhibited significant antimi-crobial activity against different pathogens like (Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Listeria monocetogenes, Salmonella typhimirum, Yersinia enterocotilica, Aspergillus niger, and Aspergillus flavus). The chemical composition of the CEO was determined by Gas chromatography-mass spec-troscopy (GC-MS) and found sixteen compounds. Moreover, the CEO nanoemulsions were characterized by a dynamic light scattering technique (DLS) and transmission electron microscope (TEM). Different CEO nano-emulsion ratios (0.50, 0.75, and 1.00%) were used as a preservative solution for the white soft cheese. Yeasts & molds and psychrotrophic counts were not detected for cheese preserved in 1.00% CEO nanoemulsion throughout the storage period until 60 days. However, observed minor yeasts & molds and psychrotrophic counts for cheese preserved in 0.50 and 0.75% of CEO nanoemulsion solutions. Organoleptic properties for cheese found that the highest total scores were given to cheeses preserved with 0.50 and 0.75% of CEO nanoemulsion solutions. These results recommended use CEO nanoemulsion as a natural preservative brined solution to preserve the quality of white soft cheese during storage.
... The oil can be found in all plant tissues but is more concentrated in the husks. In general, it can be obtained in three ways: as a byproduct of the orange juice industry, hydro-distillation of fruit components, or by cold pressing of the peels [2,[9][10][11][12][13][14][15] through distillation by steam vapor or by pressing citrus pericarps, with subsequent elimination of the aqueous phase by physical methods [16]. However, in this context, some more recent works stand out for obtaining essential oil through environmentally friendly alternatives. ...
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During the production of orange juice, more specifically after the commercial extraction of fruit juice, other waste materials are generated, consisting of peel, pieces of membranes, pulp bagasse, juice vesicles and seeds. In this way, the final destination of the waste can become a problem when not managed correctly. Therefore, there are several possibilities for using these solid residues, as they present substances of great commercial interest. In this perspective, the present work evaluates the recovery of orange essential oil from the citrus industry waste using hydrodistillation. The oil obtained was characterized by acidic index, FTIR, GC / MS, TGA and DSC. The results exhibited that oil isolated by hydrodistillation has a similarity with cold-pressed orange oil. The chemical constitution of oil obtained from waste was almost the same as the commercial orange oil analyzed. However, the thermal behaviour presents a few differences in thermal stability and vaporization temperature between analysed essential oils. Therefore, this work produces an alternative to obtain a product with quality, high yields and added value that can be used in cosmetic and pharmaceutical industries.
... 6,7 Some research suggested that the essential oil of orange peels had a considerable potential for use as an antimicrobial material. [8][9][10][11][12][13][14][15][16][17][18][19][20] Testing of antimicrobial activity against oral disease and dental bacteria, S. mutans in particular, have not been reported. Under these conditions, this study aimed at studying the effects of five essential oils of orange peel e.g. ...
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Introduction: The essential oils of orange peels had a considerable potential to be used as an antimicrobial agent. The aim of this present study is to analized chemical composition of Citrus spp. And the oral antimicrobial effect of Citrus spp. peels essential oils against Streptococcus mutans. Methods: Five orange peels species were used in this study consist of Lime (Citrus aurantifolia), Tangerine (Citrus nobilis), Sweet Orange (Citrus sinensis), Lemon (Citrus limon), and Kaffir Lime (Citrus hystrix). The isolated essential oils were analyzed using gas chromatography-mass spectroscopy (GC-MS). The Streptococcus mutans ATCC 25175 was employed against the antimicrobial effect of samples. Results: The extraction yields of hydrodistilled-essential oils from Tangerine, Kaffir Lime, Sweet Orange, Lemon, and Lime provided the extraction yields of 4.20, 2.26, 1.97, 1.74 and 0.83% yields, respectively. Major component essential oils of Citrus spp. was D-Limonene. The highest antimicrobial activity against S. mutans was Lime peel essential oil, followed by Lemon, Kaffir Lime, and Sweet Orange or Tangerine. All samples showed antimicrobial activity against S. mutans with the variation of antimicrobial action depending on the constituent of D-Limonene, β-Pinene, and α-terpineol. Conclusion: Major component chemical composition of essential oils of Citrus spp. was D-Limonene and antimicrobial activity by Lime peel essential oil due to its proportional amount of D-limonene and β-pinene and the highest antimicrobial activity.
... diacetylactis, Leuconostoc mesenteroides subsp. dextranicum y Lactobacillus plantarum (Vasek et al., 2015). ...
La microencapsulación es definida como una tecnología de empaquetamiento, aplicada con éxito en la industria alimentaria, biotecnológica y farmacéutica. Se la utiliza como método de protección de principios activos sensibles a factores externos, como los aceites esenciales. De esta manera, estos compuestos pueden ser incorporados a distintos productos donde se lleva a cabo su liberación controlada. Se aprovecha así sus propiedades antimicrobianas, conservantes, saborizantes o aromatizantes, entre otras. En este trabajo se optimizó el proceso de microencapsulación del aceite esencial de pomelo con alginato de sodio por gelificación iónica externa acoplada a una extrusión. Se utilizó alginato de sodio al 1% p/v y carga de aceite esencial del 2% p/p. Se determinó el punto óptimo con concentración de cloruro de calcio del 10% p/v con tiempo de reticulación de 45 minutos. En estas condiciones la eficiencia media fue de 95,89±0,04%, y el rendimiento medio de 56,87±1,2%.
... and antimicrobial activities. Therefore, there is great potential for their application in the food industry (Pereira et al., 2006;Vasek et al., 2015). ...
Full-text available
Background. Preparing and producing a healthy new dairy product that attracts consumers is an important issue. Assessing the effects of supplementation of milk with an essential oil to improve the quality of ultrafiltered soft buffalo cheese was the main target of this research. Materials and methods. UF soft buffalo cheese was traditionally prepared to achieve four treatments: control samples (C) had no additives and (T1, T2 and T3) were supplemented with extracted basil essential oil (BEO) at concentrations of 0.025, 0.050 and 0.075% v/v respectively. The microbiological, chemical and sensory properties of the resultant samples were determined at intervals (fresh, 15, 30 and 60 days). Results. Results revealed that a high ratio of BEO (0.075%) promoted the growth of starter culture followed by 0.050% and 0.025%. At the same time BEO inhibited mold and yeast growth in all soft UF cheese samples. The pH values decreased during the storage period as a result of starter activity. Adding BEO improved the sensory properties of UF soft buffalo cheese samples compared with control. Conclusion. Fortification of UF soft buffalo cheese with extracted basil essential oil enhanced the growth of starter culture and inhibited mold and yeast growth. It also improved the sensory properties of the final product.
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Injudicious consumption of antibiotics in the past few decades has arisen the problem of resistance in pathogenic organisms against most antibiotics and antimicrobial agents. Scenarios of treatment failure are becoming more common in hospitals. This situation demands the frequent need for new antimicrobial compounds which may have other mechanisms of action from those which are in current use. Limonene can be utilized as one of the solutions to the problem of antimicrobial resistance. Limonene is a naturally occurring monoterpene with a lemon-like odor, which mainly present in the peels of citrus plants like lemon, orange, grapefruit, etc. The study aimed to enlighten the antimicrobial properties of limonene as per previous literature. Advantageous contributions have been made by various research groups in the study of the antimicrobial properties of limonene. Previous studies have shown that limonene not only inhibits disease-causing pathogenic microbes, however, it also protects various food products from potential contaminants. This review article contains information about the effectiveness of limonene as an antimicrobial agent. Apart from antimicrobial property, some other uses of limonene are also discussed such as its role as fragrance and flavor additive, as in the formation of nonalcoholic beverages, as solvent and cleaner in the petroleum industry, and as a pesticide. Antibacterial, antifungal, antiviral, and anti-biofilm properties of limonene may help it to be used in the future as a potential antimicrobial agent with minimal adverse effects. Some of the recent studies also showed the action of limonene against COVID-19 (Coronavirus). However, additional studies are requisite to scrutinize the possible mechanism of antimicrobial action of limonene.
This study investigated physiological alterations involved in the inactivation of Levilactobacillus (L.) brevis and Leuconostoc (Lc.) mesenteroides in orange juice caused by Citrus lemon essential oil (CLEO) and C. reticulata essential oil (CREO) alone and combined with mild heat treatment (MHT). Damage in DNA, membrane integrity, membrane potential, metabolic and efflux activity of bacterial cells were measured after exposure (6 and 12 min) to CLEO or CREO (0.5 μL/mL) and/or MHT (54 °C) using flow cytometry. Limonene was the major constituent in CLEO (66.4%) and CREO (89.4%). The size of the damaged cell subpopulations increased (p < 0.05) after longer exposure time and varied with the tested essential oil and/or bacterial isolate. After exposure to CLEO and CREO alone, the cell subpopulations with damage in measured physiological functions were in a range of 19.6-66.8% and 23.8-75.9%, respectively. Exposure to CREO resulted in larger Lc. mesenteroides cell subpopulations (35.4-68.7%) with damaged DNA, permeabilized and depolarized membrane and compromised metabolic or efflux activity compared to L. brevis (23.8-58.0%). In contrast, exposure to CLEO led to higher damaged L. brevis cell subpopulations (35.1-77%) compared to Lc. mesenteroides (25.3-36.6%). Exposure to combined treatments (CLEO or CREO and MHT) affected the measured physiological functions in almost the entire L. brevis and Lc. mesenteroides cell population (up to 99%), although the damage extension on each isolate varied with tested essential oil. Results show that inactivation of L. brevis and Lc. mesenteroides cells caused by CLEO and CREO alone and combined with MHT in orange juice involves a multi-target action, which causes DNA damage, altered permeability and depolarization of membrane and compromised metabolic and efflux activities.
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El objetivo de este trabajo fue aumentar el rendimiento y selectividad en la reacción de obtención de α-terpineol a partir de limoneno, componente mayoritario del aceite esencial de pomelo, con el uso de emulsiones pickering de agua:aceite. Se utilizaron carbón activado, bentonita, sílica y alúmina como sólidos estabilizantes de la emulsión. Se caracterizaron las emulsiones midiendo su conductividad en distintas relaciones agua:aceite para determinar el punto donde ocurre la inversión de fase de la emulsión. Se prepararon las emulsiones pickering de tipo aceite/agua (o/w), estableciendo las concentraciones másicas óptimas de cada sólido. El rendimiento máximo obtenido en α-terpineol fue de un 43 % utilizando sílica, un 36 % más que en medio de reacción sin sólidos. También se logró reutilizar los sólidos dos veces sin diferencias en el rendimiento de la reacción.
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Phytochemicals has got special attention of researchers because of their value in food preservation and value addition due to consumer preference. Grapefruit (Citrus paradisi) is one of important member of family Rutaceae (citrus family). Oil was extracted through solvent extraction method using soxhlet's apparatus. The nuggets were prepared by incorporating the grapefruit peel oil at different levels 0 (control), 5, 10, 25, 50, 100% and stored for duration period (0, 7 and 14 days). Peel oil refractive index, specific gravity, pH, acid value, iodine values 1.47, 0.85, 3.67, 0.81, 99.4 were respectively calculated. The peak values for proximate analysis (protein, moisture, fat, ash contents) were 62.32, 15.29, 14.0 and 2.76 respectively were recorded in T1 (5% grapefruit peel oil) at all storage periods. In color, water activity and texture analysis were 117.77, 0.94 and 1691.7 respectively in T1 at all storage durations. Similarly in sensory evaluation of color, taste, flavor and overall acceptability found statistically that T3 (25% grapefruit oil) treatment is highly significant at all storage periods and gave maximum value for these parameters and T5 (100% grapefruit oil) gave minimum value for its effectiveness. Grapefruit peel oil was checked for its antimicrobial activity test e.g., total plate count, disc diffusion method and minimum inhibitory action against Escherichia coli on chicken nuggets. In antimicrobial analysis MIC (Minimum Inhibitory Concentration) decreased with increasing the concentration of grapefruit peel oil and in disc diffusion method, its values increased with increasing grapefruit peel oil.
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The aim of this study was to evaluate the effects of Citrus limonum and Citrus aurantium essential oils (EOs) compared to 0.2% chlorhexidine (CHX) and 1% sodium hypochlorite (NaOCl) on multi-species biofilms formed by Candida albicans, Enterococcus faecalis and Escherichia coli. The biofilms were grown in acrylic disks immersed in broth, inoculated with microbial suspension (106 cells/mL) and incubated at 37°C / 48 h. After the biofilms were formed, they were exposed for 5 minutes to the solutions (n = 10): C. aurantium EO, C. limonum EO, 0.2% CHX, 1% NaOCl or sterile saline solution [0.9% sodium chloride (NaCl)]. Next, the discs were placed in sterile 0.9% NaCl and sonicated to disperse the biofilms. Tenfold serial dilutions were performed and the aliquots were seeded onto selective agar and incubated at 37°C / 48 h. Next, the number of colony-forming units per milliliter was counted and analyzed statistically (Tukey test, p ≤ 0.05). C. aurantium EO and NaOCl inhibited the growth of all microorganisms in multi-species biofilms. C. limonum EO promoted a 100% reduction of C. albicans and E. coli, and 49.3% of E. faecalis. CHX was less effective against C. albicans and E. coli, yielding a reduction of 68.8% and 86.7%, respectively. However, the reduction of E. faecalis using CHX (81.7%) was greater than that obtained using C. limonum EO. Both Citrus limonum and Citrus aurantium EOs are effective in controlling multi-species biofilms; the microbial reductions achieved by EOs were not only similar to those of NaOCl, but even higher than those achieved by CHX, in some cases.
Antibacterial activities of eleven essential oils against Paenibacillus larvae (15 field strains and the reference BCCM/LMG 9820 strain) were studied by the disk diffusion method and the method of serial dilutions in agar. The minimal inhibitory concentration (MIC) of essential oils was determined within 1%-0.015% v/v. Highest activity: MIC ≤ 0.06-0.015% v/v was shown by essential oils of cinnamon, thyme, clove, peppermint, lemongrass, sage and oregano. Variable activity exhibited marjoram and tee tree oils. Citrus essential oils showed the lowest inhibitory effect with MIC ≥ 0.12-1.0% v/v for mandarin oil and ≥ 0.25-0.5% v/v for grapefruit oil. Established antibacterial activity against Paenibacillus larvae encourages further research to include essential oils as an alternative means in the measures for prevention and control of American foulbrood without the use of antibiotics.
The essential oils of eighteen Egyptian plants, namely, Artemisia judaica, A. monosperma, Callistemon viminals, Citrus aurantifolia, C. lemon, C. paradisi, C. sinensis, Cupressus macrocarpa, C. sempervirens, Myrtus communis, Origanum vulgare, Pelargonium graveolens, Rosmarinus officinalis, Syzygium cumini, Schinus molle, S. terebinthifolius, Thuja occidentalis and Vitex agnus-castus, were isolated by hydrodistillation. The chemical composition of the isolated oils was identified by gas chromatograph/mass spectrometer (GC/MS). The major constituents of the isolated oils were limonene (40.19%, 56.30%, 74.29% and 89.23% in C. aurantifolia, C. lemon, Citrus paradise and C. sinensis, respectively), α-pinene (37.88%, 35.49%, 26.16% and 17.26% in C. sempervirens, T. occidentalis, M. communis and S. cumini, respectively), 1,8-cineole (71.77% and 19.60% in C. viminals and R. officinalis), pulegone (77.45% in O. vulgare), β-thujone (49.83% in A. judaica), capillene (36.86% in A. monosperma), sabinene (14.93% in S. terebinthifolius), α-phellandrene (29.87% in S. molle), 4-terpeneol (20.29% in C. macrocarpa), trans-caryophyllene (15.19% in V. agnus-castus) and β-citronellol (35.92 in P. graveolens). The isolated oils were tested for their antimicrobial activity against the most economic plant pathogenic bacteria of Agrobacterium tumefaciens and Erwinia carotovora var. carotovora, and fungi of Alternaria alternata, Botrytis cinerea, Fusarium oxysporum and Fusarium solani. The isolated oils showed variable degree of antibacteabril activity against A. tumefaciens and E. carotovora var. carotovora. Based on minimum inhibitory concentration (MIC) values, the oils were more effective against E. carotovora var. carotovora than A. tumefaciens. The oil of T. occidentalis revealed the highest antibacterial activity among the tested oils showing the lowest MIC values of 400 and 350 mg/L, on A. tumefaciens and E. carotovora var. carotovora, respectively. In mycelial growth inhibition assay, most of the essential oils showed pronounced effect and the oil of A. monosperma was the most potent inhibitor with EC50 = 54, 111, 106 and 148 mg/L against A. alternata, B. cinerea, F. oxysporum and F. solani, respectively. On the other hand, the oils caused strong reduction in spore germination of fungi compared with control. The oils of A. judaica and A. monosperma caused the highest spore germination inhibition of F. oxysporum and their EC50 values were 69 and 62 mg/L, respectively. Among the tested fungi, F. oxysporum was the most susceptible fungus to all of the tested oil except the oil of S. molle. The relationship between the antimicrobial activity and the chemical composition of the isolated oils was disclosed. The findings of the present study suggest that the isolated oils have a potential to be used as antimicrobial agents.
Citrus peel extract as a natural source of antioxidant was evaluated during 6 months storage of refined corn oil at 25 and 45 °C. Extracts of citrus peel were prepared by refluxing the dried ground peel with ethanol, methanol, acetone, hexane, diethyl ether and dichloromethane. Maximum amount of citrus peel extract was obtained with methanol. Antioxidant activity of methanolic extract was assessed by measuring free fatty acid (FFA) content peroxide value (POV) and iodine value (IV) during 6 months storage of refined corn oil at 25 and 45 °C. After 6 months of storage at 45 °C, corn oil containing 1600 and 2000 ppm citrus peel extract, showed lower FFA contents (1.5% and 1.0%), and POVs (8.38 and 7.0 meq kg−1) and higher iodine values (81, 89) than the control sample (FFA 17.0% POV 101 meq kg−1 IV 47). Refined corn oil containing 200 ppm of butylated hydroxy anisole (BHA) and butylated hydroxy toluene (BHT) showed FFA contents of 2.0% and 1.8%, POVs 17.0 and 12.7 meq kg−1 and IVs 84 and 87, respectively, after 6 months of storage at 45 °C. These results show that methanolic extract of citrus exhibited very strong antioxidant activity, which was almost equal to synthetic antioxidants (BHA and BHA). Therefore, the use of citrus peel extract is recommended as a natural antioxidant to suppress development of rancidity in oils and fats.
The aim of this study was to evaluate the antimicrobial efficacy of selected plant essential oil (EO) combinations against four food-related microorganisms. Ten EOs were initially screened against Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Saccharomyces cerevisiae using agar disk diffusion and broth dilution methods. The highest efficacy against all the tested strains was shown when testing the oregano EO. EOs of basil and bergamot were active against the Gram-positive bacteria (S. aureus and B. subtilis), while perilla EO strongly inhibited the growth of yeast (S. cerevisiae). The chemical components of selected EOs were also analyzed by GC/MS. Phenols and terpenes were the major antimicrobial compounds in oregano and basil EOs. The dominant active components of bergamot EO were alcohols, esters and terpenes. For perilla EO, the major active constituents were mainly ketones. The checkerboard method was then used to investigate the antimicrobial efficacy of EO combinations by means of the fractional inhibitory concentration index (FICI). Based on an overall consideration of antimicrobial activity, organoleptic impact and cost, four EO combinations were selected and their MIC values were listed as follows: oregano–basil (0.313–0.313μl/ml) for E. coli, basil–bergamot (0.313–0.156μl/ml) for S. aureus, oregano–bergamot (0.313–0.313μl/ml) for B. subtilis and oregano–perilla (0.313–0.156μl/ml) for S. cerevisiae. Furthermore, the mechanisms of the antimicrobial action of EO combinations to the tested organisms were studied by the electronic microscopy observations of the cells and the measurement of the release of cell constituents. The electron micrographs of damaged cells and the significant increase of the cell constituents' release demonstrated that all EO combinations affected the cell membrane integrity.
The essential oil obtained by steam distillation of solids and effluents from the commercial oil extraction of grapefruit was analyzed by GC/MS. Twenty-eight components were identified, which constituted 98% of the oil. The major constituent was limonene (70.9%).