Accepted Manuscript The preservative potential of Amomum tsaoko essential oil against E. coil, its antibaterial property and mode of action
Na Guo, Yu-Ping Zang, Qi Cui, Qing-Yan Gai, Jiao Jiao, Wei Wang, Yuan-Gang Zu, Yu-Jie Fu PII:
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05 October 2016
05 December 2016
07 December 2016
Please cite this article as: Na Guo, Yu-Ping Zang, Qi Cui, Qing-Yan Gai, Jiao Jiao, Wei Wang, Yuan-Gang Zu, Yu-Jie Fu, The preservative potential of Amomum tsaoko essential oil against E. coil, its antibaterial property and mode of action, Food Control (2016), doi: 10.1016/j.foodcont. 2016.12.013
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ACCEPTED MANUSCRIPT Highlight Highlights
Amomum tsaoko essential oil AEO with edible and medicinal function is a popular spice in south-west China.
Amomum tsaoko essential oil AEO can effectively act against foodborne bacteria at low concentrations.
The antibacterial mechanism of Amomum tsaoko essential oil AEO against E. coli was the change in permeability and integrity of the disrupted cell membranes.
Amomum tsaoko essential oil AEO may have useful applications as an alternative natural food preservative and additive in food field.
ACCEPTED MANUSCRIPT 1
The preservative potential of Amomum tsaoko essential oil against E. coil, its
antibaterial property and mode of action 1,2,3,
Wang 1,2,3, Yuan-Gang Zu 1,2,3, Yu-Jie Fu 1,2,3*
5 6 7
Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast
Forestry University, 150040 Harbin, PR China
Engineering Research Center of Forest Bio-preparation, Ministry of Education,
Northeast Forestry University, 150040 Harbin, PR China
Resources, Northeast Forestry University, 150040 Harbin, PR China
*Corresponding author: Yu-jie Fu, Ph. D, Professor, Vice Director. Key Laboratory of
Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Box 332,
Hexing Road 26, Harbin 150040, P. R. China
E-mail: [email protected]
Collaborative Innovation Center for Development and Utilization of Forest
19 20 21
Amomum tsaoko is widely distributed in south-west China as a spice. In present study,
the antibacterial activity of Amomum tsaoko essential oil (AEO) against foodborne
pathogens was evaluated. The antibacterial activity was determined by measuring the
zones of inhibition (ZOI), minimum inhibitory concentration (MIC), minimum
bactericidal concentration (MBC), and the time-kill assay. Results showed the
susceptibility of foodborne bacteria Escherichia coli (E. coli) was excellent with the
lowest MIC and MBC values of 3.13 and 6.25 mg/mL, respectively. The probable
antibacterial mechanism of Amomum tsaoko essential oil AEO was the change in
permeability and integrity of the disrupted cell membranes leading to leakage of 1
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nucleic acids and proteins. Detection of the kinetics of E. coli deactivation in situ
showed that Amomum tsaoko essential oil AEO has good preservative activities in
foods. It is necessary to consider that Amomum tsaoko essential oil AEO will become
a promising antibacterial additive for food preservative.
35 36 37
Chemical compounds studied in this article
Crystal violet (PubChem CID: 11057); Tetracycline (PubChem CID: 54675776);
Tween 80 (PubChem CID: 5281955); Glutaraldehyde (PubChem CID: 3485); Ethanol
(PubChem CID: 702); 3, 3’-Diethyloxacarbocyanine iodide (PubChem CID:
6432767); Propidium iodide (PubChem CID: 104981)
42 43 44
Amomum tsaoko essential oil; Foodborne bacteria; Antibacterial mechanism;
Membrane integrity; Food preservative
47 48 49 50
In recent years there has been a rising concern about food safety for consumers
and food industry. Food products contaminated with pathogens can not only lead to
reduce the quality and quantity of food products (Soliman & Badeaa, 2002), but also
generate illness and disease (Jacob, Mathiasen & Powell, 2010). Meat is a rich source
of nutrients including animal proteins, essential amino acids, fatty acids, minerals,
trace elements and vitamins in daily life (Singh et al., 2014). The abundant nutrient
compositions of meat provide it an ideal environment for the multiplication of meat
spoilage microorganisms and foodborne pathogens (Babuskin et al., 2015). E. coli
Escherichia coli (E. coli) pathogenic strains has have been linked to foodborne 2
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illnesses since 1982. It has They have been found in a variety of foodstuffs including
milk, yogurt, water, salad vegetables, fruits, fruit juices cider and meat products
(Kornacki & Marth, 1982). In order to prevent and control pathogenic
microorganisms in foods, synthetic chemicals have been used to control microbial
growth and the incidence of food poisoning in the past few years. However, many
consumers are concerned about the potential adverse side effects of synthetic
chemicals in foods, it has been proved that some of the synthetic chemicals had
undesirable biological effects on human health, leading to immeasurable risks (Osman
& Abdulrahman, 2003). Thus, it is of vital importance to use safe and natural
antibacterial agents, particularly those from plants and fruits for the food preservation
(Tiwari et al., 2009).
Essential oils extracted from the scented plants are classified as the generally
recognized as safe (GRAS) substances for food preservative (Lv et al., 2011). It is a
volatile oily liquid generated by the secondary metabolism of plants, and can be used
as natural antimicrobials (Diao et al., 2013.) There are many reports on the
antibacterial activities of essential oils, and the use of essential oils as antimicrobial
agents in food products (Sagdic et al., 2003, & Salgueiro et al., 2010).
The herbs of Zingiberaceae family are always using for food additives and
conventional drug treatments (Sabulal et al., 2006). And there are about 85 species of
genus Zingiber herbs distributed in East Asia and tropical Australia. Amomum tsaoko
Crevost et Lemarie which was a member of Zingiberaceae family is widely
distributed in south-west China. The dried fruit of Amomum tsaoko is a well-known
commercial spice as the a food additives (Zhang, Lu, & Jiang, 2014). The essential oil
from Amomum tsaoko possesses superior antioxidant, anti-tumour, antibacterial and
antifungal efferts abilities (Zhang et al, 2015; Li et al, 2011, and Qiu et al, 1999). The
compositions of Amomum tsaoko essential oil AEO can be divided into five classes,
including monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene
hydrocarbons, oxygenated sesquiterpenes and others. 1, 8-cineole is the most
important constituent in Amomum tsaoko essential oil AEO (45.24%) (Yang et al.,
2010), which can be used as flavoring as a flavoring agent for food products 3
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(Vincenzi, mancini, & Dessi, 1996).
A series of studies has have demonstrated the antibacterial activity of Amomum
tsaoko Amomum tsaoko fruit and AEO. However, to the best of our knowledge, little
is known about the possible antibacterial mechanism of Amomum tsaoko essential oil
AEO in the food field. In the present study, in vitro antibacterial activity and basic
mechanism of Amomum tsaoko essential oil AEO towards foodborne bacteria (E.
coli), and the application of Amomum tsaoko essential oil AEO against E. coil in pork
soup model were investigated, which could provide scientific datas for Amomum
tsaoko essential oil AEO to be an alternative natural food preservative and additive.
2. Materials and methods
2.1 Chemicals and Essential oil
Crystal violet, Propidium iodide (PI) and 3, 3’-Diethyloxacarbocyanine iodide
(DiOC2(3)) were purchased from Sigma Chemicals (Shanghai, China). Tween 80 and
glutaraldehyde were purchased from Aladdin Chemicals Co. (Shanghai, China).
Nutrient Broth (NB), Lysogeny Broth (LB) and Plate Count Agar (PLA) were
purchased from Aobox Biotechnology Co. (Beijing, China).The essential oil was
extracted in my group according to the method described by Viuda-Martos et al.
(2011) in my group. The essential oil of all air-dried fruit of Amomum tsaoko samples
was prepared using hydro-distillation for 3 h and a Lab HEAT Clevenger-type
apparatus. The extracted essential oil was dried with anhydrous sodium sulfate and
stored in vials under dark conditions at 4°C prior to use.
2.2. Bacteria cultures
The antibacterial activities of Amomum tsaoko essential oil AEO were tested
against four different bacteria. Two Gram-positive strains were Staphylococcus
aureus ATCC 25923 and Bacillus subtilis ATCC 6051. Two Gram-negative strain
were Escherichia coli ATCC 25922 and Salmonella typhimurium ATCC 14028. The
strains were provided from the Institute of Applied Microbiology, Heilongjiang
Academy of Science (China), which was maintained on an agar plates at 4°C and
subcultured every one month. All bacteria were overnight activated in nutrient broth
(NB) NB medium at 37°C to a mid-log phase. Before each experiment, the turbidity 4
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of the cell suspensions was measured at 600 nm and adjusted to the required
concentration using the McFarland standard (Firuzi et al., 2010).
2.3 Antimicrobial activity
2.3.1 Agar disk diffusion assay
The ntibacterial antibacterial activity of Amomum tsaoko essential oil AEO
against four bacteria was tested by the agar disk diffusion method (Lv et al., 2011).
Briefly, overnight cultures of bacteria were adjusted in NB medium to contain 107
cells/mL. Sterile discs (6 mm diameter) impregnated with 10 μL uL of the diluted oil
aliquots (300 mg/mL) were air dried at room temperature for 10 min to allow the
diffusion of the oil, and placed on the seeded agar plates. The plates were incubated at
37°C for 24 h. After incubation, bacteria inhibitions were visually evaluated as the
diameter of the zones of inhibition (ZOI) surrounding the disks (disk diameter
included) and recorded in millimeter. Tetracycline served as a positive control and
Tween 80 at a final concentration of 0.5% (v/v) was used as a negative control.
2.3.2 Measurement of MIC and MBC
The minimum inhibitory concentration (MIC) and minimum bacterial
concentration (MBC) values were determined using the serial two fold dilutions
method with minor modifications (Ogata et al., 2000) (CLSI, Clinical and Laboratory
Standards Institute, 2006, M7–A7). Amomum tsaoko essential oil AEO were dissolved
in the sterile Tween 80 solution (5%, w/v) which has be mentioned that the
concentration of Tween 80 solution did not have any antibacterial activity (Delamare
et al., 2007). 2-Fold serial dilutions of essential oil were prepared in sterile NB
medium ranging from 0.78 to 50 mg/mL. The same volumes of exponentially growing
strains suspension were co-cultured with the essential oils in 96-well microplate. The
plates were then incubated at 37°C for 24 h and were visually read for the absence or
presence of turbidity. The MIC was defined as the lowest concentration which
showed no visible growth or turbidity (Mushtaq et al., 2016). The MBC was defined
as the lowest concentration which no growth was observed after sub-culturing 10 μL
of the MIC test solutions on PCA at 37 °C for 24 h (Pavithra et al., 2009).at 37 °C for
24 h. The MIC was defined as the lowest concentration which inhibits bacteria 5
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growth. The MBC was defined as the lowest concentration that could kill 99.9% of
treated cells on the agar plate after incubation
2.3.3 Time-kill curves
Time-kill assays of E. coli strains were investigated by measuring the reduction
in the numbers of CFU per milliliter over 3 h (Zu et al., 2010). Amomum tsaoko
essential oil AEO (corresponding to control, MIC and MBC) was incubated with
equivalent amounts of the exponential phase of E. coli strains. All samples were
maintained at 37°C under agitation condition. After 0, 20, 40, 60, 80, 100, 120, 140,
160, 180 min from the time of incubation, 100 μL samples were removed for colony
counting by decimal dilution in NB medium and plating out on PCA. The experiment
was carried out three times.100 μL samples were removed for analysis and diluted
several times for colony counting.
2.4 Antibacterial mechanism
2.4.1 Scanning electron microscope assay
Scanning electron microscopy (SEM) was used to observe the morphological
changes of the E. coli strains (Yong et al., 2015). After incubation in lysogeny broth
McFarland standard and treated with Amomum tsaoko essential oil AEO at different
concentrations (corresponding to control, MIC and MBC) for 2 h. After incubation,
the suspensions were centrifuged at 1500 g for 10 min and washed twice with 0.1 M
phosphate buffer solution (PBS, pH 7.4). Then E. coli was fixed in 5% glutaraldehyde
for 4 h in dark place. Following three washes with PBS, all samples were dehydrated
in a series of a sequential graded ethanol (30%, 50%, 70%, 90%, and 100%). Finally,
all samples were sputter-coated with platinum before viewing by SEM (Quanta–200
environmental scanning electron microscope system (FEI Company, Hillsboro, USA))
2.4.2 Atomic force microscope assay
LB medium at 37°C for overnight, E. coli strains were adjusted to 0.5–1
The changes in bacterial morphology induced by Amomum tsaoko essential oil
AEO were further observed by atomic force microscopy (Braga and Ricci, 1998).
After incubation in LB medium at 37°C for overnight, E. coli strains were harvested
and treated with Amomum tsaoko essential oil AEO at different concentrations 6
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(corresponding to control, MIC and MBC) for 2 h. For AFM analysis, 10 μL of the
samples was applied on a freshly cleaved mica surface and dried in vacuum before
AFM study. Then, the mica surfaces were washed softly with deionized water three
times and air-dried for examining.
2.4.3 Crystal violet assay
The change of membrane permeability was determined by crystal violet assay
(Vaara & Vaara, 1981). Overnight cultures of E. coli strains were harvested and
treated with Amomum tsaoko essential oil AEO at different concentrations
(corresponding to control, 1/2MIC, MIC, 2MIC and 4MIC) for 2 h. Then strains were
harvested by centrifugation at 9300 g for 5 min and then incubated with 10 μg/mL
crystal violet in the dark for 15 min. After centrifuging, the absorbance of supernatant
was determined by measuring the OD 590 nm using a UV–VIS spectrophotometer
(UV-5500PC Spectrophotometer (METASH Company, Shanghai, China)). The
crystal violet uptake was calculated using following formula:
% of take up = (OD of the sample) / (OD of crystal violet solution) × 100
2.4.4 Membrane potential disruption assay
3, 3’ - Diethyloxacarbocyanine iodide (DiOC2(3)) was used to determine the
changes of Membrane potential (Novo et al., 2000). Overnight cultures E. coli strains
were mixed with different concentrations of Amomum tsaoko essential oil AEO
(corresponding to control, MIC, and MBC) and incubated for 2 h. Strains were
subsequently collected and washed twice with PBS. Then suspensions were incubated
with 50 μM DiOC2(3), in the dark for 10 min. Flow cytometry was performed on E.
coli strains per run by using the 488 nm beam from an argon ion laser to excite the
DiOC2(3), the green fluorescence was detected through a 530 nm, 20 nm bandwidth
band-pass filter, and the red fluorescence was detected through a 610 nm,19 nm
bandwidth band-pass filter. The depolarizing the bacteria membrane was measured as
the intensity ratio of the red fluorescence to the green fluorescence (Silverman,
Perlmutter, & Shapiro, 2003).
2.4.5 Membrane damages assay
The red fluorescent nucleic acid stain Propidium iodide (PI) was used to detect 7
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the integrity of cell membrane (Liu et al., 2015). Different concentrations of Amomum
tsaoko essential oil AEO (corresponding to control, MIC and MBC) were added to E.
coli strains for 1, 2 and 3 h. Strains were harvested by centrifugation at 8000 g for 10
min, washed twice with PBS. Then, 30 μM PI was added and incubated in the ice bath
under dark place for 15 min. The fluorescence intensity was detected in the excitation
wavelength of 530 nm and emission wavelength between 550 and 700 nm with a 5-
nm slit. All samples were measured using F–4500 fluorescence spectrophotometer
(HITACHI Company, Tokyo, Japan).
2.4.6 Measurement the Leakage of Cell constituents
The release of cell constituents into supernatant was examined according to the
method described by Diao et al., (2014). Bacteria strains were prepared as described
above. E. coli strains were mixed with different concentrations of Amomum tsaoko
essential oil AEO (corresponding to control, MIC and MBC) were incubated at 37°C
in an environmental incubator shaker for 1, 2 and 3 h. The loss of materials absorbing
at 260 nm was determined with a UV–visible spectrophotometer. Correction was
made for the absorption of the suspension with PBS containing the same
concentration of Amomum tsaoko essential oil AEO after 2 min of contact with E. coil
strains. And then the concentrations of proteins in supernatant were determined
according to the method described by Xu et al., (2010).
2.5 In situ antibacterial assay in pork soup
Pork soup used in this study was purchased from the local market. It was prepared
according to the manufacturer instructions in sterile conditions. It contained pork
powder, edible lard, onion, salt, yeast extract, carrot, monosodium glutamate, citric
acid, starch and spices which was prepared according to the manufacturer instructions
(Unilever Co., Beijing, China). Briefly, 6 grams of the soup power was dispensed in
distilled water with portions of 300 mL into 500 mL screw capped flask and then
sterilized by autoclave. After cooling, The the food preserving properties was
evaluated with the method according to Bukvički et al., (2014) with some
modifications. The cooked soup were inoculated with overnight cultures E. coli
strains which were adjusted to approximately 1 × 106 cells/mL with pork soup. Every 8
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sample mixed with different concentrations of Amomum tsaoko essential oil AEO
ranging from 1.56 to 25 mg/mL thoroughly. All the samples were divided in two
groups: one group was kept at 25°C and the other at 4°C. The inhibition percentage of
the samples at different temperatures, 25°C (1) and 4°C (2) were measured with the
reduction of the numbers of bacterial colonies (Colony Forming Unit, CFU) using the
%inhibition=100-(CFUsample/CFUblank) × 100
%inhibition=100-(CFUsample/CFUblank) × 100-Tinhibition
The numbers of bacterial colonies were measured by the spread plate method,
where CFUsample and CFUblank is the CFU of the antibacterial samples and the
blank sample incubation at 25°C, respectively. Inhibition corresponded to temperature
inhibition of cells at 4°C, measured according to the formula (3):
Where CFUTgrowth and CFUT0growth presented the growth of strain at 4°C in
medium, after and before incubation, respectively.
2.6 Sensory evaluation
256 257 258 259 260 261 262 263 264 265 266 267
Sensory properties of adding AEO to pork soup were evaluated by a sensory acceptance test as described previously (Moosavy et al., 2008) The prepared soups were divided to seven equal parts, and the essential oil was added in ranging from 1.56 to 25 mg/mL. The analysis was carried out at each sampling time (0 h, 12 h, 24 h, 36 h, and 48 h) with 25℃ and 4℃. The sensory acceptance test was performed by a panel of seven judges who were experienced in the sensory analysis of food, mainly from staff of Engineering Research Center of Forest Bio-preparation (Northeast Forestry University, China). Each panelist evaluated the samples based on a 9-point hedonic scale where 9 = like extremely, 8 = like very much, 7 = like moderately, 6 = like slightly, 5 = neither like nor dislike, 4 = dislike slightly, 3 = dislike moderately, 2 = dislike very much, and 1 = dislike extremely, for overall evaluations contained various characteristics such as color, odor and taste.
2.6 2.7 Statistical analysis
All values were expressed as means ± standard deviation (SD) of three
experiments. Data were analyzed by using one-way ANOVA test. The photographs of
SEM, AFM and figures were only the representative. In all cases, a value of ρ<0.05
was considered statistically significant.
3. Results and discussion 9
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3.1 ZOI, MIC, and MBC of the Essential Oil
The ZOI, MIC, and MBC values of Amomum tsaoko essential oil AEO against
different bacteria strains are presented in Table 1. Results showed that the Amomum
tsaoko essential oil AEO had antibacterial effects on all tested strains. The ZOI, MIC,
and MBC values for Gram-positive bacterial strains were in the range of 19.7–24.5
mm, 3.13–6.25 mg/mL and 3.13–6.25 mg/mL, respectively. And they were in the
range of 3.13–6.25 mm, 3.13–6.25 mg/mL and 6.25–12.5 mg/mL for Gram-negative
bacterial strains, respectively. Among these bacteria, the sensitivities to the Amomum
tsaoko essential oil AEO were different from different bacteria tested, and the
susceptibility of E. coli was excellent with the lowest MIC and MBC values of 3.13
and 6.25 mg/mL. The MBC values of the essential oil against S. typhimurium strains
were 12.5 mg/mL, which was the highest concentration of essential oil tested in this
To some extent, the Gram-negative bacteria are generally less sensitive than the
Gram-positive ones to the essential oil (Gill & Holley, 2006). It is possible due to the
significant differences in the outer layers of Gram-negative and Gram-positive
bacteria. The resistance of Gram-negative bacteria towards antibacterial substances is
related to the hydrophilic surface of their outer membrane. It is rich in
lipopolysaccharide molecules which can be impermeable to lipophilic compounds as
Randrianarivelo et al. (2009) had reported that some Gram-positive bacteria were less
or equally sensitive to Gram-negative bacteria. The antibacterial activity of essential
oil probably depended on the type of essential oil more than the structure of the
bacteria (Dorman et al., 2000). In this study, when compared to Gram-positive
bacteria, the MIC of Gram-negative E. coli is the lowest values.
3.2 Effect of Amomum tsaoko essential oil AEO on the rate of kill of E. coil
On the basis of the results of MIC and MBC assay, a A time-kill assays was used
to describe the viability of E. coli strains. As can be seen from Fig. 1, two
concentrations of AEO with MBC and MIC were tested for the ability to kill E. coli
within 3 h. It showed that low concentrations of AEO were not sufficient to kill E. 10
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coil. At MBC, it could Amomum tsaoko essential oil exhibited a strong and rapid
bactericidal effect on E. coli, and it can completely inactivate the bacteria within 1 h
and prolonged antibacterial activity over 3 h. These results suggested that the AEO
exhibited a strong and rapid bactericidal effect on E. coli and antibacterial activity of
AEO antibacterial activity of Amomum tsaoko essential oil was in concentration and
time-dependent manners. The rapid and sustaining antibacterial activity of Amomum
tsaoko essential oil AEO can provided showed a great potential in food applications
as natural preservatives.
3.3 Morphological analysis of E. coli
SEM was used to visually observe the morphology changes of E. coli cells after
Amomum tsaoko essential oil AEO treatment (Fig. 2). The untreated E. coli cells
showed the distinctive striated cell wall (Fig. 2-A). Their connatural morphology (rod
shape) was retained. After 2 h of MIC treatment, the boundary of the cell was
wrinkled and irregular (Fig. 2-B). And at the MBC level, some cells were damaged,
lysing to debris (Fig. 2-C). Similar to the SEM image, AFM image in Fig. 3 showed
that untreated E. coli cells seemed to be intact (Fig. 3-A, a). However, Amomum
tsaoko essential oil AEO caused the irregularity in the surface of E. coli with MIC
treatment (Fig. 3-B, b), and even appeared to lyse with MIC treatment following the
release of their cellular contents (Fig. 3-C, c).
Results of morphological changes showed that Amomum tsaoko essential oil
AEO impaired membrane structure of E. coli with the leakage of cytosolic
components. This is partly consistent with the report of Zeng et al., (2011). They
reported that essential oil could lyse cells, and the cell walls and membranes were
broken with the decreases of heterogeneity in electron density in cytoplasm. The
results of SEM and AFM graphs reflected the morphological alterations of the
bacteria membrane, sufficiently.
3.4 The effects of Amomum tsaoko essential oil AEO on cell membrane
3.4.1 Crystal violet study
Crystal violet which poorly penetrates the membrane, can easily enter the
damaged membrane (Li et al., 2013). Uptake of crystal violet by E. coli was 36.7% in 11
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the absence of Amomum tsaoko essential oil AEO, but increased to 89.9% and 92.7%
after 2MIC and 4MIC treatment, respectively (Fig. 4). The significant enhancement in
the uptake of crystal violet revealed that Compared to control cells, a significant
enhancement in the uptake of crystal violet was observed in E. coli treated with
Amomum tsaoko essential oil EO. This result showed that Amomum tsaoko essential
oil AEO could alter the membrane permeability of E. coli cells and make the cells
hyperpermeable to solutes, which are generally less permeable.
3.4.2 Measurement of membrane potential
Across the membrane, the electrochemical gradient of protons is essential for
bacteria to maintain the synthesis of ATP and the transportation across the membrane
(Xu et al., 2008). The loss of the membrane potential often occurs with an increase of
permeability of the membrane. To confirm if the Amomum tsaoko essential oil AEO
can affect the membrane potential of bacteria, a membrane potential-sensitive dye
DiOC2(3) was monitored by measuring the uptake of the membrane-lipophilic
impermeant fluorescent indicator. Representative flow cytometric results are shown in
Fig. 5. Cells treated with MIC and MBC of Amomum tsaoko essential oil AEO, led to
the depolarization of membrane. The percentage of membrane potential disruption
increased from 7.06 to 78.23%. The depolarization of membrane potential of E. coil
indicated that the reduction of the energy level of bacteria and the depolarization and
rupture of the membrane in concentration-dependent Amomum tsaoko essential oil
AEO (Caron & Badley, 1995). As a result, the bacteria can’t couldn’t maintain the
normal activities, and following followed to swell breaking.
3.4.3 PI-DNA bonding assay
The ability of Amomum tsaoko essential oil AEO to cause membrane
permeabilization was determined by propidium iodide (PI) PI uptake assay. The
increased fluorescence caused by PI–DNA interaction could quantitate the strength of
membrane permeabilizatio permeabilization following the essential oil-induced
membrane leakage. Compared to the control, with the increased of the concentrations,
the fluorescence intensity (A U) was increased from 11.2 to 18.5 at 1 h (Fig. 6-A),
from 12.2 to 21.5 at 2 h (Fig. 6-B) and from 13.0 to 15.4 at 3h (Fig. 6-C) in Fig. 6-D. 12
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The increase of fluorescence intensity further proved the damage of the cell
membrane. The maximum fluorescence was appeared at 2 h with the MBC treatment.
The fluorescence intensity decreased in the next 1 h.
3.5 3.4.4 Leakage of cytoplasmic materials
The leakage of cytoplasmic material was could reflect the severe and irreversible
damage to the cytoplasmic membrane. Measurement of specific cell leakage markers
such as 260 nm absorbing materials and proteins could indicate the changes of
membrane integrity compared to unexposed cells (Bajpai, Sharma, & Baek, 2013). In
Table 2, compared to control, after treatment with Amomum tsaoko essential oil AEO
at the concentration of MIC and MBC, the concentration of cell constituents (OD 260
nm) and proteins in suspensions increased by 6.5, 10.2 times and 8.9, 29.5 times at 1
h, 6.2, 10.8 times and 9.1, 20.5 times at 2 h, and 5.7, 6.7 times and 7.1, 15.6 times at 3
h, respectively. These increase of nucleic acids and proteins concentrations in
suspensions suggested that essential oil damaged cytoplasmic membrane and caused
the leakage of intracellular constituents subsequently. Our findings were in agreement
with Jenie et al., 2008 and Oonmetta et al., 2006. The amount of nucleic acids at 3 h
was lower than 1 and 2 h, showed that the Amomum tsaoko essential oil could damage
nucleic acid materials after passed through the cytoplasmic membrane.
3.6 3.5 In situ antibacterial activity in pork soup
Regarding the receivable antibacterial activity of Amomum tsaoko essential oil
AEO against all the tested bacteria, the antibacterial activity of Amomum tsaoko
essential oil AEO against E. coli was tested under 4°C and 25°C in pork soup. The
antibacterial activity was better under refrigerated conditions, 4°C (Table 3). At
temperature of 4°C, the inhibition percentage was stabilized for all the concentrations
during period of storage and the concentration inhibition was in the range of 91.38–
98.52%.With the highest tested concentrations (25 mg/mL), 100% inhibition was
achieved in 12 h regardless of the incubation temperature. No inhibition was found at
the lowest concentrations (0.78 mg/mL) at room temperature. The effect was also
dose dependent, decreasing at lower doses with period of storage. To avoid
undesirable sensory changes and other harmful characteristics, the application of a 13
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low amount of natural antimicrobial ingredients should be explored to control
spoilage (Stojković et al., 2013). It’s a sustainable way to get over the use of
antibiotics contributes which increased antibiotic resistance and the subsequent
transmission of these resistances to the environment. Overall, the activity of Amomum
tsaoko essential oil AEO provides a strong evidence that it is a green and efficient
way in the control of foodborne bacteria.
3.6 Sensory analysis
Sensory evaluation showed that the treatments containing AEO were pleasant
than the control in sensory attributes during storage period as a whole (Table 3). The
soup containing 3.13 and 6.25 mg/mL of the essential oil had the most favorable
acceptance. Nevertheless, concentrations of 1.56 and 12.5 mg/mL of the essential oil
were also acceptable. But, it is noted that the acceptance was rising from 7.1 to 7.5 at
the concentrations of 25 mg/mL of the essential oil in the 25°C within 24 h. The AEO
was a volatile oily liquid with the antibacterial and antioxidant activity. It could be
taken as a food additive on products. Too much of the essential oil would add less
pleasant odour but more preservative property on food. Food with long time storage
could choose high concentrations of AEO.
Based on the present study, the Amomum tsaoko essential oil AEO exhibited
strong and rapid antibacterial activities against foodborne bacteria (E. coli). It can be
proposed that the mechanism of action for Amomum tsaoko essential oil AEO against
E. coli was the change in permeability and integrity of the disrupted cell membranes
leading to leakage of nucleic acids and proteins. The ability of the Amomum tsaoko
essential oil AEO to disrupt the morphology of the E. coli was clearly shown by
electron microscopy. At the end, in situ control of E. coil was successfully taken as a
model with the application of Amomum tsaoko essential oil AEO (Graphical abstract).
These findings lead to the conclusion that taking good advantage of Amomum tsaoko
essential oil AEO at low different amounts can have economic application in food
preservatives. Amomum tsaoko essential oil AEO possesses a good potential as an
effectively natural antibacterial agent in the food field. 14
ACCEPTED MANUSCRIPT 424
The authors gratefully acknowledge the financial supports by National Key
Research Development Program of China [2016YFD0600805], Application
Technology Research and Development Program of Harbin [2013AA3BS014],
Fundamental Research Funds for the Central Universities [2572015EA04], and
Special Fund of National Natural Science Foundation of China .
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593 594 595 596 597 598 599 600 601 602 603 20
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Fig.1 Time–kill kinetics of Amomum tsaoko essential oil AEO (control, MIC and
MBC) against E. coli.
Fig. 2. Scanning electron microphotographs of E. coli. A, images of untreated E. coli;
B and C, images of E. coli treated with different concentrations of Amomum tsaoko
essential oil AEO, MIC and MBC.
Fig. 3. Topographic (A-C) and phase imaging (a-c) atomic force microscopy images
of E. coli. A (a), images of untreated E. coli; B (b) and C (c), images of E. coli treated
with different concentrations of Amomum tsaoko essential oil AEO, MIC and MBC.
Fig. 4. Effect of Amomum tsaoko essential oil AEO on membrane permeability of E.
coil after treatment. The concentrations were corresponded to 1/2MIC, MIC, 2MIC
and 4MIC, the control did not contain the test sample. Values of each column are
means ± SD (n=3)
Fig. 5. Analysis of the membrane potential of E. coli after treatment with Amomum
tsaoko essential oil AEO. The concentrations were corresponded to MIC (B) and
MBC (C), the control did not contain the test sample (A). (D) Columns show mean
values of three experiments (±S.D.).
Fig. 6. The fluorescence spectra of PI in cells treated with the Amomum tsaoko
essential oil AEO at 1, 2 and 3 h. The concentrations were MIC and MBC, the control
did not contain the test sample. Values of each curve are means ± SD (n=3)
ACCEPTED MANUSCRIPT Table 1. Zone of Inhibition (ZOI), antibacterial (MIC), and bactericidal (MBC) activities of the Amomum tsaoko essential oil AEO against tested bacteria. Bacteria
S. aureus B. subtilis S. typhimurium E. coli
24.5±0.9c 19.7.±1.5 17.5±0.8
Essential oil AEO MIC(mg/mL) MBC(mg/mL) 3.13 ± 0.00 3.13 ± 0.00 6.25 ± 0.00 6.25 ± 0.00 6.25 ± 0.00 12.5 ± 0.00
ZOI(mm)b 21 20 0
22.1±0.7 3.13 ± 0.00 6.25 ± 0.00 0 a Tested at a concentration of 3 mg/disc. b Tested at a concentration of 30 µg/disc. c Values represent means of three independent replicates ± SD. d NA, not active.
Tetracycline MIC(μg/mL) MBC(μg/mL) 6.25 6.25 6.25 12.5 d NA NA NA
ACCEPTED MANUSCRIPT Table 2. The release of cell constituents of E. coil after treatment with Amomum tsaoko essential oil AEO. Cell constituents' release 3h 1h
2h OD value
2h Protein (μg/mL)
Values represent means of three independent replicates ± SD.
ACCEPTED MANUSCRIPT Table 3. The antibacterial activity of Amomum tsaoko essential oil AEO in pork soup against foodborne E. coli (mean ± SD). Concentration (mg/mL)
Percentage of inhibition of E. coli 0h
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00
Values represent means of three independent replicates ± SD.
ACCEPTED MANUSCRIPT Table 4. The average of sensory acceptability of pork soup in different concentrations of AEO during 25°C and 4°C. Concentration (mg/mL) 25
Mean rating ± SD 0h
Values represent means of three independent replicates ± SD.