The preservative potential of Amomum tsaoko essential oil against E. coil, its antibacterial property and mode of action

The preservative potential of Amomum tsaoko essential oil against E. coil, its antibacterial property and mode of action

Accepted Manuscript The preservative potential of Amomum tsaoko essential oil against E. coil, its antibaterial property and mode of action Na Guo, Y...

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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:

S0956-7135(16)30686-7

DOI:

10.1016/j.foodcont.2016.12.013

Reference:

JFCO 5382

To appear in:

Food Control

Received Date:

05 October 2016

Revised Date:

05 December 2016

Accepted Date:

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

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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.

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The preservative potential of Amomum tsaoko essential oil against E. coil, its

2

antibaterial property and mode of action 1,2,3,

Yu-Ping Zang

1,2,3,

Qi Cui

1,2,3,

3

Na Guo

4

Wang 1,2,3, Yuan-Gang Zu 1,2,3, Yu-Jie Fu 1,2,3*

Qing-Yan Gai

1,2,3,

Jiao Jiao

1,2,3,

Wei

5 6 7

1.

Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast

8

Forestry University, 150040 Harbin, PR China

9

2.

Engineering Research Center of Forest Bio-preparation, Ministry of Education,

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Northeast Forestry University, 150040 Harbin, PR China

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3.

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Resources, Northeast Forestry University, 150040 Harbin, PR China

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*Corresponding author: Yu-jie Fu, Ph. D, Professor, Vice Director. Key Laboratory of

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Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Box 332,

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Hexing Road 26, Harbin 150040, P. R. China

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Tel: +86-451-82190535.

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Fax: +86-451-82190535.

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E-mail: [email protected]

Collaborative Innovation Center for Development and Utilization of Forest

19 20 21

Abstract

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Amomum tsaoko is widely distributed in south-west China as a spice. In present study,

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the antibacterial activity of Amomum tsaoko essential oil (AEO) against foodborne

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pathogens was evaluated. The antibacterial activity was determined by measuring the

25

zones of inhibition (ZOI), minimum inhibitory concentration (MIC), minimum

26

bactericidal concentration (MBC), and the time-kill assay. Results showed the

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susceptibility of foodborne bacteria Escherichia coli (E. coli) was excellent with the

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lowest MIC and MBC values of 3.13 and 6.25 mg/mL, respectively. The probable

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antibacterial mechanism of Amomum tsaoko essential oil AEO was the change in

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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

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showed that Amomum tsaoko essential oil AEO has good preservative activities in

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foods. It is necessary to consider that Amomum tsaoko essential oil AEO will become

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a promising antibacterial additive for food preservative.

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Chemical compounds studied in this article

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Crystal violet (PubChem CID: 11057); Tetracycline (PubChem CID: 54675776);

39

Tween 80 (PubChem CID: 5281955); Glutaraldehyde (PubChem CID: 3485); Ethanol

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(PubChem CID: 702); 3, 3’-Diethyloxacarbocyanine iodide (PubChem CID:

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6432767); Propidium iodide (PubChem CID: 104981)

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Keywords

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Amomum tsaoko essential oil; Foodborne bacteria; Antibacterial mechanism;

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Membrane integrity; Food preservative

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1. Introduction

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In recent years there has been a rising concern about food safety for consumers

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and food industry. Food products contaminated with pathogens can not only lead to

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reduce the quality and quantity of food products (Soliman & Badeaa, 2002), but also

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generate illness and disease (Jacob, Mathiasen & Powell, 2010). Meat is a rich source

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of nutrients including animal proteins, essential amino acids, fatty acids, minerals,

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trace elements and vitamins in daily life (Singh et al., 2014). The abundant nutrient

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compositions of meat provide it an ideal environment for the multiplication of meat

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spoilage microorganisms and foodborne pathogens (Babuskin et al., 2015). E. coli

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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

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milk, yogurt, water, salad vegetables, fruits, fruit juices cider and meat products

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(Kornacki & Marth, 1982). In order to prevent and control pathogenic

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microorganisms in foods, synthetic chemicals have been used to control microbial

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growth and the incidence of food poisoning in the past few years. However, many

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consumers are concerned about the potential adverse side effects of synthetic

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chemicals in foods, it has been proved that some of the synthetic chemicals had

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undesirable biological effects on human health, leading to immeasurable risks (Osman

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& Abdulrahman, 2003). Thus, it is of vital importance to use safe and natural

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antibacterial agents, particularly those from plants and fruits for the food preservation

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(Tiwari et al., 2009).

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Essential oils extracted from the scented plants are classified as the generally

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recognized as safe (GRAS) substances for food preservative (Lv et al., 2011). It is a

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volatile oily liquid generated by the secondary metabolism of plants, and can be used

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as natural antimicrobials (Diao et al., 2013.) There are many reports on the

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antibacterial activities of essential oils, and the use of essential oils as antimicrobial

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agents in food products (Sagdic et al., 2003, & Salgueiro et al., 2010).

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The herbs of Zingiberaceae family are always using for food additives and

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conventional drug treatments (Sabulal et al., 2006). And there are about 85 species of

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genus Zingiber herbs distributed in East Asia and tropical Australia. Amomum tsaoko

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Crevost et Lemarie which was a member of Zingiberaceae family is widely

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distributed in south-west China. The dried fruit of Amomum tsaoko is a well-known

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commercial spice as the a food additives (Zhang, Lu, & Jiang, 2014). The essential oil

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from Amomum tsaoko possesses superior antioxidant, anti-tumour, antibacterial and

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antifungal efferts abilities (Zhang et al, 2015; Li et al, 2011, and Qiu et al, 1999). The

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compositions of Amomum tsaoko essential oil AEO can be divided into five classes,

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including monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene

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hydrocarbons, oxygenated sesquiterpenes and others. 1, 8-cineole is the most

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important constituent in Amomum tsaoko essential oil AEO (45.24%) (Yang et al.,

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2010), which can be used as flavoring as a flavoring agent for food products 3

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(Vincenzi, mancini, & Dessi, 1996).

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A series of studies has have demonstrated the antibacterial activity of Amomum

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tsaoko Amomum tsaoko fruit and AEO. However, to the best of our knowledge, little

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is known about the possible antibacterial mechanism of Amomum tsaoko essential oil

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AEO in the food field. In the present study, in vitro antibacterial activity and basic

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mechanism of Amomum tsaoko essential oil AEO towards foodborne bacteria (E.

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coli), and the application of Amomum tsaoko essential oil AEO against E. coil in pork

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soup model were investigated, which could provide scientific datas for Amomum

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tsaoko essential oil AEO to be an alternative natural food preservative and additive.

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2. Materials and methods

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2.1 Chemicals and Essential oil

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Crystal violet, Propidium iodide (PI) and 3, 3’-Diethyloxacarbocyanine iodide

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(DiOC2(3)) were purchased from Sigma Chemicals (Shanghai, China). Tween 80 and

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glutaraldehyde were purchased from Aladdin Chemicals Co. (Shanghai, China).

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Nutrient Broth (NB), Lysogeny Broth (LB) and Plate Count Agar (PLA) were

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purchased from Aobox Biotechnology Co. (Beijing, China).The essential oil was

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extracted in my group according to the method described by Viuda-Martos et al.

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(2011) in my group. The essential oil of all air-dried fruit of Amomum tsaoko samples

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was prepared using hydro-distillation for 3 h and a Lab HEAT Clevenger-type

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apparatus. The extracted essential oil was dried with anhydrous sodium sulfate and

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stored in vials under dark conditions at 4°C prior to use.

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2.2. Bacteria cultures

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The antibacterial activities of Amomum tsaoko essential oil AEO were tested

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against four different bacteria. Two Gram-positive strains were Staphylococcus

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aureus ATCC 25923 and Bacillus subtilis ATCC 6051. Two Gram-negative strain

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were Escherichia coli ATCC 25922 and Salmonella typhimurium ATCC 14028. The

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strains were provided from the Institute of Applied Microbiology, Heilongjiang

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Academy of Science (China), which was maintained on an agar plates at 4°C and

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subcultured every one month. All bacteria were overnight activated in nutrient broth

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(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

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concentration using the McFarland standard (Firuzi et al., 2010).

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2.3 Antimicrobial activity

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2.3.1 Agar disk diffusion assay

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The ntibacterial antibacterial activity of Amomum tsaoko essential oil AEO

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against four bacteria was tested by the agar disk diffusion method (Lv et al., 2011).

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Briefly, overnight cultures of bacteria were adjusted in NB medium to contain 107

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cells/mL. Sterile discs (6 mm diameter) impregnated with 10 μL uL of the diluted oil

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aliquots (300 mg/mL) were air dried at room temperature for 10 min to allow the

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diffusion of the oil, and placed on the seeded agar plates. The plates were incubated at

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37°C for 24 h. After incubation, bacteria inhibitions were visually evaluated as the

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diameter of the zones of inhibition (ZOI) surrounding the disks (disk diameter

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included) and recorded in millimeter. Tetracycline served as a positive control and

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Tween 80 at a final concentration of 0.5% (v/v) was used as a negative control.

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2.3.2 Measurement of MIC and MBC

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The minimum inhibitory concentration (MIC) and minimum bacterial

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concentration (MBC) values were determined using the serial two fold dilutions

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method with minor modifications (Ogata et al., 2000) (CLSI, Clinical and Laboratory

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Standards Institute, 2006, M7–A7). Amomum tsaoko essential oil AEO were dissolved

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in the sterile Tween 80 solution (5%, w/v) which has be mentioned that the

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concentration of Tween 80 solution did not have any antibacterial activity (Delamare

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et al., 2007). 2-Fold serial dilutions of essential oil were prepared in sterile NB

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medium ranging from 0.78 to 50 mg/mL. The same volumes of exponentially growing

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strains suspension were co-cultured with the essential oils in 96-well microplate. The

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plates were then incubated at 37°C for 24 h and were visually read for the absence or

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presence of turbidity. The MIC was defined as the lowest concentration which

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showed no visible growth or turbidity (Mushtaq et al., 2016). The MBC was defined

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as the lowest concentration which no growth was observed after sub-culturing 10 μL

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of the MIC test solutions on PCA at 37 °C for 24 h (Pavithra et al., 2009).at 37 °C for

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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

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treated cells on the agar plate after incubation

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2.3.3 Time-kill curves

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Time-kill assays of E. coli strains were investigated by measuring the reduction

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in the numbers of CFU per milliliter over 3 h (Zu et al., 2010). Amomum tsaoko

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essential oil AEO (corresponding to control, MIC and MBC) was incubated with

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equivalent amounts of the exponential phase of E. coli strains. All samples were

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maintained at 37°C under agitation condition. After 0, 20, 40, 60, 80, 100, 120, 140,

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160, 180 min from the time of incubation, 100 μL samples were removed for colony

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counting by decimal dilution in NB medium and plating out on PCA. The experiment

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was carried out three times.100 μL samples were removed for analysis and diluted

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several times for colony counting.

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2.4 Antibacterial mechanism

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2.4.1 Scanning electron microscope assay

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Scanning electron microscopy (SEM) was used to observe the morphological

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changes of the E. coli strains (Yong et al., 2015). After incubation in lysogeny broth

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(LB)

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McFarland standard and treated with Amomum tsaoko essential oil AEO at different

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concentrations (corresponding to control, MIC and MBC) for 2 h. After incubation,

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the suspensions were centrifuged at 1500 g for 10 min and washed twice with 0.1 M

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phosphate buffer solution (PBS, pH 7.4). Then E. coli was fixed in 5% glutaraldehyde

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for 4 h in dark place. Following three washes with PBS, all samples were dehydrated

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in a series of a sequential graded ethanol (30%, 50%, 70%, 90%, and 100%). Finally,

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all samples were sputter-coated with platinum before viewing by SEM (Quanta–200

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environmental scanning electron microscope system (FEI Company, Hillsboro, USA))

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2.4.2 Atomic force microscope assay

LB medium at 37°C for overnight, E. coli strains were adjusted to 0.5–1

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The changes in bacterial morphology induced by Amomum tsaoko essential oil

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AEO were further observed by atomic force microscopy (Braga and Ricci, 1998).

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After incubation in LB medium at 37°C for overnight, E. coli strains were harvested

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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

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samples was applied on a freshly cleaved mica surface and dried in vacuum before

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AFM study. Then, the mica surfaces were washed softly with deionized water three

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times and air-dried for examining.

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2.4.3 Crystal violet assay

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The change of membrane permeability was determined by crystal violet assay

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(Vaara & Vaara, 1981). Overnight cultures of E. coli strains were harvested and

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treated with Amomum tsaoko essential oil AEO at different concentrations

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(corresponding to control, 1/2MIC, MIC, 2MIC and 4MIC) for 2 h. Then strains were

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harvested by centrifugation at 9300 g for 5 min and then incubated with 10 μg/mL

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crystal violet in the dark for 15 min. After centrifuging, the absorbance of supernatant

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was determined by measuring the OD 590 nm using a UV–VIS spectrophotometer

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(UV-5500PC Spectrophotometer (METASH Company, Shanghai, China)). The

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crystal violet uptake was calculated using following formula:

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% of take up = (OD of the sample) / (OD of crystal violet solution) × 100

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2.4.4 Membrane potential disruption assay

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3, 3’ - Diethyloxacarbocyanine iodide (DiOC2(3)) was used to determine the

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changes of Membrane potential (Novo et al., 2000). Overnight cultures E. coli strains

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were mixed with different concentrations of Amomum tsaoko essential oil AEO

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(corresponding to control, MIC, and MBC) and incubated for 2 h. Strains were

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subsequently collected and washed twice with PBS. Then suspensions were incubated

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with 50 μM DiOC2(3), in the dark for 10 min. Flow cytometry was performed on E.

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coli strains per run by using the 488 nm beam from an argon ion laser to excite the

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DiOC2(3), the green fluorescence was detected through a 530 nm, 20 nm bandwidth

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band-pass filter, and the red fluorescence was detected through a 610 nm,19 nm

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bandwidth band-pass filter. The depolarizing the bacteria membrane was measured as

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the intensity ratio of the red fluorescence to the green fluorescence (Silverman,

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Perlmutter, & Shapiro, 2003).

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2.4.5 Membrane damages assay

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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

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tsaoko essential oil AEO (corresponding to control, MIC and MBC) were added to E.

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coli strains for 1, 2 and 3 h. Strains were harvested by centrifugation at 8000 g for 10

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min, washed twice with PBS. Then, 30 μM PI was added and incubated in the ice bath

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under dark place for 15 min. The fluorescence intensity was detected in the excitation

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wavelength of 530 nm and emission wavelength between 550 and 700 nm with a 5-

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nm slit. All samples were measured using F–4500 fluorescence spectrophotometer

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(HITACHI Company, Tokyo, Japan).

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2.4.6 Measurement the Leakage of Cell constituents

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The release of cell constituents into supernatant was examined according to the

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method described by Diao et al., (2014). Bacteria strains were prepared as described

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above. E. coli strains were mixed with different concentrations of Amomum tsaoko

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essential oil AEO (corresponding to control, MIC and MBC) were incubated at 37°C

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in an environmental incubator shaker for 1, 2 and 3 h. The loss of materials absorbing

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at 260 nm was determined with a UV–visible spectrophotometer. Correction was

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made for the absorption of the suspension with PBS containing the same

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concentration of Amomum tsaoko essential oil AEO after 2 min of contact with E. coil

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strains. And then the concentrations of proteins in supernatant were determined

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according to the method described by Xu et al., (2010).

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2.5 In situ antibacterial assay in pork soup

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Pork soup used in this study was purchased from the local market. It was prepared

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according to the manufacturer instructions in sterile conditions. It contained pork

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powder, edible lard, onion, salt, yeast extract, carrot, monosodium glutamate, citric

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acid, starch and spices which was prepared according to the manufacturer instructions

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(Unilever Co., Beijing, China). Briefly, 6 grams of the soup power was dispensed in

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distilled water with portions of 300 mL into 500 mL screw capped flask and then

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sterilized by autoclave. After cooling, The the food preserving properties was

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evaluated with the method according to Bukvički et al., (2014) with some

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modifications. The cooked soup were inoculated with overnight cultures E. coli

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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

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ranging from 1.56 to 25 mg/mL thoroughly. All the samples were divided in two

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groups: one group was kept at 25°C and the other at 4°C. The inhibition percentage of

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the samples at different temperatures, 25°C (1) and 4°C (2) were measured with the

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reduction of the numbers of bacterial colonies (Colony Forming Unit, CFU) using the

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following equations:

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%inhibition=100-(CFUsample/CFUblank) × 100

(1)

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%inhibition=100-(CFUsample/CFUblank) × 100-Tinhibition

(2)

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The numbers of bacterial colonies were measured by the spread plate method,

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where CFUsample and CFUblank is the CFU of the antibacterial samples and the

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blank sample incubation at 25°C, respectively. Inhibition corresponded to temperature

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inhibition of cells at 4°C, measured according to the formula (3):

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Tinhibition=100-(CFUTgrowth/CFUT0growth) ×100

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(3)

Where CFUTgrowth and CFUT0growth presented the growth of strain at 4°C in

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medium, after and before incubation, respectively.

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2.6 Sensory evaluation

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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.

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2.6 2.7 Statistical analysis

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All values were expressed as means ± standard deviation (SD) of three

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experiments. Data were analyzed by using one-way ANOVA test. The photographs of

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SEM, AFM and figures were only the representative. In all cases, a value of ρ<0.05

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was considered statistically significant.

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3. Results and discussion 9

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3.1 ZOI, MIC, and MBC of the Essential Oil

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The ZOI, MIC, and MBC values of Amomum tsaoko essential oil AEO against

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different bacteria strains are presented in Table 1. Results showed that the Amomum

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tsaoko essential oil AEO had antibacterial effects on all tested strains. The ZOI, MIC,

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and MBC values for Gram-positive bacterial strains were in the range of 19.7–24.5

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mm, 3.13–6.25 mg/mL and 3.13–6.25 mg/mL, respectively. And they were in the

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range of 3.13–6.25 mm, 3.13–6.25 mg/mL and 6.25–12.5 mg/mL for Gram-negative

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bacterial strains, respectively. Among these bacteria, the sensitivities to the Amomum

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tsaoko essential oil AEO were different from different bacteria tested, and the

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susceptibility of E. coli was excellent with the lowest MIC and MBC values of 3.13

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and 6.25 mg/mL. The MBC values of the essential oil against S. typhimurium strains

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were 12.5 mg/mL, which was the highest concentration of essential oil tested in this

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study.

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To some extent, the Gram-negative bacteria are generally less sensitive than the

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Gram-positive ones to the essential oil (Gill & Holley, 2006). It is possible due to the

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significant differences in the outer layers of Gram-negative and Gram-positive

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bacteria. The resistance of Gram-negative bacteria towards antibacterial substances is

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related to the hydrophilic surface of their outer membrane. It is rich in

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lipopolysaccharide molecules which can be impermeable to lipophilic compounds as

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barriers

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Randrianarivelo et al. (2009) had reported that some Gram-positive bacteria were less

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or equally sensitive to Gram-negative bacteria. The antibacterial activity of essential

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oil probably depended on the type of essential oil more than the structure of the

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bacteria (Dorman et al., 2000). In this study, when compared to Gram-positive

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bacteria, the MIC of Gram-negative E. coli is the lowest values.

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3.2 Effect of Amomum tsaoko essential oil AEO on the rate of kill of E. coil

to

the

penetration

of

numerous

antibiotic

molecules.

However,

300

On the basis of the results of MIC and MBC assay, a A time-kill assays was used

301

to describe the viability of E. coli strains. As can be seen from Fig. 1, two

302

concentrations of AEO with MBC and MIC were tested for the ability to kill E. coli

303

within 3 h. It showed that low concentrations of AEO were not sufficient to kill E. 10

ACCEPTED MANUSCRIPT 304

coil. At MBC, it could Amomum tsaoko essential oil exhibited a strong and rapid

305

bactericidal effect on E. coli, and it can completely inactivate the bacteria within 1 h

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and prolonged antibacterial activity over 3 h. These results suggested that the AEO

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exhibited a strong and rapid bactericidal effect on E. coli and antibacterial activity of

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AEO antibacterial activity of Amomum tsaoko essential oil was in concentration and

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time-dependent manners. The rapid and sustaining antibacterial activity of Amomum

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tsaoko essential oil AEO can provided showed a great potential in food applications

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as natural preservatives.

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3.3 Morphological analysis of E. coli

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SEM was used to visually observe the morphology changes of E. coli cells after

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Amomum tsaoko essential oil AEO treatment (Fig. 2). The untreated E. coli cells

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showed the distinctive striated cell wall (Fig. 2-A). Their connatural morphology (rod

316

shape) was retained. After 2 h of MIC treatment, the boundary of the cell was

317

wrinkled and irregular (Fig. 2-B). And at the MBC level, some cells were damaged,

318

lysing to debris (Fig. 2-C). Similar to the SEM image, AFM image in Fig. 3 showed

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that untreated E. coli cells seemed to be intact (Fig. 3-A, a). However, Amomum

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tsaoko essential oil AEO caused the irregularity in the surface of E. coli with MIC

321

treatment (Fig. 3-B, b), and even appeared to lyse with MIC treatment following the

322

release of their cellular contents (Fig. 3-C, c).

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Results of morphological changes showed that Amomum tsaoko essential oil

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AEO impaired membrane structure of E. coli with the leakage of cytosolic

325

components. This is partly consistent with the report of Zeng et al., (2011). They

326

reported that essential oil could lyse cells, and the cell walls and membranes were

327

broken with the decreases of heterogeneity in electron density in cytoplasm. The

328

results of SEM and AFM graphs reflected the morphological alterations of the

329

bacteria membrane, sufficiently.

330

3.4 The effects of Amomum tsaoko essential oil AEO on cell membrane

331

3.4.1 Crystal violet study

332

Crystal violet which poorly penetrates the membrane, can easily enter the

333

damaged membrane (Li et al., 2013). Uptake of crystal violet by E. coli was 36.7% in 11

ACCEPTED MANUSCRIPT 334

the absence of Amomum tsaoko essential oil AEO, but increased to 89.9% and 92.7%

335

after 2MIC and 4MIC treatment, respectively (Fig. 4). The significant enhancement in

336

the uptake of crystal violet revealed that Compared to control cells, a significant

337

enhancement in the uptake of crystal violet was observed in E. coli treated with

338

Amomum tsaoko essential oil EO. This result showed that Amomum tsaoko essential

339

oil AEO could alter the membrane permeability of E. coli cells and make the cells

340

hyperpermeable to solutes, which are generally less permeable.

341

3.4.2 Measurement of membrane potential

342

Across the membrane, the electrochemical gradient of protons is essential for

343

bacteria to maintain the synthesis of ATP and the transportation across the membrane

344

(Xu et al., 2008). The loss of the membrane potential often occurs with an increase of

345

permeability of the membrane. To confirm if the Amomum tsaoko essential oil AEO

346

can affect the membrane potential of bacteria, a membrane potential-sensitive dye

347

DiOC2(3) was monitored by measuring the uptake of the membrane-lipophilic

348

impermeant fluorescent indicator. Representative flow cytometric results are shown in

349

Fig. 5. Cells treated with MIC and MBC of Amomum tsaoko essential oil AEO, led to

350

the depolarization of membrane. The percentage of membrane potential disruption

351

increased from 7.06 to 78.23%. The depolarization of membrane potential of E. coil

352

indicated that the reduction of the energy level of bacteria and the depolarization and

353

rupture of the membrane in concentration-dependent Amomum tsaoko essential oil

354

AEO (Caron & Badley, 1995). As a result, the bacteria can’t couldn’t maintain the

355

normal activities, and following followed to swell breaking.

356

3.4.3 PI-DNA bonding assay

357

The ability of Amomum tsaoko essential oil AEO to cause membrane

358

permeabilization was determined by propidium iodide (PI) PI uptake assay. The

359

increased fluorescence caused by PI–DNA interaction could quantitate the strength of

360

membrane permeabilizatio permeabilization following the essential oil-induced

361

membrane leakage. Compared to the control, with the increased of the concentrations,

362

the fluorescence intensity (A U) was increased from 11.2 to 18.5 at 1 h (Fig. 6-A),

363

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

ACCEPTED MANUSCRIPT 364

The increase of fluorescence intensity further proved the damage of the cell

365

membrane. The maximum fluorescence was appeared at 2 h with the MBC treatment.

366

The fluorescence intensity decreased in the next 1 h.

367

3.5 3.4.4 Leakage of cytoplasmic materials

368

The leakage of cytoplasmic material was could reflect the severe and irreversible

369

damage to the cytoplasmic membrane. Measurement of specific cell leakage markers

370

such as 260 nm absorbing materials and proteins could indicate the changes of

371

membrane integrity compared to unexposed cells (Bajpai, Sharma, & Baek, 2013). In

372

Table 2, compared to control, after treatment with Amomum tsaoko essential oil AEO

373

at the concentration of MIC and MBC, the concentration of cell constituents (OD 260

374

nm) and proteins in suspensions increased by 6.5, 10.2 times and 8.9, 29.5 times at 1

375

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

376

h, respectively. These increase of nucleic acids and proteins concentrations in

377

suspensions suggested that essential oil damaged cytoplasmic membrane and caused

378

the leakage of intracellular constituents subsequently. Our findings were in agreement

379

with Jenie et al., 2008 and Oonmetta et al., 2006. The amount of nucleic acids at 3 h

380

was lower than 1 and 2 h, showed that the Amomum tsaoko essential oil could damage

381

nucleic acid materials after passed through the cytoplasmic membrane.

382

3.6 3.5 In situ antibacterial activity in pork soup

383

Regarding the receivable antibacterial activity of Amomum tsaoko essential oil

384

AEO against all the tested bacteria, the antibacterial activity of Amomum tsaoko

385

essential oil AEO against E. coli was tested under 4°C and 25°C in pork soup. The

386

antibacterial activity was better under refrigerated conditions, 4°C (Table 3). At

387

temperature of 4°C, the inhibition percentage was stabilized for all the concentrations

388

during period of storage and the concentration inhibition was in the range of 91.38–

389

98.52%.With the highest tested concentrations (25 mg/mL), 100% inhibition was

390

achieved in 12 h regardless of the incubation temperature. No inhibition was found at

391

the lowest concentrations (0.78 mg/mL) at room temperature. The effect was also

392

dose dependent, decreasing at lower doses with period of storage. To avoid

393

undesirable sensory changes and other harmful characteristics, the application of a 13

ACCEPTED MANUSCRIPT 394

low amount of natural antimicrobial ingredients should be explored to control

395

spoilage (Stojković et al., 2013). It’s a sustainable way to get over the use of

396

antibiotics contributes which increased antibiotic resistance and the subsequent

397

transmission of these resistances to the environment. Overall, the activity of Amomum

398

tsaoko essential oil AEO provides a strong evidence that it is a green and efficient

399

way in the control of foodborne bacteria.

400

3.6 Sensory analysis

401

Sensory evaluation showed that the treatments containing AEO were pleasant

402

than the control in sensory attributes during storage period as a whole (Table 3). The

403

soup containing 3.13 and 6.25 mg/mL of the essential oil had the most favorable

404

acceptance. Nevertheless, concentrations of 1.56 and 12.5 mg/mL of the essential oil

405

were also acceptable. But, it is noted that the acceptance was rising from 7.1 to 7.5 at

406

the concentrations of 25 mg/mL of the essential oil in the 25°C within 24 h. The AEO

407

was a volatile oily liquid with the antibacterial and antioxidant activity. It could be

408

taken as a food additive on products. Too much of the essential oil would add less

409

pleasant odour but more preservative property on food. Food with long time storage

410

could choose high concentrations of AEO.

411

4. Conclusion

412

Based on the present study, the Amomum tsaoko essential oil AEO exhibited

413

strong and rapid antibacterial activities against foodborne bacteria (E. coli). It can be

414

proposed that the mechanism of action for Amomum tsaoko essential oil AEO against

415

E. coli was the change in permeability and integrity of the disrupted cell membranes

416

leading to leakage of nucleic acids and proteins. The ability of the Amomum tsaoko

417

essential oil AEO to disrupt the morphology of the E. coli was clearly shown by

418

electron microscopy. At the end, in situ control of E. coil was successfully taken as a

419

model with the application of Amomum tsaoko essential oil AEO (Graphical abstract).

420

These findings lead to the conclusion that taking good advantage of Amomum tsaoko

421

essential oil AEO at low different amounts can have economic application in food

422

preservatives. Amomum tsaoko essential oil AEO possesses a good potential as an

423

effectively natural antibacterial agent in the food field. 14

ACCEPTED MANUSCRIPT 424

Acknowledgements

425

The authors gratefully acknowledge the financial supports by National Key

426

Research Development Program of China [2016YFD0600805], Application

427

Technology Research and Development Program of Harbin [2013AA3BS014],

428

Fundamental Research Funds for the Central Universities [2572015EA04], and

429

Special Fund of National Natural Science Foundation of China [31270618].

430

References

431

Babuskin, S., Babu, P. A. S., Sivarajan, M., & Sukumar, M. (2015). Evaluation and

432

predictive modeling the effects of spice extracts on raw chicken meat stored at

433

different temperatures. Journal of Food Engineering, 166, 29-37.

434

Bajpai, V. K., Sharma, A., & Baek, K. H. (2013). Antibacterial mode of action of

435

Cudrania tricuspidata fruit essential oil, affecting membrane permeability and surface

436

characteristics of food-borne pathogens. Food Control, 32(2), 582-590.

437

Braga, P. C., & Ricci, D. (1998) Atomic force microscopy: application to

438

investigation of Escherichia coli morphology before and after exposure to cefodizime.

439

Antimicrob Agents Chemother, 42(1):18-22.

440

Bukvički, D., Stojković, D., Soković, M., Vannini, L., Montanari, C., Pejin, B., &

441

Marin, P. D. (2014). Satureja horvatii essential oil: In vitro antimicrobial and

442

antiradical properties and in situ control of Listeria monocytogenes in pork meat.

443

Meat science, 96(3), 1355-1360.

444

Caron, G., & Badley, R. A. (1995). Viability assessment of bacteria in mixed

445

populations using flow cytometry. Journal of microscopy, 179(1), 55-66.

446

CLSI, Clinical and Laboratory Standards Institute, 2006. Methods for dilution

447

antimicrobial susceptibility tests for bacteria that grow aerobically. Approved

448

standard. M7-A7. 7th edition Wayne, PA

449

Delamare, A. P. L., Moschen-Pistorello, I. T., Artico, L., Atti-Serafini, L., &

450

Echeverrigaray, S. (2007). Antibacterial activity of the essential oils of Salvia

451

officinalis L. and Salvia triloba L. cultivated in South Brazil. Food chemistry, 100(2),

452

603-608.

453

De Vincenzi, M., Mancini, E., & Dessi, M. R. (1996). Monographs on botanical 15

ACCEPTED MANUSCRIPT 454

flavouring substances used in foods. Part V. Fitoterapia, 67(3), 241-251.

455

Diao, W. R., Hu, Q. P., Zhang, H., & Xu, J. G. (2014). Chemical composition,

456

antibacterial activity and mechanism of action of essential oil from seeds of fennel

457

(Foeniculum vulgare Mill.). Food Control, 35(1), 109-116.

458

Diao, W. R., Hu, Q. P., Feng, S. S., Li, W. Q., & Xu, J. G. (2013). Chemical

459

composition and antibacterial activity of the essential oil from green huajiao

460

(Zanthoxylum schinifolium) against selected foodborne pathogens. Journal of

461

agricultural and food chemistry, 61(25), 6044-6049.

462

Dorman, H. J. D., & Deans, S. G. (2000). Antimicrobial agents from plants:

463

antibacterial activity of plant volatile oils. Journal of applied microbiology, 88(2),

464

308-316.

465

Firuzi, O., Asadollahi, M., Gholami, M., & Javidnia, K. (2010). Composition and

466

biological activities of essential oils from four Heracleum species. Food chemistry,

467

122(1), 117-122.

468

Gill, A. O., & Holley, R. A. (2006). Disruption of Escherichia coli, Listeria

469

monocytogenes and Lactobacillus sakei cellular membranes by plant oil aromatics.

470

International journal of food microbiology, 108(1), 1-9.

471

Goni, P., López, P., Sánchez, C., Gómez-Lus, R., Becerril, R., & Nerín, C. (2009).

472

Antimicrobial activity in the vapour phase of a combination of cinnamon and clove

473

essential oils. Food Chemistry, 116(4), 982-989.

474

Gutierrez, J., Barry-Ryan, C., & Bourke, P. (2009). Antimicrobial activity of plant

475

essential oils using food model media: efficacy, synergistic potential and interactions

476

with food components. Food microbiology, 26(2), 142-150.

477

Jacob, C., Mathiasen, L., & Powell, D. (2010). Designing effective messages for

478

microbial food safety hazards. Food Control, 21(1), 1-6.

479

Jenie, B. S. L., Priosoeryanto, B. P., Syarief, R., & Rekso, G. T. (2008). Mode of

480

action Temu kunci (Kaempferia pandurata) essential oil on E. coli K1. 1 cell

481

determined by leakage of material cell and salt tolerance assays. HAYATI Journal of

482

Biosciences, 15(2), 56-60.

483

Kabara, J. J. (1991). Phenols and chelators. Food preservatives, Blackie, Glasgow, 16

ACCEPTED MANUSCRIPT 484

200-214.

485

Kordali, S. Kordali, R. Kotan, A. Mavi, A. Cakir, A. Ala, & A. Yildirim. (2005).

486

Determination of the chemical composition and antioxidant activity of the essential

487

oil of Artemisia dracunculus and of the antifungal and antibacterial activities of

488

Turkish Artemisia absinthium, A. drancunculus, Artemisia santonicum, and Artemisia

489

spicigera essential oils. Journal of Agricultural and Food Chemistry, 53, 9452–9458

490

Kornacki, J. L., & Marth, E. H. (1982). Foodborne illness caused by Escherichia coli:

491

a review. Journal of Food Protection, 45(11), 1051–1067.

492

Li, N., Luo, M., Fu, Y. J., Zu, Y. G., Wang, W., Zhang, L. & Sun, Y. (2013). Effect of

493

Corilagin on Membrane Permeability of Escherichia coli, Staphylococcus aureus and

494

Candida albicans. Phytotherapy Research, 27(10), 1517-1523.

495

Li, W., Wang, P. J., Shigematsu, M., & Lu, Z. G. (2011, March). Chemical

496

composition and antimicrobial activity of essential oil from Amomum tsao-ko

497

cultivated in Yunnan area. In Advanced Materials Research, 183, 910-914.

498

Lv, F., Liang, H., Yuan, Q., & Li, C. (2011). In vitro antimicrobial effects and

499

mechanism of action of selected plant essential oil combinations against four food-

500

related microorganisms. Food Research International, 44(9), 3057-3064.

501

Moosavy, M. H., Basti, A. A., Misaghi, A., Salehi, T. Z., Abbasifar, R., Mousavi, H.

502

A. E., Alipour, M., Razavi, N. E., Gandomi, H., & Noori, N. (2008). Effect of Zataria

503

multiflora Boiss. essential oil and nisin on Salmonella typhimurium and

504

Staphylococcus aureus in a food model system and on the bacterial cell membranes.

505

Food Research International, 41(10), 1050-1057.

506

Mushtaq, S., Aga, M. A., Qazi, P. H., Ali, M. N., Shah, A. M., Lone, S. A., Shah, A.,

507

Hussain, A., Rasool, F., Dar, H., Shah, Z. H., & Shah, Z. H. (2016). Isolation,

508

characterization and HPLC quantification of compounds from Aquilegia fragrans

509

Benth: Their in vitro antibacterial activities against bovine mastitis pathogens.

510

Journal of ethnopharmacology, 178, 9-12.

511

Novo, D. J., Perlmutter, N. G., Hunt, R. H., & Shapiro, H. M. (2000). Multiparameter

512

Flow cytometric analysis of antibiotic effects on membrane potential, membrane

513

permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus. 17

ACCEPTED MANUSCRIPT 514

Antimicrobial agents and chemotherapy, 44(4), 827-834.

515

Ogata, M., Hoshi, M., Urano, S., & Endo, T. (2000). Antioxidant activity of eugenol

516

and related monomeric and dimeric compounds. Chemical and pharmaceutical

517

bulletin, 48(10), 1467-1469.

518

Oonmetta-Aree, J., Suzuki, T., Gasaluck, P., & Eumkeb, G. (2006). Antimicrobial

519

properties and action of galangal (Alpinia galanga Linn.) on Staphylococcus aureus.

520

LWT-Food Science and Technology, 39(10), 1214-1220.

521

Osman, K. A., & Abdulrahman, H. T. (2003). Risk assessment of pesticide to human

522

and the environment. Saudi journal of biological sciences, 10, 81-106.

523

Qiu, S., Shou, D., Chen, L., Dai, H., & Liu, K. (1999). Pharmacological comparison

524

between volatile oil and water extract. China journal of Chinese materia medica,

525

24(5), 297-9.

526

Pavithra, P. S., Sreevidya, N., & Verma, R. S. (2009). Antibacterial activity and

527

chemical composition of essential oil of Pamburus missionis. Journal of

528

ethnopharmacology, 124(1), 151-153.

529

Randrianarivelo, R., Sarter, S., Odoux, E., Brat, P., Lebrun, M., Romestand, B. &

530

Danthu, P. (2009). Composition and antimicrobial activity of essential oils of

531

Cinnamosma fragrans. Food Chemistry, 114(2), 680-684.

532

Sabulal, B., Dan, M., Kurup, R., Pradeep, N. S., Valsamma, R. K., & George, V.

533

(2006). Caryophyllene-rich rhizome oil of Zingiber nimmonii from South India:

534

Chemical characterization and antimicrobial activity. Phytochemistry, 67(22), 2469-

535

2473.

536

Sagdic, O., Karahan, A. G., Ozcan, M., & Ozkan, G. (2003). Note: effect of some

537

spice extracts on bacterial inhibition. Food Science and Technology International,

538

9(5), 353-358.

539

Salgueiro, L., Martins, A. P., & Correia, H. (2010). Raw materials: the importance of

540

quality and safety. A review. Flavour and fragrance journal,25(5), 253-271.

541

Silverman, J. A., Perlmutter, N. G., & Shapiro, H. M. (2003). Correlation of

542

daptomycin bactericidal activity and membrane depolarization in Staphylococcus

543

aureus. Antimicrobial agents and chemotherapy, 47(8), 2538-2544. 18

ACCEPTED MANUSCRIPT 544

Singh, P., Sahoo, J., Chatli, M. K., & Biswas, A. K. (2014). Shelf life evaluation of

545

raw chicken meat emulsion incorporated with clove powder, ginger and garlic paste

546

as natural preservatives at refrigerated storage (4±1° C).International Food Research

547

Journal, 21(4), 1363-1373.

548

Soliman, K. M., & Badeaa, R. I. (2002). Effect of oil extracted from some medicinal

549

plants on different mycotoxigenic fungi. Food and chemical toxicology, 40(11), 1669-

550

1675.

551

Stojković, D., Reis, F. S., Ferreira, I. C., Barros, L., Glamočlija, J., Ćirić, A., &

552

Soković, M. (2013). Tirmania pinoyi: chemical composition, in vitro antioxidant and

553

antibacterial activities and in situ control of Staphylococcus aureus in chicken soup.

554

Food Research International, 53(1), 56-62.

555

Tiwari, B. K., Valdramidis, V. P., O’Donnell, C. P., Muthukumarappan, K., Bourke,

556

P., & Cullen, P. J. (2009). Application of natural antimicrobials for food preservation.

557

Journal of agricultural and food chemistry, 57(14), 5987–6000.

558

Vaara, M., & Vaara, T. (1981). Outer membrane permeability barrier disruption by

559

polymyxin

560

Antimicrobial agents and chemotherapy, 19(4), 578-583.

561

Viuda-Martos, M., Mohamady, M. A., Fernández-López, J., ElRazik, K. A., Omer, E.

562

A., Pérez-Alvarez, J. A., & Sendra, E. (2011). In vitro antioxidant and antibacterial

563

activities of essentials oils obtained from Egyptian aromatic plants. Food Control,

564

22(11), 1715-1722.

565

Wang, S. H., Chang, M. H., & Chen, T. C. (2004). Shelf-life and microbiological

566

profiler of chicken wing products following sous vide treatment. International

567

Journal of Poultry Science, 3(5), 326-332.

568

Jiangsu New Medical College. (1977). Dictionary of Chinese Materia Medica.

569

Xu, J. G., Hu, Q. P., Wang, X. D., Luo, J. Y., Liu, Y., & Tian, C. R. (2010). Changes

570

in the main nutrients, phytochemicals, and antioxidant activity in yellow corn grain

571

during maturation. Journal of agricultural and food chemistry, 58(9), 5751-5756.

572

Xu, J., Zhou, F., Ji, B. P., Pei, R. S., & Xu, N. (2008). The antibacterial mechanism of

573

carvacrol and thymol against Escherichia coli. Letters in Applied Microbiology,

in

polymyxin-susceptible

and-resistant

19

Salmonella

typhimurium.

ACCEPTED MANUSCRIPT 574

47(3), 174-179.

575

Yang, Y., Yue, Y., Runwei, Y., & Guolin, Z. (2010). Cytotoxic, apoptotic and

576

antioxidant activity of the essential oil of Amomum tsao-ko. Bioresource technology,

577

101(11), 4205-4211.

578

Yong, A. L., Ooh, K. F., Ong, H. C., Chai, T. T., & Wong, F. C. (2015). Investigation

579

of antibacterial mechanism and identification of bacterial protein targets mediated by

580

antibacterial medicinal plant extracts. Food chemistry, 186, 32–36.

581

Zeng, W. C., Zhu, R. X., Jia, L. R., Gao, H., Zheng, Y., & Sun, Q. (2011). Chemical

582

composition, antimicrobial and antioxidant activities of essential oil from Gnaphlium

583

affine. Food and chemical Toxicology, 49(6), 1322-1328.

584

Zhang, T. T., Lu, C. L., & Jiang, J. G. (2014). Bioactivity evaluation of ingredients

585

identified from the fruits of Amomum tsaoko Crevost et Lemaire, a Chinese spice.

586

Food & function, 5(8), 1747-1754.

587

Zhang, T. T., Lu, C. L., & Jiang, J. G. (2015). Antioxidant and anti-tumour evaluation

588

of compounds identified from fruit of Amomum tsaoko Crevost et Lemaire. Journal of

589

Functional Foods, 18, 423-431.

590

Zu, Y. G., Liu, X. L., Fu, Y. J., Wu, N., Kong, Y., & Wink, M. (2010). Chemical

591

composition of the SFE-CO2 extracts from Cajanus cajan (L.) Huth and their

592

antimicrobial activity in vitro and in vivo. Phytomedicine, 17(14), 1095-1101.

593 594 595 596 597 598 599 600 601 602 603 20

ACCEPTED MANUSCRIPT 604

Figure legends:

605

Fig.1 Time–kill kinetics of Amomum tsaoko essential oil AEO (control, MIC and

606

MBC) against E. coli.

607 608

Fig. 2. Scanning electron microphotographs of E. coli. A, images of untreated E. coli;

609

B and C, images of E. coli treated with different concentrations of Amomum tsaoko

610

essential oil AEO, MIC and MBC.

611 612

Fig. 3. Topographic (A-C) and phase imaging (a-c) atomic force microscopy images

613

of E. coli. A (a), images of untreated E. coli; B (b) and C (c), images of E. coli treated

614

with different concentrations of Amomum tsaoko essential oil AEO, MIC and MBC.

615 616

Fig. 4. Effect of Amomum tsaoko essential oil AEO on membrane permeability of E.

617

coil after treatment. The concentrations were corresponded to 1/2MIC, MIC, 2MIC

618

and 4MIC, the control did not contain the test sample. Values of each column are

619

means ± SD (n=3)

620 621

Fig. 5. Analysis of the membrane potential of E. coli after treatment with Amomum

622

tsaoko essential oil AEO. The concentrations were corresponded to MIC (B) and

623

MBC (C), the control did not contain the test sample (A). (D) Columns show mean

624

values of three experiments (±S.D.).

625 626

Fig. 6. The fluorescence spectra of PI in cells treated with the Amomum tsaoko

627

essential oil AEO at 1, 2 and 3 h. The concentrations were MIC and MBC, the control

628

did not contain the test sample. Values of each curve are means ± SD (n=3)

629 630

21

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

ZOI(mm)a

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

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

1h

2h OD value

2h Protein (μg/mL)

3h

Control

0.02±0.01

0.02±0.06

0.03±0.03

0.75±0.11

0.79±0.13

0.95±0.20

MIC

0.12±0.01

0.14±0.02

0.17±0.03

6.70±0.81

7.16±0.45

6.70±0.77

MBC

0.20±0.02

0.25±0.03

0.21±0.03

22.14±2.67

16.21±1.91

14.81±2.88

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)

25.0

12.5 AEO

6.25

3.13

1.56

Temp. (°C)

Percentage of inhibition of E. coli 0h

12 h

24 h

36 h

48 h

+25

0.00±0.00

100.00 ± 0.00

100.00 ± 0.00

100.00 ± 0.00

100.00 ± 0.00

+4

0.00±0.00

100.00 ± 0.00

100.00 ± 0.00

100.00 ± 0.00

100.00 ± 0.00

+25

0.00±0.00

98.52±0.76

97.21±1.21

97.31±0.77

96.03±1.02

+4

0.00±0.00

100.00 ± 0.00

100.00 ± 0.00

100.00 ± 0.00

100.00 ± 0.00

+25

0.00±0.00

76.38±1.62

63.09±0.83

50.48±2.23

31.82±3.39

+4

0.00±0.00

96.27±0.33

95.74±1.25

95.88±0.98

96.41±1.01

+25

0.00±0.00

33.41±2.04

28.24±0.97

20.33±2.31

15.60±2.29

+4

0.00±0.00

91.38±1.87

93.02±0.78

93.11±1.32

94.07±1.37

+25

0.00±0.00

0.00±0.00

0.00±0.00

0.00±0.00

0.00±0.00

+4

0.00±0.00

93.33±2.12

94.21±1.74

93.33±0.76

93.01±2.01

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

12.5

6.25

3.13

1.56

0

Temp. (°C)

Mean rating ± SD 0h

12 h

24 h

36 h

48 h

+25

7.1±0.4

7.8±0.4

7.5±0.2

6.7±0.0

6.3±0.5

+4

7.1±0.3

7.4±0.3

7.6±0.3

7.3±0.2

7.1±0.1

+25

8.1±0.3

7.9±0.3

7.7±0.4

7.1±0.2

6.3±0.2

+4

8.1±0.2

8.0±0.1

7.9±0.3

7.6±0.3

7.3±0.2

+25

8.6±0.1

8.1±0.3

7.7±0.3

7.0±0.5

6.1±0.3

+4

8.6±0.3

8.5±0.2

8.3±0.1

7.9±0.4

7.3±0.4

+25

8.6±0.2

8.0±0.4

7.5±0.5

6.7±0.3

5.9±0.3

+4

8.6±0.2

8.5±0.3

8.2±0.3

7.9±0.3

7.2±0.2

+25

8.5±0.1

7.8±0.2

7.1±0.4

6.3±0.3

5.0±0.2

+4

8.5±0.2

8.3±0.2

8.1±0.1

7.7±0.2

7.2±0.1

+25

8.3±0.2

7.3±0.4

6.3±0.3

5.2±0.4

3.8±0.3

+4

8.3±0.2

7.8±0.3

7.1±0.2

6.3±0.3

5.2±0.5

Values represent means of three independent replicates ± SD.