cinnamon oil packaging on chicken sample for inactivation and inhibition of Listeria monocytogenes and Salmonella Typhimurium, and post-processing film properties

Accepted Manuscript Application of high-pressure processing and polylactide/cinnamon oil packaging on chicken sample for inactivation and inhibition of Listeria monocytogenes and Salmonella Typhimurium, and post-processing film properties Jasim Ahmed, Mehrajfatema Mulla, Yasir Ali Arfat PII:

S0956-7135(17)30069-5

DOI:

10.1016/j.foodcont.2017.02.023

Reference:

JFCO 5458

To appear in:

Food Control

Received Date: 3 October 2016 Revised Date:

5 January 2017

Accepted Date: 12 February 2017

Please cite this article as: Ahmed J., Mulla M. & Arfat Y.A., Application of high-pressure processing and polylactide/cinnamon oil packaging on chicken sample for inactivation and inhibition of Listeria monocytogenes and Salmonella Typhimurium, and post-processing film properties, Food Control (2017), doi: 10.1016/j.foodcont.2017.02.023. 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.

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Application of high-pressure processing and polylactide/cinnamon oil packaging on chicken sample for inactivation and inhibition of Listeria monocytogenes and Salmonella

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Typhimurium, and post-processing film properties

Jasim Ahmed*, Mehrajfatema Mulla, and Yasir Ali Arfat

Food and Nutrition Program, Environment & Life Sciences Research Center,

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Kuwait Institute for Scientific Research

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P.O. Box 24885, Safat, 13109, Kuwait

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* Corresponding author.

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E-mail addresses: [email protected], [email protected] (J. Ahmed).

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Abstract:

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The synergistic effects of combined high-pressure (HP) treatment and active packaging

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consisting of polylactide (PLA), polyethylene glycol (PEG) and cinnamon oil (CIN) on the

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inactivation and inhibition of Listeria monocytogenes and Salmonella Typhimurium in chicken

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samples during the refrigerated storage were assessed. The target was to decrease the treatment

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pressure intensity of the traditional HP technology and cinnamon oil concentration so that the

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process becomes more economically viable. The CIN concentration was varied from 7 to 17% in

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the plasticized film (PLA/PEG 80/20). It was observed that the most effective treatment was the

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combination of a pressure treatment at a level of 300 MPa, and packaging the chicken sample in

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an active packaging containing 17% CIN, which reduced the population of the pathogens to a

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safe level during 21 days of refrigerated storage. Furthermore, the impact of HP and CIN on the

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thermal, rheological and mechanical properties of those films was evaluated. Time-temperature

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superposition principle indicated that the mechanical properties of those films remained intact

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after the HP-treatment, and at high temperature. Therefore, the developed films could be used for

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packaging of chicken samples under high-pressure, and high-temperature with maintaining the

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packaging integrity and the food safety at the highest level.

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Keywords: High-pressure processing, Chicken meat, Cinnamon oil, Time-temperature-

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superposition, Listeria monocytogenes, Salmonella Typhimurium.

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1. Introduction

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Poultry meat is an important food commodity, and the production has increased significantly

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from 58.5 million tonnes in 2000 to about 108 million tonnes in 2013 (FAO, 2016). Among

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chicken meat producing countries, the US occupies the top position which accounts for 18.5% of

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the total world production followed by the China (16.8%) and Brazil (11.8%) (FAO, 2012). It

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has been projected that the poultry meat production will attain a target of 134.5 million tons in

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the next 10 years, and secures the top the chart among all types of meat production. The poultry

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meat is highly perishable, and consider is an optimum medium for the growth of

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microorganisms. There is a high chance of contamination with pathogens in the poultry

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processing units which might occur during post-processing manipulation from equipment or food

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handlers. Poultry meat is mostly infected by Salmonella and causes outbreaks of food-borne

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disease (Bryan & Doyle, 1995 and Newell et al., 2010). Incidences of Listeria monocytogenes,

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Campylobacter jejuni, and Escherichia coli contamination in chicken are a public health issue

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(Altekruse, Stern, Fields, & Swerdlow, 1999; Rodrigo, Adesiyun, Asgarali, & Swanston, 2005;

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Anang, Rusul, Bakar, & Ling, 2007). It has been reported that Salmonella causes for 11% of

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illnesses, 35% of total hospitalizations, and 28% of deaths linked with foodborne illnesses each

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year in the US (Scallan et al., 2011).

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Numerous approaches have been tested to control the growth of microorganisms during

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transportation and storage of the fresh poultry meat. Active packaging is one of the emerging

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areas where the antimicrobial agents (e.g. essential oils, nisin, nanoparticles) are embedded on

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the packaging materials so those agents can interact with the packaged food in a desirable way to

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control the growth of microbes. Essential oils (EO) which are rich in the bioactive compounds

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such as phenolics and terpenoids have drawn great attention due to their proven health benefits

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(Burt, 2004). On the other hand, the green packaging based on biodegradable materials have

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lesser impact on the environment. Therefore, the research in the field of developing eco-friendly

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packaging materials from natural polymers is on verge in order to get a partial alternative to the

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plastic packaging. Polylactides (PLA) is a biodegradable polymer obtained from sugar beet or

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corn starch, and has a commercial success in developing biomedical devices, and packaging.

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A series of publication come out from our laboratory on the development of packaging materials

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based on plasticized PLA [using Polyethylene glycol (PEG) as a plasticizer], metallic

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nanoparticles (e.g. ZnO, Ag-Cu and graphene oxide), and essential oils (cinnamon, garlic, clove)

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either individually or in a combination. Additionally, the applicability of those composite

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materials have been examined for a wide range of food packaging. A complete zone of inhibition

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against C. jejuni was exhibited by PLA/cinnamon and PLA/clove films (PLA/PEG/CIN and

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PLA/PEG/CLO) at the highest concentration (1.6 mL per 2 g PLA/PEG blend) (Ahmed,

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Hiremath, & Jacob, 2016a). It was also observed that an incorporation of 50% cinnamon oil

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(CIN) in the PLA-based packaging material completely inhibited the growth of L.

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monocytogenes and S. Typhimurium in chicken samples (Ahmed, Mulla, & Arfat, 2016b). Based

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on our previous works, it is inferred that a complete inactivation of pathogens required a

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significant quantity of EO. However, a higher oil concentration has a detrimental effect on the

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sensory quality of the chicken sample, and it also increased the manufacturing cost substantially.

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To address those challenges, a combination of biodegradable antimicrobial film with a lower

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concentration of EO, and high-pressure processing a novel technology can be explored leading to

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a complete inhibition of pathogens in the chicken samples.

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Inactivation of the vegetative cells including Listeria monocytogenes, Staphylococcus aureus,

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Escherichia coli, and Salmonella Typhimurium have been achieved through the application of

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HP at the pressure level of 500 to 600 MPa (Jofré, Aymerich, Grébol, & Garriga 2009;

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Rendueles et al., 2011). Cheah and Ledward (1995) reported that a pressure level above 300 MPa

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induced the lipid oxidation as well as irreversible changes including the denaturation of

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myoglobin in muscle proteins. Additionally, a larger change in the packaging materials including

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PLA and PET-based films were observed at 500 MPa for 15 min at 50 °C (Galotto, Ulloa,

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Guarda, Gavara, & Miltz, 2009). These studies forecast that a lower pressure level is required

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during the HP treatment so that the product quality as well the mechanical properties of the

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packaging materials can sustain. The synergistic effects of antimicrobial alginate films

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containing enterocins and a pressure level of 400 MPa for 10 min achieved the inactivation of L.

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monocytogenes and Salmonella spp. on cooked ham (Marcos, Aymerich, Monfort, & Garriga,

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2008). Similarly, a combined effect of polyamide/polyethylene/coriander oil film and a pressure

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level of 500 MPa for 1 min caused a complete inhibition of L. monocytogenes for 60 days in

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ready-to-eat chicken breast samples (Stratakos, Delgado-Pando, Linton, Patterson, & Koidis,

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2015). It has been reported that HP-induced sublethally injured pathogens are more susceptible

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to antimicrobial compounds (Kalchayanand, Sikes, Dunne, & Ray, 1994), so the use of HP-

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treatment in combination with active packaging could be an interesting alternative. Therefore, it

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is hypothesized that a combination of lower concentration of EO in PLA-based film in

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conjunction with lower intensity of pressure could achieve the microbial inactivation effectively

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with excellent quality retention of meat, and above all, augment the commercial applicability of

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the developed film and the HP-treatment for the poultry industry.

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To date, to our knowledge the application HP-treatment and EO-based active packaging on

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chicken sample inoculated with both Gram-positive and Gram-negative pathogens has not been

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investigated. Therefore, an attempt was made to examine the potential for inactivation of L.

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monocytogenes and S. Typhimurium in chicken meat by high-pressure treatment combined with

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the PLA/cinnamon oil based active packaging, and their impact on the survival of those

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microorganisms at refrigerated storage for up to 21 days. Additionally, the thermomechanical

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properties of the films were tested before and after the pressure treatment based on the film

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which showed the least survival of pathogens.

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2. Materials and Methods

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2.1. Materials

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Polylactide (PLA 4043D), in pellet form, was purchased from NatureWorks (Minnetonka, USA)

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and dried overnight at 60°C for 24 hours in a vacuum oven before use. Poly ethylene glycol

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(PEG) (Mw = 1,500 g/mol) and Sri Lanka origin cinnamon oil (CIN) with the highest purity

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(density 1.03 g/mL at 25 °C; RI: n20/D 1.533) were obtained from Sigma (St. Louis, USA).

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Dichloromethane (DCM) was procured from Fisher Scientific (Loughborough, UK). Fresh whole

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chicken was purchased from a local poultry processor, transported to the laboratory with 2 hours

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under the refrigeration, and kept refrigerated before the use.

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2.2. Bacterial strains and media

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Listeria monocytogenes (ATCC 19114) strain in the lyophilized pellets form and Salmonella

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enterica sv Typhimurium (ATCC 14028) strains inoculation culture loops were obtained from

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MediMark Europe (Grenoble, France), and Remel Europe (Dartford, UK), respectively. Brain

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heart infusion agar (BHIA) and Tryptic soya broth (TSB) were procured from Conda

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Laboratories (Torrejón de Ardoz, Spain) and TM Media (Bhiwadi, India), respectively.

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Polymyxin-Acriflavin-Lithium chloride-Ceftazidime-Aesculin-Mannitol (PALCAM) agar base,

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PALCAM selective supplement and Xylose lysine deoxycholate agar (XLD) were purchased

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from Oxoid (Basingstoke, UK).

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2.3 Preparation of inoculums

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L. monocytogenes and S. Typhimurium strains were revived initially in TSB at 37°C for 18–24 h

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before subcultures were made by streaking on BHIA plates. A standardized inoculum containing

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108 CFU/mL was made freshly with the help of the 0.5 McFarland turbidity standards by direct

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suspension of a single colony from the respective BHIA plate in buffered peptone water.

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2.4. Preparation of antimicrobial-plasticized PLA films with CIN

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Antimicrobial PLA/PEG/CIN films were prepared following our earlier method (Ahmed et al.

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2016a). Briefly, the antimicrobial films were solvent casted with PLA (1.6g), PEG (0.4g) using

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DCM as solvent, and designated as PLA/PEG (80/20). A range of cinnamon oil 0.16, 0.24, 0.32

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and 0.40 mL were blended into the solvent to obtain the antimicrobial films and the actual

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concentration of the oil were 7, 11, 14 and 17%. Those films were designated as

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PLA/PEG/CIN1, PLA/PEG/CIN2, PLA/PEG/CIN3 and PLA/PEG/CIN4, respectively. The film

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without addition of cinnamon oil is considered as a control, and termed as the PLA/PEG.

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2.5. Chicken sample preparation

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The cut chicken pieces (~2 g cubes) were sterilized by exposing to the UV light for 15 min prior

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to test. The chicken samples were then randomly divided into 2 sets (in two petri dishes) and

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each petri dish was discretely contaminated by transferring 5 ml of bacterial inoculum (L.

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monocytogenes and S. Typhimurium) with concentration of 108 CFU/mL to achieve a final

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counts of about 106 CFU/g. The inoculums were spread over the discs using sterile spreaders and

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the samples were left for 10 min to allow the inoculums to soak and attach onto the chicken

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surfaces. The contaminated chicken samples were wrapped in UV pre-sterilized PLA/PEG

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(control film) and the PLA/PEG/CIN films with various CIN concentrations, and put into Nasco

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Whirl-Pak™ bags (20 wrapped samples per each bag), heat sealed and kept refrigerated before

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the HP-treatment. Samples without the pressure treatment considered as a control against the HP-

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treated each packaging material.

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2.6. High-pressure treatment

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Chicken samples were pressure treated in an AVURE QFP 2L-700 HP unit (Avure

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Technologies®, USA). The internal diameter of pressure vessel was 100 mm with a maximum

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capacity and vessel pressure of 2 L and 690 MPa respectively. Demineralized water was used as

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pressure-transmitting fluid. The temperature of the pressure transmitting fluid, and the

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temperature of the water jacket surrounding the pressure vessel was monitored by two

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thermocouples located at the top and midway down the treatment chamber respectively.

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Pressurization was performed at selected pressure levels (200, 250 and 300 MPa for 10 min) at

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23 °C. The ‘come-up’ and depressurization times were excluded in the treatment time. The

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average pressurization rate was 20–25s per 100 MPa and depressurization time was

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approximately <5 s. The initial temperature of the water was 20 °C, and it reached to 26 °C at the

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highest pressure level of 300 MPa due to adiabatic heating.

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After the HP-treatment, samples were immediately transferred to refrigerated storage at 4±0.5 °C

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for 21 days. Samples without the HP-treatment were also stored at similar condition. Samples

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were taken for performing bacterial enumeration at 0, 7, 14 and 21 days from and examined for

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the survival of L. monocytogenes and S. Typhimurium.

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2.7. Enumeration of L. monocytogenes and S. Typhimurium

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The counts of both bacterial strains were determined by serial dilution method. Samples from

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each packaging materials (control and HP-treated) were opened aseptically, prior to sampling

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and the pathogens were extracted from the chicken samples by adding it to a centrifuge tube

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containing 9 ml buffered peptone water (BPW) followed by homogenization in vortex for 2 min.

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Further 10 fold dilutions were prepared in 9 ml BPW. 0.1 ml of each diluted homogenate was

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then transferred on PALCAM agar for L. monocytogenes and XLD agar for S. Typhimurium,

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respectively. Plates were incubated at 37°C for 48 h (L. monocytogenes) and 24 h (S.

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Typhimurium) in incubator chamber, respectively. Results were expressed as log CFU/g. All

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microbiological experiments were conducted in triplicates.

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2.8. Effect of pressure treatment on film properties

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2.8.1. Color and thickness

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Color of the film surface was measured using a Mini EZ Scan portable colorimeter (model

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4500L HunterLab, Reston, VA, USA) consisting a D65 illuminant and 10° observation angle. A

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CIE Lab scale was used to measure the degree of lightness (L), redness (+a) or greenness (−a),

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and yellowness (+b) or blueness (−b) of the films. The colorimeter was placed on film samples

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positioned on a white standard plate (L*= 93.52, a*= -1.32, b*=-0.14) and L*, a* and b* values

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were recorded. For each film five readings were taken.

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Film thickness was measured using a micrometer (Mitutoyo, Model MCD-1"PXF, Mituyoto

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Corp., Kawasaki-shi, Japan) with an accuracy of 0.001mm at 10 random locations around the

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film, and a mean value was calculated. Precision of thickness measurement was ±5%.

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2.8.2. Mechanical properties

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Tensile strength (TS), tensile modulus (TM) and elongation at break (EAB) of the films were

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measured from stress–strain curve obtained using a Texture Analyser TA.XT2 plus (Stable

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Micro Systems, Haslemere, England) with a 50 N load cell equipped with tensile grips (A/TG

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model) (D882, ASTM, 2001). Grip separation was set at 50 mm and the cross-head speed was 50

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mm/min. TS and EAB were measured from ten samples from each type of film and treatment.

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At least five specimens were tested for each set of samples.

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2.8.3. Oxygen transmission rate (OTR)

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For each film, the oxygen transmission rate (OTR) was measured at 23 °C and 50±1% RH by an

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Extra-Solution PermeO2 instrument (Capannori, LU, Italy) (D 3985, ASTM 1995). Films with an

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open testing area of 50 cm2 were placed into the test cell and exposed to a mixture of gas (99%

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N2 + 1% H2) flow on one side and the pure oxygen on the other side. Experiments were

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conducted in triplicates.

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2.8.4. Attenuated total reflectance-Fourier transformation infrared (ATR- FTIR)

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spectroscopy

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FTIR spectra of the films were obtained using an attenuated total reflectance-Fourier transform

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infrared (ATR-FTIR) spectrophotometer (Nicolet™ iS™5 with OMNIC™ spectra software,

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Thermo Scientific, Waltham, MA, USA). Film was placed onto a diamond crystal and the

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analysis was performed in the wavenumber of 600-4000 cm-1, with 32 scans recorded at 4 cm-1

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resolution.

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2.8.5. Differential scanning calorimetry (DSC) Measurement

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Thermal analysis of films samples was carried out using a TA Q2000 differential scanning

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calorimeter (DSC) (TA Instruments, New Castle, DE) equipped with refrigerated cooling system

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with indium under an ultra-high purity nitrogen atmosphere (flow rate 50 mL/min). About 8 mg

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samples were run at a 10 ℃/min heating/cooling ramp in 2 heating-cooling cycles (-60 ℃ to 180

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℃). The melting temperature (Tm), the crystallization temperature (Tc) and the glass transition

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temperature (Tg) were analyzed from the 2nd heating/cooling cycle. The enthalpy (area under the

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curve) and degree of crystallinity (χc) was determined from the instrument software (Universal

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Analysis version 4.5A, TA Instruments, New Castle, DE, USA). Thermal scans for each sample

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were carried out in triplicates.

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2.8.6. Rheological measurement

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A Discovery Hybrid Rheometer HR-3 (TA Instruments, New Castle, DE, USA) attached with an

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electrically heated plate (EHP) was employed to measure the melt rheology of PLA/PEG and

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PLA/PEG/CIN films before and after HP-treatment at selected temperature (140, 150, 160 and

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170 °C). Film samples were placed between the plates (measuring plate diameter 25 mm) in a

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500-µm gap for 5 min so that the residual stresses would relax before the rheological

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measurement. The EHP system accurately controlled the sample temperature by means of a

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platinum resistance thermometer positioned at the center and in contact with the opposite face of

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the lower plate. The frequency-sweep measurement (0.1–10 Hz) was carried out at a constant

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strain of 0.03% within a linear viscoelastic range (LVR) at selected temperatures. All rheological

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measurements were carried out in duplicates and rheological parameters were obtained directly

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from the manufacturer supplied computer software (TRIOS, TA Instruments, New Castle, DE,

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USA).

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2.9. Statistical analysis

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Data were subjected to analysis of variance (ANOVA) and mean comparisons were carried out

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by Duncan’s multiple range test (Steel & Torrie, 1980). Statistical analysis was performed using

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the Statistical Package for Social Science (SPSS 17.0 for windows, SPSS Inc., Chicago, IL,

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U.S.A.).

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3. Results and Discussion

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3.1. Antimicrobial properties of PLA/PEG/CIN films and high pressure treatment

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From our earlier studies (Ahmed, Hiremath & Jacob, 2017), it was observed that PLA/PEG/CIN

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films showed a strong inhibition against both Gram-positive and Gram-negative bacteria when

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the oil concentration was 45% in the composite, and furthermore, the HP results confirmed that a

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pressure level of 350 MPa was adequate for a complete inhibition of both S. Typhimurium and L.

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monocytogenes. Therefore, a pressure level below 350 MPa and a PLA to CIN ratio of 1: 0.25 or

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lower were tested for both test organisms in chicken samples during refrigerated storage.

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Figures 1 and 2 represent the survival of L. monocytogenes and S. Typhimurium in chicken

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samples packed in selected PLA-based packaging materials in conjunction with and without the

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HP-treatment under refrigeration storage for 21 days. L. monocytogenes and S. Typhimurium

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mean initial counts in inoculated chicken samples were 6.11 and 5.43 log CFU/g, respectively.

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Refrigeration of chicken sample packed in PLA/PEG at the ambient pressure allowed the growth

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of both L. monocytogenes and S. Typhimurium to a value of 6.74 and 5.55 CFU/g at day 21 of

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storage (Fig. 1a and 2a). The growth of those organisms were restricted when cinnamon oil was

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incorporated in the composite films. The initial numbers of L. monocytogenes and S.

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Typhimurium reduced significantly to 4.72 and 4.05 log CFU/g, respectively when packed in the

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PLA/PEG/CIN4 films, and stored for 21 days under the refrigeration. This is consistent with the

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study carried out by Quintavalla & Vicini (2002), they observed that the effectiveness of

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antimicrobial packaging is highly responsible on the gradual release of the antimicrobial

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compounds from the film to the food. Qin et al., (2015) found that mushrooms packed in

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PLA/poly (caprolactone) film containing 9wt% cinnamaldehyde showed pronounced effect in

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reducing the microbial counts.

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After pressurization at 200 MPa, reductions of S. Typhimurium in chicken samples were 0.37

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and 0.62 log CFU/g in PLA/PEG and PLA/PEG/CIN4 films, respectively and the corresponding

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values for L. monocytogenes were 0.11 and 1.35 log CFU/g (Fig. 1b and 2b). Counts of L.

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monocytogenes and S. Typhimurium were significantly lowered during 21 days storage, and

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almost 2 log CFU/g reduction was observed for both test organisms for chicken samples packed

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in PLA/PEG/CIN3 and PLA/PEG/CIN4 after 21 days (Fig. 1b and Fig. 2b). The counts of L.

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monocytogenes decreased drastically, and even a complete inactivation was achieved after 21

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days refrigerated storage when the sample treated at 250 MPa in PLA/PEG/CIN4 film, however,

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only 3 log CFU/g reduction was occurred for S. Typhimurium at the similar condition (Fig. 1c

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and 2c). It indicates Listeria was more sensitive to the combined treatment (HP +

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PLA/PEG/CIN4) against Salmonella.

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treatment at 200 and 250 MPa but the counts remained relatively stable throughout storage, thus

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exerting a bacteriostatic effect. The inactivation of pathogens was significantly influenced by the

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storage time period. It was observed that the inactivation of microbes ranged between 0.36 and

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2.86 log CFU/g for HP-treated samples in active packaging film during 21 days storage. A

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combination of PLA/PEG/CIN films and the pressure treatment at 300 MPa showed a

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pronounced effect on microbial inactivation during refrigerated storage. A complete inhibition of

Although, the reduction was not complete after HP-

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S. Typhimurium was observed in PLA/PEG/CIN4 films after 21 day storage. Remarkably, both

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test organisms inactivated immediately when the chicken samples packed in PLA/PEG/CIN4

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films followed by the HP-treatment at 300 MPa. No further growth of those organisms occurred

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during the entire storage period (Figure 1d and 2d). It was earlier observed that that L.

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monocytogenes has ability to revive/grow in the cooked chicken at refrigeration temperature,

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even when HP-treatment was adopted, and therefore, additional hurdles are required in order to

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inhibit the growth in the chicken breast during storage (Stratakos et al., 2015). In this study, the

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inactivated or even injured cells could not able to revive because of the antimicrobial activity

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exerted by the cinnamon oil evaporation from the composite packaging. It was observed that

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about 5% CIN lost from the film during 21 days refrigerated storage (data not shown). The

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inactivation achieved by the combination of HP-treatment and active packaging was

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considerably higher than the sum of the inactivation obtained with individual treatment (e.g.

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Figures 1a and 1d), so a synergistic effect was obvious. Similar inactivation of L. monocytogens

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in contaminated chicken breast has been reported by Stratakos et al., (2015); they observed that

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the combination of HP-treatment and active packaging resulted in a synergistic effect reducing

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the counts of the pathogen below the detection limit throughout 60 days storage at 4 °C. The

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counts of L. monocytogenes reached to below the detection level in the sliced cooked ham

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packed with alginate film containing enterocins followed by pressurization at 400 MPa for 10

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min and stored for 3 months at 6°C (Marcos et al., 2008). Aymerich, Jofré, Garriga, & Hugas,

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(2005) have also observed that the combination of HPP (400 MPa for 10 min) with interleaves

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containing nisin led to the absence of Listeria monocytogenes and Salmonella in sliced cooked

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ham. The possible mechanism of the inactivation of microorganisms by EO/HP combination

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could be through the disruption of cell membrane being the site of action for both methods

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(Stratakos et al., 2015).

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3.2. Characterization of films

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Based on microbiological data it was inferred that the chicken samples packed in a composite

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film containing 17% CIN and pressure treated at 300 MPa resulted complete inactivation of

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pathogens. Therefore, the properties of packaging materials after the HP-treatment of 300 MPa

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were evaluated for thermomechanical and barrier properties as discussed below.

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3.2.1. Color and thickness of the films

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Incorporation of cinnamon oil enhanced more greenness and yellowness in the composite

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PLA/PEG/CIN film which is mostly attributed by coloring pigment of the oil. These results are

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in close agreement with earlier reports by Ahmed et al., (2016a). HP-treatment did not show any

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significant change in color values except for L* value of 300 MPa PLA/PEG film sample. The

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average thickness value of PLA/PEG and PLA/PEG/CIN4 film was 0.048 and 0.049 mm,

303

respectively, and those values unaffected by HP-treatment.

304

3.2.2. Oxygen transmission rate

305

The oxygen transmission rate (OTR) values doubled (726 ml/m2 day) for PLA/PEG/CIN

306

composite film over PLA/PEG films (328 ml/m2 day) (P < 0.05). Such an enormous increase in

307

OTR value has been attributed by incorporation of the EO that acts as an effective plasticizer and

308

reduces the resistance of the PLA/PEG film for oxygen transmission. This result was in

309

agreement with Rojas-Graü et al. (2006) who reported that plant essential oils incorporated into

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apple puree edible films increase OTR of resulting films as nonpolar substances such as lipids

311

and essential oils act as poor gas barriers. Several researchers reported a similar OTR values for

312

PLA/EO films (Arrieta et al., 2013; Martino et al., 2009). The effect of HP-treatment on OTR

313

values of studied films are reported in Table 1 and an increasing trend in the OTR values was

314

observed (P < 0.05). It is believed that a change in the structure could alter the gas permeability

315

of the films (Galotto et al., 2010).

316

3.2.3. Mechanical properties

317

Mechanical properties provide reliable information on the application of the developed

318

polymeric films. It can be seen from Table 1, the PLA/PEG film had higher TS, TM and a lower

319

EAB than the PLA/PEG/CIN4 film. The addition of cinnamon oil lowers the polymer-polymer

320

chain interaction, and provide a flexible domain in film resulting a decrease in TS, TM and

321

increase in EAB (Ahmed et al., 2016b; Qin, Yang & Xue, 2015). HP-treatment did not shown

322

any significant change in the studied films. A similar observation has been made by Caner

323

(2002)

324

PET/PVDC/nylon/HDPE/PE,

325

PET/EVA/LLDPE/PP/nylon/PP and PET/EVA/PET after HPP at 600 and 800 MPa for 5, 10 and

326

20min at 45°C. Similarly Ochiai and Nakagawa (1992) also measured the tensile strength

327

properties of PP/PVDC/PP after pressurization at 400 MPa for 10min and observed no change in

328

tensile properties due to the HPP.

329

3.2.4. FTIR spectroscopy

330

FTIR spectroscopy of PLA/PEG and PLA/PEG/CIN4 films before and after the pressure

331

treatment is shown in Figure 3 to identify the possible chemical changes occur during the HP-

332

treatment. The bands at 2,885 of the PLA/PEG spectrum were attributed to the stretching

various

flexible

films:

PET/SiOx/LDPE,

PE/nylon/EVOH/PE,

PET/AL2O3/LDPE,

PE/nylon/PE,

metallized

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vibration of functional group of ─CH2 of PEG and bands at 947 and 842 cm−1 were because of

334

the crystalline phase of PEG (Chen, Peng, Chiang, & Huang, 2015). Several peaks were

335

observed for PLA: the peak at 1,750 cm−1 corresponded to the C═O stretching of ester group

336

(Shirai et al., 2013); the peaks at 1180 and 1090 cm-1 attributed to the symmetric C-O-C and

337

−C−O− stretching of the PLA ester groups, respectively (Agarwal, Koelling, & Chalmers, 1998);

338

peaks at 1452 and 1361 cm−1 corresponded to the asymmetric and symmetric ─CH3 deformation

339

vibrations of PLA, respectively (Chieng, Ibrahim, Yunus, & Hussein, 2013). FTIR spectra of

340

PLA/PEG/CIN4 films showed the presence of additional peaks attributed by the cinnamon oil

341

against the PLA/PEG film. Medium weak bands were observed in the wavelengths of 1600 to

342

1658 cm-1 that could be due to the C═C stretching of existing alkenes from oil. Furthermore

343

absorption peak was detected at 1514 cm-1, representing aromatic domain with NH bending

344

(Ahmed et al., 2016b; Jackson, Watson, Halliday, & Mantsch, 1995). FTIR spectra were similar

345

for the HP treated films when compared to their respective controls, suggesting no alteration in

346

the molecular structure of films. No change in the FTIR spectra was reported by (Schauwecker,

347

Balasubramaniam, Sadler, Pascall, & Adhikari, 2002) when working with HP treatment of

348

EVOH based materials at 690 MPa for 10 min at 95 ºC.

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3.2.5. Thermal properties

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Effect of HP-treatment on thermal properties of PLA/PEG and PLA/PEG/CIN4 films are

352

presented in Table 2. The glass transition temperature, Tg and the melting temperature, Tm and

353

the crystallization temperature, Tc of the oil incorporated composite films dropped significantly

354

from the PLA/PEG film due to the enhanced polymer chain mobility caused by plasticizing

355

effect of cinnamon oil. The Tg and Tm values of PLA/PEG dropped drastically from 16.7 to -1.4

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°C, and 148 to 137 °C, respectively. Similar decrease in Tg and Tm with addition of essential oil

357

has been reported earlier by researchers (Armentano et al., 2015; Kulinski & Piorkowska, 2005).

358

A lower Tc value for PLA/PEG/CIN4 was because of the CIN essential oil which increases the

359

ability of PLA for cold crystallization (Armentano et al., 2015). After HP-treatment, thermal

360

parameters of the PLA/PEG and PLA/PEG/CIN4 samples did not change much with the pressure

361

treatment. The melting and crystallization enthalpy values indicated that PLA/PEG/CIN4 was

362

pressure resistant whereas PLA/PEG showed pressure sensitivity, and supported rheometric

363

observation. These results are in good agreement with the observation made by Schauwecker,

364

Balasubramaniam, Sadler, Pascall, & Adhikari, 2002; they observed a similar heat flow curves

365

for control and HP-treated (690 MPa at 95 ºC for 10 min) multilayer nylon/EVOH/polyethylene

366

film.

367

3.2.6. Rheology

368

Mechanical spectra of the untreated and the HP-treated films at 150 °C are illustrated in Figure 4.

369

Both the elastic modulus, G′ and viscous modulus, G″ increased with frequency. It can be seen

370

that the G″ predominates over the G′ indicating a predominating liquid-like property of the

371

studied films. A gradual decrease in phase angle (δ) with frequency indicated a decrease in

372

liquid-like behavior of the melt especially at higher frequency range. An incorporation of CIN

373

into PLA/PEG matrix acts as an additional plasticizer, and reduced both moduli significantly

374

(Figure 4a and 4b). Essential oil induced plasticization of PLA/PEG matrix has been reported

375

earlier (Lee, Lee, & Song, 2015, Ahmed et al. 2016a, b). After HP-treatment at 300 MPa, both

376

moduli increased for PLA/PEG only, and the corresponding phase angle, δ decreased especially

377

at higher frequency range (Fig. 4a) whereas the moduli and the phase angle remained constant

378

for CIN oil incorporated films (Fig. 4b). The reason for an abnormal increment of the moduli for

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PLA/PEG is not clear. Possibly, an excessive pressure application could enhance the interaction

380

between PLA and PEG in the film and restricted the chain mobility. On the contrary, essential oil

381

acts as a protecting barrier during HP-treatment of the film, and retained the mechanical property

382

intact.

383

Oscillatory frequency sweep data were obtained for the PLA/PEG and PLA/PEG/CIN4 films

384

before and after HP-treatment at selected temperatures. Frequency sweep of PLA/PEG/CIN4 at

385

140, 150, 160 and 170 °C are shown in Figure 5a and 5c. The composites exhibited a shear

386

thinning behavior, and the G″ decreased with increasing temperature as evidenced from the

387

slopes of the linear regression of the power-type relationship of ln ω vs ln G′′. The slopes ranged

388

from 0.72 to 0.88 and 0.80 to 0.92 for PLA/PEG and PLA/PEG/CIN4 in the temperature range of

389

140 to 170 ℃, respectively. It clearly indicated both composites exhibited the Newtonian liquid-

390

like behavior. However, the additional plasticization attributed by CIN in the composite was duly

391

supported by the slope values which approaching to unity.

392

Since the rheological properties of polymer melts mostly depend on the process temperature, the

393

time–temperature superposition (TTS) principle was employed to broaden the observations

394

horizon of viscoelastic properties to generate the master curves with a reference temperature.

395

For thermorheological materials, log-log plots of G″ (since predominating liquid-like property)

396

as a function of frequency can be superimposed by horizontal shifts log (aT) and vertical shifts

397

log (bT) versus log (ωT) axis:

398

Mathematically, TTS principle can be written as:

399

bT G′′(aTω,Tref ) = bT G′′(ω,T )

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(3)

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where Tref is the reference temperature (K) and T is the measurement temperature (K). Activation

401

energy was calculated from the TTS Arrhenius equation.

402

The linear viscoelastic master curves for both PLA/PEG and PLA/PEG/CIN4 are generated by

403

the time-temperature superposition (TTS) principle and shifted to a temperature (Tref) of 160 oC.

404

TTS generated master curves for PLA/PEG/CIN4 are illustrated in Figure 5 b and d. TTS

405

parameters of show that the rheograms of pressure treated and untreated superimposed well at

406

the Tref. Both aT and bT values for were insignificantly different between HP-treated and

407

untreated film composites. It indicates that the polymer chains remained unaffected by both

408

pressure and temperature. Fitting an Arrhenius equation, it was observed that the HP-treated

409

PLA/PEG/CIN4 film had slightly higher activation energy (Ea) of 133 kJ/mol than the untreated

410

film (118 kJ/mol) which indicated that HP-treated film was relatively stronger and required

411

much energy to melt against untreated sample. However, a reverse trend was noticed for the

412

PLA/PEG film where the Ea for the control sample was 343 kJ/mol, and it dropped abnormally to

413

223 kJ/mol for HP-treated sample. Such a significant drop in the Ea value supported a possible

414

change in PLA/PEG polymeric chain entanglement under HP-treatment. Therefore, a distinct

415

difference was observed between two films under high-pressure.

416

4. Conclusions

417

The combination of cinnamon oil incorporated plasticized polylactide film and moderate HP-

418

treatment exhibited a synergistic inactivation effect against L. monocytogenes and S.

419

Typhimurium in fresh chicken meat samples. No revival of those test organisms during 3 weeks

420

of refrigerated storage. The addition of cinnamon oil in the composite film further reduced the

421

pressure level to achieve a similar log reduction when HP applied alone. An increasing

422

concentration of CIN drastically reduced the pressure intensity for the inactivation during the

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entire refrigerated storage for 3 weeks. Structural characterization of PLA/PEG/CIN4 films

424

treated at 300 MPa for 10 min revealed that the HP-treatment did not influence the

425

thermomechanical properties of the post-processed films. CIN incorporation in the composite

426

films enhanced the flexibility and oxygen transmission rate compared to the control plasticized

427

PLA films due to plasticization effect attributed by the oil. This work demonstrates that the

428

combination of HP-treatment and active packaging can be used effectively for the extension of

429

the shelf-life of fresh chicken meat during refrigerated storage.

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The authors express their gratitude to the Kuwait Foundation for Advancement of

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Sciences (KFAS) and the Kuwait Institute for Scientific Research for providing the grant for the

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research work (Grant number FB 087C).

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food packaging materials. Innovative Food Science & Emerging Technologies, 6, 51-58. Marcos, B., Aymerich, T., Monfort, J. M., & Garriga, M. (2008). High-pressure processing and

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antimicrobial biodegradable packaging to control Listeria monocytogenes during storage of

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Martino, V. P., Jiménez, A., & Ruseckaite, R. A. (2009). Processing and characterization of poly

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(lactic acid) films plasticized with commercial adipates. Journal of Applied Polymer Science,

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PLA/PCL/cinnamaldehyde antimicrobial packaging on physicochemical and microbial quality

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Rojas-Graü, M. A., Avena-Bustillos, R. J., Friedman, M., Henika, P. R., MartínBelloso, O., &

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9267. Scallan E., Hoekstra R.M., Angulo F.J., Tauxe R.V., Widdowson M.A., Roy S.L., Griffin P.M.

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Color parameters

L*

a*

Thickness (mm)

b*

∆E

Oxygen transmission rate (ml/m2 day)

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Table 1- Effect of high-pressure treatment on visual color, mechanical properties and oxygen permeability of PLA/PEG and PLA/PEG/CIN4 films. Tensile strength (MPa)

Tensile modulus (MPa)

EAB (%)

91.20±0.05c

-1.43±0.02a

0.21±0.03b

1.19±0.47b

0.048±0.004a

328.31±18.00d

18.28±0.90a

817.75±23.9a

41.70±3.70b

300 MPa PLA/PEG

91.85±0.60b

-1.40±0.01a

0.20±0.02b

1.22±0.34b

0.048±0.005a

367.18±16.50c

18.26±0.20a

816.5±26.0a

43.18±5.76b

0.101 MPa PLA/PEG/CIN4

92.50±0.02a

-1.70±0.03b

1.71±0.01a

2.55±0.04a

0.049±0.003a

725.63±20.00b

10.08±0.14b

442±26.8b

100.55±4.51a

300 MPa PLA/PEG/CIN4

91.97±0.01ab

-1.75±0.05b

1.62±0.10a

2.62±0.07a

0.049±0.004a

771.58±18.50a

9.82±0.90b

442±29.5b

104.64±5.59a

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Table 2- Thermal properties of pressure treated and pressure untreated PLA/PEG and PLA/PEG/CIN4 films

0.101MPa PLA/PEG

16.66±0.23a

147.95±0.06a

16.5±1.66c

77.52±0.59a

11.81±1.67b

% Crystallinity (% Xc) 14.10±0.03b

300MPa PLA/PEG

16.87±0.74a

148.32±0.17a

19.24±0.07b

78.54±0.10a

16.04±0.07a

16.08±0.08a

0.101MPa PLA/PEG/CIN4

-1.44±0.01b

136.97±0.14c

22.39±0.21a

62.20±0.91b

10.26±0.17bc

11.03±0.39c

300MPa PLA/PEG/CIN4

-1.36±0.00b

137.59±0.42b

23.50±0.07a

60.79±0.78c

9.60±0.30c

9.86±0.25d

Tm (°C)

Hm (Jg-1)

Tc (°C)

Values are given as mean ± SD (n = 3).

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Figure 1. Growth of L. monocytogenes in chicken samples packed with PLA/PEG, PLA/PEG/CIN1, PLA/PEG/CIN2, PLA/PEG/CIN3, PLA/PEG/CIN4 films and treated at a) 0.101 MPa b) 200 MPa c) 250 MPa and d) 300 MPa and stored at 4°C for 21 days.

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Figure 2. Growth of S. Typhimurium in chicken samples packed with PLA/PEG, PLA/PEG/CIN1, PLA/PEG/CIN2, PLA/PEG/CIN3, PLA/PEG/CIN4 films and treated at a) 0.101 MPa b) 200 MPa c) 250 MPa and d) 300 MPa and stored at 4°C for 21 days.

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Figure 3. Effect of high-pressure treatment on FTIR spectra of PLA/PEG and PLA/PEG/CIN4 films.

0.101MPa PLA/PEG

1750

300MPa PLA/PEG 2

0.101MPa PLA/PEG/CIN4

0.5

0 3000

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2885

1

1180

1514

300MPa PLA/PEG/CIN4 1.5

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1500

1000

500

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Figure 4. Mechanical spectra of untreated and high-pressure treated films melted at 150 °C a. PLA/PEG (80/20), and b. PLA/PEG/CIN4.

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Figure 5. Effect of temperature and applicability of Time-temperature superposition (TTS) principle for the viscous modulus of untreated and high-pressure treated PLA/PEG/CIN films: a and b. Untreated; c and d. 300 MPa pressure treated.

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Highlights

High-pressure and biodegradable active packaging used for chicken meat.

•

Cinnamon oil significantly enhanced the microbial inactivation.

•

No revival of pathogens occur in post-process sample during storage.

•

Packaging materials remained intact after high-pressure treatment.

AC C

EP

TE D

M AN U

SC

RI PT

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