Atmospheric cold plasma inactivation of Escherichia coli and Listeria monocytogenes in tender coconut water: Inoculation and accelerated shelf-life studies

Food Control 106 (2019) 106678

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Atmospheric cold plasma inactivation of Escherichia coli and Listeria monocytogenes in tender coconut water: Inoculation and accelerated shelflife studies

T

Nikhil Kumar Mahnota,b, Charu Lata Mahantaa, Brian E. Farkasb, Kevin M. Keenerb,c,d,∗, N.N. Misrad a

Department of Food Engineering and Technology, School of Engineering, Tezpur University, Assam, India Department of Food Sciences, Purdue University, West Lafayette, IN, USA Department of Food Science and Human Nutrition, Iowa State University, Ames, IA, USA d Center for Crops Utilization Research, Iowa State University, Ames, IA, USA b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Barrier discharge Nonthermal Tender coconut water Accelerated shelf-life

In the current work we evaluated the effect of atmospheric cold plasma (ACP) generated in air and M65 (65% O2, 30% CO2, 5%N2) as working gases for inactivation of Escherichia coli and Listeria monocyotogenes in tender coconut water. A 5 log10 reduction in the population of both microbes was achieved in tender coconut water with addition of 400 ppm citric acid and plasma treatment with M65 gas for 120 s at 90 kV, followed by a 24 h post-treatment storage under refrigerated conditions. Optical emission spectroscopy was utilized in identifying reactive gas species of nitrogen and oxygen which were accounted for cellular leakage and morphological changes in microbes on plasma treatment. The plasma treatments on tender coconut water caused a lowering of pH and small changes in Hunter color parameters (a* and b*), while total soluble solids and total titratable acidity did not change significantly. Accelerated shelf-life studies (ASLS) carried out at 10 °C, 20 °C and 30 °C compared three batches: Batch 1(tender coconut water), Batch 2 (Tender coconut water + citric acid + ascorbic acid) and Batch 3 (Tender coconut water + citric acid + Plasma treatment with M65 + ascorbic acid). ASLS revealed that the rate constants for parameters namely percent transmission, ascorbic acid content, total titratable acidity and total color change decreased in the order Batch 1 > Batch 2 > Batch 3. Estimation of rate constants following Arrhenius model for Batch 3 at 5 °C was comparable to experimental results. A shelf-life of 48 days was predicted for Batch 3 at 5 °C considering 75% ascorbic acid content degradation. Thus, ACP was concluded to be a novel technology for tender coconut water processing.

1. Introduction In recent times atmospheric cold plasma technology is being explored as a nonthermal intervention for decontamination and shelf-life extension of foods. From the research standpoint, ample work has been carried out revealing the promising applications of cold plasma technology in the field of microbial decontamination in liquid food products, mostly fruit juices of apple, orange, pomegranate, tomato (Almeida, Cavalcante, Cullen, Frias, Bourke, Fernandes, 2015; Shi et al., 2011; Surowsky, Frohling, Gottschalk, Schluter, & Knorr, 2014; Kovacevi, Putnik, Dragovic-Uzelac, Pedisic, Rezek-Jambrak, & Herceg, 2016; Ma & Lan, 2015), milk (Korachi et al., 2015) and coconut liquid endosperm (Gabriel et al., 2016). Of these, only a few studies

employing plasma jets, dielectric barrier discharge, and radio–frequency technology of plasma generation were effective in inactivating 5-log reduction in Escherichia coli, Staphylococcus aureus, Candida albicians, Citrobacter freundii in different fruit juices were reported (Montenegro, Ruan, Ma, & Chen, 2002; Surowsky et al., 2014). The U.S. Food and Drug Administration (FDA, 2001) suggests a need of 5-log10 reduction performance standard against hardy pathogenic microbes for an effective juice processing. Within this context, E. coli and L. monocytogenes have been reported to be responsible for disease outbreaks from the consumption of fruit and vegetable juices (Han & Linton, 2004; Vojdani, Beuchat, & Tauxe, 2008). Tender coconut water (TCW), not to be confused with coconut milk, is the inner clear liquid portion also called the liquid endosperm of the

∗

Corresponding author. Department of Food Sciences, Purdue University, West Lafayette, IN, USA. E-mail addresses: [email protected] (N.K. Mahnot), [email protected] (C.L. Mahanta), [email protected] (B.E. Farkas), [email protected] (K.M. Keener), [email protected] (N.N. Misra). https://doi.org/10.1016/j.foodcont.2019.06.004 Received 8 March 2019; Received in revised form 10 May 2019; Accepted 3 June 2019 Available online 18 June 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. (A) Schematic of the experimental set-up for ACP treatment and the optical emission spectroscopy of the light emitted. (B) Optical emission spectrum for air and M65 plasma generated at 90 kV.

2. Materials and methods

fruit which is considered to possess great nutritive importance, with multiple health benefits (Falck, Thomas, Flack, Tutuo & Clem, 2000; Alleyne, Roache, Thomas, & Shirley, 2005). Chemical constituents of tender coconut water include sugars, amino acids, vitamins and a considerable amount of macro and micro-nutrients (Yong, Ge, Ng, & Tan, 2009), thus making it a very good medium for microbial growth. As the market demand for tender coconut water as a beverage is on the rise, the need for effective processing is timely and much sought after by juice processors. While thermal treatment is the primary go to for processing of TCW but heating results in loss of nutritional properties as well as flavor (Haseena, Kasturi, & Padmanabhan, 2010). Works on non-thermal technologies like microfiltration and dense phase carbon dioxide technologies have been carried out as mentioned in the works of Mahnot, Kalita, Mahanta, and Chaudhuri (2014). These technologies have their own drawbacks like larger processing times, higher cost as well as use of sophisticated equipment. A notable recent development within the evolution of non-thermal food processing technologies is the application of cold plasma for food decontamination (Misra, Yepez, Xu, & Keener, 2019). Plasma is an ensemble of several excited atomic, molecular, ionic, and radical species, co-existing with numerous others, including electrons, positive and negative ions, free radicals, gas atoms, molecules in the ground or excited state, quanta of electromagnetic radiation (UV photons and visible light) (Misra et al., 2018). In our recent publication, we demonstrated an effective reduction of the population of Salmonella enterica serovar Typhimurium LT2 by 5 log10 in TCW upon addition of 400 ppm citric acid and cold plasma treatment (Mahnot, Mahanta, Keener, & Misra, 2019). In this work, we further our work by studying the safety and shelf-life of tender coconut water subjected to cold plasma treatment. The primary objective of the current study was to effectively achieve a 5 log10 reduction performance standard on Escherichia coli and Listeria monocytogenes in tender coconut water using atmospheric cold plasma (ACP) from a dielectric barrier discharge. A further objective of the work was to assess the shelf-life using accelerated shelf-life testing.

2.1. Materials Whole tender coconuts were obtained from a market in West Lafayette Indiana, USA. The nuts were cracked open in the lab and the liquid endosperm was extracted and stored in glass bottles for further use. Ascorbic acid was obtained from Mallinckrodt, U.K. Citric acid was obtained from EMD, USA, meta-phosphoric acid and sodium hydroxide were obtained from Fisher Scientific, USA, while peptone, MacConkey Sorbitol agar, Oxford medium base and tryptic soy agar (TSA) were obtained from Difco, USA, Modified Listeria selective supplement was obtained from Oxoid, UK and Dichloran Rose Bengal Chloramphenicol (DRBC) Agar from EMD, USA.

2.2. Microbial methodology The microbes were selected one from gram negative group i.e. Escherichia coli ATCC25922 and the other from gram positive group, Listeria monocytogenes ATCC19115, which were grown for overnight and 24 h respectively at 37 °C in TSB media in a shaking incubator to reach the stationary phase. For preparation of inoculated samples, to every 100 ml of tender coconut water 125 μl of the media containing either E. coli or L. monocytogenes were added and shaken well to have a homogenous mixture. The added microbes were not mixed together, and 25 ml of the TCW or TCW with added citric acid was taken for plasma treatment. The microbial enumeration was carried out using serial dilution technique with 0.1% peptone water and cell counts were made using drop plate technique on MacConkey Sorbitol agar for E. coli. For L. monocytogenes, counts were obtained on Oxford medium base with Listeria selective supplement added as per specification. Briefly 5 ml of 30% ethanol was added to each supplement vial and mixed until dissolved and to every 500 ml of the sterile oxford medium base the alcohol dissolved vial was added aseptically. For the drop plate technique, briefly 10 μl of the diluted or non-diluted samples were spotted and the plates were incubated at 37 °C for 24 h–48 h to enumerate visible colonies. For shelf-life testing, total plate count and total fungal counts were enumerated using spread plate method on TSA media and 2

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experimental setup comprised of a collimating lens of 5 mm diameter coupled to an optical fiber (Ocean Optics, USA.) of core diameter of 400 μm which was lined in parallel to the TCW containing package (See Fig. 1(A)). The lens-fiber assembly was placed at a distance of 15 cm from the package. The characteristic light emission was transferred by the fiber to a custom-built high-resolution spectrometer (10 μm grating size, HR2000 + Ocean Optics, USA), controlled through the OceanView software with an integration time of 5 s and averaging of 6 scans. Spectral data acquisition also involved background noise correction. The electronic transitions of the gaseous species were identified using previously published reports and National Institute of Standards and Technology (NIST) database (http://physics.nist.gov/PhysRefData/ ASD/lines_form.html) (Kramida et al., 2015).

DRBC agar respectively. 2.3. Experimental setup for cold plasma treatment The Atmospheric Cold Plasma (ACP) treatment was carried out using a dielectric barrier discharge (DBD) set-up, as shown in Fig. 1(A). The system comprised of a high voltage transformer (with input voltage of 120 V at 60 Hz), a voltage regulator (also called, variac; 0–100%, output voltage controlled within 0–120 kV), two donut shaped electrodes made of aluminum, with the interelectrode distance set to 5 cm. The dielectric barriers constituted of 1 mm thick polypropylene plastic boards between which a polypropylene box (16.8 cm × 26.9 cm × 4 cm) used as the sample holding container was sandwiched. The cold plasma treatment was carried out using two working gases viz., dry air (78% N2, 21% O2 and traces of other gases) and modified air M65 (65% O2, 30% CO2, 5% N2). The gases were filled into the box at a flow rate of 0.6 L/min for 120 s; the boxes were further enclosed inside high barrier flexible bags (Cryovac BH4670T, Sealed Air, USA) to prevent loss of any reactive gaseous species formed from the electrical discharge. The liquid samples were kept in polystyrene containers at the centre the polypropylene box before the barrier bags were sealed with a heat sealer (Food Saver, USA). The plasma treatment voltage was fixed at 90 kV and the treatments were carried out under ambient temperature conditions.

2.7. Scanning electron microscopy (SEM) The effect of ACP treatment on the morphological characteristics of E. coli and L. monocytogenes in TCW with 400 ppm citric acid was studied using electron microscopy. The ACP treatment was carried out using M65 gas for 120 s at 90 kV and post-treatment, the samples were stored under refrigerated conditions for 24 h. Untreated inoculated samples were taken as control. For SEM, briefly 1 ml of inoculated controls and treated samples were taken and primarily fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 2 h at 4 °C and few drops of the fixed cells were incubated on cover slips coated with 0.1% poly-L-lysine. They were then post-fixed in 1% osmium tetroxide and the dehydration step included a graded ethanol series (30%, 50%, 70%, 80%, 90% and 100%) and finally the slips were transferred into hexamethyldisilazane and air dried. The dried specimens were coated with platinum in a Cressington 208HR sputter coater (Ted Pella, Inc, USA) and imaged in a FEI Nova NanoSEM 200 (FEI Ltd, USA) at 5 kV for observation.

2.4. ACP treatment of tender coconut water for microbial reduction TCW sample inoculated with the microbes were treated with ACP with both air and M65 as the working gas for 120 s at 90 kV and then stored under refrigeration for 24 h; subsequently, microbial recoveries were carried out. As a next step, citric acid at a concentration of 400 ppm was added to TCW prior to plasma treatments, and microbial recoveries. Additionally, we ran an experiment for inactivation of E. coli OH157:H7 in TCW using ACP with and without addition of 400 ppm citric acid under similar treatment conditions. The inoculations and recovery were done as mentioned for E. coli ATCC25922. Non-treated samples were used as controls. This was done to confirm the efficacy against the pathogenic strain.

2.8. Cell membrane integrity The cell membrane integrity was examined by monitoring the release of UV absorbing materials at 260 nm and 280 nm (Mahnot et al., 2019). For cell leakage studies, TCW, and TCW with 400 ppm citric acid (TCW + CA) samples were treated with ACP in air and M65 gas for 120 s at 90 kV followed by 24 h post-treatment refrigerated storage. In the next step, the samples were centrifuged at 13,200 g for 10 min in a centrifuge (Avanti J-26XP, Beckman Coulter, Inc. USA) and 200 μL of the supernatant was carefully transferred into microtitre plates. The absorbance was measured at 260 nm and 280 nm using a microtiter plate reader (Epoch, BioTek, USA).

2.5. Quality of ACP treated TCW TCW treated with ACP in air and M65 as the working gases at 90 kV without (TCW) and with addition of 400 ppm citric acid (TCW + CA) were analyzed for parameters, viz. pH, total soluble solids as °Brix, total titratable acidity and color. The analyses were carried immediately after the treatment for 120 s and also after 24 h of post storage under refrigerated condition. The pH was measured using a pH meter (Spectrum Technologies, USA), total soluble solids (TSS) measured using pocket refractometer (Atago, USA), total titratable acid (TTA) was estimated according to the method as in Sadasivam and Manickam (2007) and expressed as citric acid equivalent and hunter L*, a* and b* for color values were measured using LabScan XE, USA. For shelf-life testing, parameters like TTA, total color change (ΔE) were monitored. The total color change was estimated using the following equation ΔE = [(ΔL∗)2+(Δa∗)2+(Δb∗)2]1/2. In addition, the light transmission was measured at 610 nm using a spectrophotometer (Hitachi U 1100 Spectrometer, USA) as prescribed by Jackson, Gordon, Wizzard, McCook, and Rolle (2004) and ascorbic acid concentration was estimated using 2, 6-Dichlorophenol-indephenol dye titration method (Ranganna, 1986).

2.9. Accelerated shelf life testing (ASLT) of plasma treated TCW 2.9.1. Samples and sampling ASLT was carried out with three different sample batches - (1) Batch 1: untreated TCW; (2) Batch 2: TCW with 400 ppm citric acid and 200 ppm ascorbic acid; and (3) Batch 3: TCW with 400 ppm citric acid treated with ACP in M65 gas (90 kV, 120 s, 24 h post-treatment refrigerated storage), aseptically filled into storage vials of 30 ml capacity, with addition of 200 ppm ascorbic acid. For ASLT, the samples were not inoculated with the microbes under study. All batches were stored at 10 °C, 20 °C and 30 °C to accelerate the physicochemical changes. To validate the extrapolation ability of the ASLT model developed, a separate batch of the Batch 3 sample was stored at 5 °C and analyzed every 10 days till 30th day. The analysis for different batches at different temperatures were done on different days taking into consideration two points. Firstly, uncertainty of microbial stability of untreated samples would speed up the deterioration and make the product microbially unacceptable. Secondly, storage at higher temperatures would change product characteristic much faster than refrigerated storage. Briefly, for the Batch 1

2.6. Optical emission spectroscopy (OES) of plasma OES was carried out to identify the electronic transitions in the gaseous species. The spectral monitoring was done while the plasma treatment of the samples was carried out in air and M65 at 90 kV. The 3

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microbial population based cut-off value for shelf-life prediction was ruled out. Thus, ascorbic acid degradation was employed as the cut-off criteria to ascertain product failure. Being an important nutritional and quality indicator, ascorbate is widely utilized for shelf-life prediction of juices (Polydera, Stofors, & Taokuis, 2003; Sapie & Hwa, 2014).

sampling at 10 °C was done every three days till 6th day, at 20 °C every two days till 4th day and at 30 °C it was done on 0th, 1st and 2nd day. For the Batch 2 at 10 °C sampling was done every four days till 8th day, for 20 °C every three days till 6th day and for 30 °C for every two days till 4th day. And Batch 3, stored at 5 °C, was sampled on 0th, 10th and 21st day, at 20 °C sampled after every 5 days till 15th day.

2.10. Statistical analysis 2.9.2. Kinetics calculation For the ASLT, first the experimental data were plotted against time and equations for the parameters in test were obtained via least-squares regression. The reaction order for the parameters were determined by employing both zero and first-order equations. For zero order reaction, [Pt] = -k*t + [P0]

Single samples were pulled per sampling point for all the experiments. All the experiments were carried out in triplicates and the data were presented as mean ± standard deviation. The error bars denote one standard deviation. The data presented here are analyzed for variance and the significance of differences between treatments were assessed by Duncan multiple sample comparison test. The significance levels were tested at P < 0.05 using SPSS software (IBM® SPSS® Statistics ver. 20).

(1)

For 1st order reaction, [Pt] = [P0] exp(-k*t)

(2) 2.11. Key resource table

where, P0 and Pt are the parameters under consideration at initial time and at time ‘t’, respectively; ‘k’ is the rate constant of the reaction (or reaction velocity). Activation energies (Ea) were calculated based on the temperature dependence of the parameters simulated by Arrhenius equation as under ln(k) = ln(A) - (Ea/RT)

Resource

(3)

where Ea, R, T and A are activation energy of each reaction (Jmol ), Universal gas constant (8.314 J mol−1 K−1), absolute temperature (T) and pre-exponential factor (day−1), respectively. For prediction of time needed for changes in parameters equation (4) was used for parameters following zero order reaction. For parameters following first order reaction equation (5) was applied. (4)

where, ΔCQ is the change in parameter and Tpred is the time predicted to incur these changes. Tpred = ln (PF/PI)/k

Identifier

Chemical 2, 6- Dichlorophenol aluminum ascorbate Ascorbic acid Chloramphenicol Citric acid CO2 Dichloran ethanol glutaraldehyde hexamethyldisilazane L-lysine meta-phosphoric acid osmium tetroxide platinum Rose Bengal sodium cacodylate sodium hydroxide Sorbitol ProteinPeptide PF PI

−1

Tpred = ΔCP / k

Source

(5)

Where PF/PI is the ratio of parameter change i.e. final to initial. From the above calculations the theoretical value of rate constants for the third batch of samples when stored at 5 °C were estimated, and compared against the experimental findings at 5 °C. Finally, the shelflife predictions were made based on the time at which 75% of the ascorbic acid degradation occurred. Considering that the ACP treated samples with citric acid resulted in no microbial recovery, the use of a

Table 1 Microbial reduction upon high voltage cold plasma treatment with air and M65 as a working gas in tender coconut water without (TCW) and with 400 ppm citric acid (TCW + CA) at 90 kV for 120 s and 24 h post refrigerated storage for both air and M65 as a working gas. E. coli ATCC 25922

Initial (log cfu/ml) Air

Control (log cfu/ml) M65

a

Air a

Log reduction M65

b

Air b

M65 c

TCW TCW + CA

6.77 ± 0.10 6.84 ± 0.00a

6.77 ± 0.10 6.84 ± 0.00a

7.02 ± 0.14 6.89 ± 0.03b

6.96 ± 0.16 6.92 ± 0.03b

2.23 ± 0.33 N.C

L. monocytogenes ATCC19115

Initial (log cfu/ml) Air

M65

Control (log cfu/ml) Air

M65

Log reduction Air

a

a

b

b

c

N.C N.C

M65

TCW TCW + CA

6.47 ± 0.00 6.47 ± 0.00a

6.47 ± 0.00 6.47 ± 0.00a

6.6 ± 0.15 6.54 ± 0.08b

6.5 ± 0.28 6.60 ± 0.00b

2.03 ± 0.05 2.39 ± 0.21c

N.C N.C

E. coli OH157:H7

Initial (log cfu/ml) Air

M65

Control (log cfu/ml) Air

M65

Log reduction Air

M65

TCW TCW + CA

a

5.81 ± 0.04 6.39 ± 0.12a

a

b

5.81 ± 0.04 6.39 ± 0.12a

6.13 ± 0.02 6.54 ± 0.28b

*N.C − No Counts i.e., no microbial colony growth observed post treatment. Different letters (a,b,c …) in the same row denote significant difference at p < 0.05. 4

c

6.10 ± 0.06 6.38 ± 0.12b

d

1.09 ± 0.17 4.09 ± 0.00d

1.79 ± 0.21e N.C

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Fig. 2. Changes in physicochemical properties of tender coconut water without and with addition of 400 ppm citric acid with ACP using both air and M65 at zero hour and 24 h post-refrigerated storage. Different letters (a–g) on the bars indicate significant differences at P < 0.05.

3. Results and discussion

respectively. Similar improved microbial reduction in gram negative bacteria has been reported with M65 gas in orange juice (Xu, Garner, Tao, & Keener, 2017). L. monocytogenes inoculated TCW samples subjected to ACP treatment resulted in 2.03 ± 0.05 log10 reduction with air and no microbial recoveries were obtained with M65 plasma. Further, inoculated samples of TCW with 400 ppm of citric acid (TCW + CA) subjected to ACP treatment with both air and M65 led to no microbial recoveries during enumeration tests. The addition of

3.1. Microbial reduction in tender coconut water Treating TCW with ACP in air as well as M65 gas caused the inactivation of both E. coli and L. monocytogenes (Table 1). Microbial recoveries showed that for E. coli a 1.09 ± 0.17 log10 and 1.79 ± 0.21 log10 reduction was achieved using air plasma and M65 gas plasma, 5

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Fig. 3. SEM micrographs of E. coli (A) before and (C) after ACP treatment, and that for L. monocytogenes (B) before and (D) after ACP treatment. Cell leakage before and after ACP treatment for (A) E. coli and (B) L. monocytogenes at 280 nm and 260 nm. Different letters (a–d) and (x–z) on the bars indicate significant differences at P < 0.05.

2014). Further, citric acid aided in increasing the effectiveness of inactivation through ACP using both air and M65, as was confirmed in our recent work (Mahnot et al., 2019). Acidification is yet another impact of plasma treatment leading to microbial stress and inactivation. It is reported that generation of nitrous acids and nitric acid from NO via NO2 lead to such acidification (Oehmigen et al., 2010). Drastic inactivation of microbes with ACP combined with citric acid addition is possible due to the following phenomenon put forward by Mahnot et al. (2019): firstly, electroporation effect on ACP treatment which might aid in better citric acid diffusion through the bacterial plasma membrane leading to inactivation; secondly, generation of a peroxy form of citric acid on ACP treatment on reaction with hydrogen peroxides and this peroxy-organic acid/organic acid systems are supposed to have better

400 ppm citric acid to coconut water as a preservative during storage of coconut water was suggested in previous work by Mahnot et al. (2014), and its success with ACP was established in our recent work (Mahnot et al., 2019). For L. monocytogenes, air plasma resulted in a 2.39 ± 0.21 log10 reduction, whereas with M65 ACP treatment no colonies were recovered. This showed that M65 was a better option for microbial reduction in ACP as compared to air. The major reason for microbial reduction on ACP treatment is due to generation of reactive gas species of nitrogen and oxygen as detected through optical emission spectroscopy Fig. 1(B) of the current ACP application with air and M65 gases. Additionally, generation of ozone, nitrous gases and hydrogen peroxide on plasma treatment have also been widely reported to cause microbial inactivation (Muhammad et al., 2019; Liao et al., 2018; Surowsky et al., 6

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Fig. 4. Changes of physicochemical properties during storage period at different temperatures at (-◊-) 10 °C, (-○-) 20 °C, (-□-) 30 °C and (-Δ-) 5 °C. (A), (B) and (C) for percent transmission, (D), (E) and (F) for ascorbic acid content, (G), (H) and (I) for total titratable acidity, (J), (K) and (L) for total color change of the Batches 1, 2 and 3 respectively.

3.2. Optical emission spectroscopy (OES) of plasma

antimicrobial effects than that of parent organic acids; and thirdly, hydrogen peroxides. With the use of both ACP and citric acid we could achieve more than 5 log10 reductions in microbial counts which meets FDA standard for juice processors (FDA, 2001). Thereby, proving the potential of ACP technology as a non-thermal decontamination intervention for juice processing. It is worth noting that citric acid alone did not inactivate either of the microbes when untreated inoculated samples were enumerated after 24 h of post refrigeration storage (Table 1). The data also shows that E. coli was easier to be inactivated than L. monocytogenes using ACP in air, indicating that Gram negative bacteria are more susceptible to microbial inactivation than Gram positives. Similar result of differential sensitivity of gram positive and negative bacteria was reported by Prochnow, Clauson, Hong, and Murphy (2016). The outer membrane of lipopolysaccharide and thin layer of peptidoglycan in Gram-negative bacteria has been suggested to be more susceptible than the relatively stable peptidoglycan layer in Gram positive bacteria (Laroussi, Mendis, & Rosenberg, 2003).

Fig. 1(B) shows the emission spectra for ACP during treatment of TCW in air and M65 gas (inset in Fig. 1(B)). The spectrum for air depicted the presence of peaks of strong emission in the wavelength range from 315 nm to 405 nm which were identified as transitions from second positive system of nitrogen, N2(C–B).The first negative system of N2+(B-X) transition were also observed near 426.5 nm and at a wavelength of 399.7 nm with a relatively lower intensity. For ACP generated in M65 gas, the emission spectrum showed only singlet oxygen atom peaks at 777.8 nm and 844.7 nm with respective transitions from 2s22p3(4s0)3p 5P→2s22p3(4s0)3s 5S0 and 2s22p3(4s0)3P→2s22p3(4s0)3p 3 P. These spectral peaks are typical of such high voltage ACP sources, and have been reported in earlier work of Misra, Keener, Bourke, and Cullen (2015) and Misra et al. (2014). The very low intensity peaks of nitrogen second positive system in spectrum of ACP with M65 gas was likely due to the unavoidable leakage of small quantities of air into the package during sample preparation. These spectra confirmed the 7

Food Control 106 (2019) 106678 1.885 (0.993) 0.414 (0.850) 0.277 (0.623) 1.101 (0.993) 0.314 (0.850) 0.196 (0.625) 0.345 (0.972) 0.097 (0.466) 0.098 (0.985)

3.3. Effect on quality parameters of tender coconut water A summary of the results for all the quality parameters is provided in Fig. 2. ACP treatment of TCW with either air or M65 at 90 kV for 120 s did show a significant change in pH between the samples both at 0 h and 24 h after a treatment time of 120 s. While comparing the change of pH between samples at 24 h after treatment, firstly we observed the pH of TCW was reduced from 6.29 ± 0.06 to 6.00 ± 0.03 and 5.54 ± 0.10 when air and M65 were respectively used as the inducer gases. Secondly, the addition of citric acid at 400 ppm concentration to TCW reduced the pH of coconut water from 6.29 ± 0.06 to 5.35 ± 0.03. Further, the pH of the same samples (TCW + CA) on ACP treatment and 24 h storage further reduced to 5.27 ± 0.02 and 5.18 ± 0.08, respectively, for air and M65 as inducer gases. The reduction in pH can be attributed to ACP induced generation of nitric and nitrous acids as well as due to concentration of reactive plasma gas species as reported in previous literature for water as well as other juices (Liu et al., 2015; Muhammad et al., 2019; Xu et al., 2017). No significant changes in TTA were observed for ACP treated TCW with either air or M65 at 0 h as well as after 24 h of storage. While TCW with citric acid, upon ACP treatment with both air and M65 showed a small decrease in the TTA, no significant changes were observed between measurement at 0 h and 24 h post-treatment. Addition of citric acid significantly increases the TTA of tender coconut water although taste change is a concern, preliminary sensory tests with varying concentration of citric acid and previous works suggests a positive effect on sensory properties (Mahnot et al., 2014). An in-depth sensory analysis is needed with citric acid addition as well as post ACP treatment considering that taste is a very important criteria for consumer's decision on processed beverages. ACP treatment of TCW with both air and M65 did not show any changes in the TSS as compared to untreated TCW which had a TSS of 7.50 ± 0.01oBrix while a negligible increase to 7.60 ± 0.01 °Brix was noted on adding 400 ppm of citric acid. Similar results were also reported in our previous study (Mahnot et al., 2019). The ACP treatment did not show any major impact on the L* of the sample for any treatment. While the a* values shifted towards greener color as suggested by the decreased values on ACP treatment of tender coconut water with air and M65. Citric acid addition increased the a* values of TCW on ACP treatment in air and M65 gas, as compared to untreated TCW with and without added citric acid. No statistically significant changes were observed between measurements at 0 h and 24 h after treatment of a sample. Hunter b∗ value for TCW decreased significantly on ACP treatment with M65 with added citric acid as compared to untreated tender coconut water. The changes in a* and b∗ values might be attributed to oxidation or polymerization of tender coconut water constituents like phenols, proteins or due to some nonenzymatic reaction like ascorbic acid degradation, as reported in literature for various juices, fruits and vegetables upon ACP treatment (Liao et al., 2018; Pankaj, Wan, Colonna, & Keener, 2017; Pankaj, Wan, & Keener, 2018). The total color difference (ΔE) values were not noticeable and varied from 0.37 to 1.7 for all the samples as compared to untreated tender coconut water which lie below the ΔE≈ 2.3 beyond which just noticeable differences can be observed (Mokrzycki & Tatol, 2011). Based on all these quality indicators, it can be concluded that ACP does not significantly alter the quality of TCW, except for a reduction in pH.

0.183 (0.982) 0.064 (0.969) 0.014 (0.940) 0.073 (0.969) 0.034 (0.835) 0.007 (0.983) 0.704 (0.912) 0.094 (0.910) 0.071 (0.899) 0.302 (0.994) 0.086 (0.966) 0.062 (0.904)

** Values under bracket are the coefficient of regression (R2).

0.095 (0.979) 0.078 (0.900) 0.034 (0.986) 0.327 (0.999) 0.302 (0.994) 0.017 (0.838) 0.237 (0.999) 0.127 (0.869) 0.009 (0.863) 0.054 (0.996) 0.062 (0.911) 0.003 (0.765) Batch 1 Batch 2 Batch 3

30 °C 20 °C

occurrence of collision reactions that would lead to formation of reactive oxygen and nitrogen species (RONS) that would diffuse into the TCW to form very low quantities of antimicrobial species, as measured earlier (Mahnot et al., 2019). The RONS are chemically very active as they cause oxidative stress (Weidinger & Kozlov, 2015) and studies have already confirmed their antimicrobial nature effected from cell damage (Laroussi & Leipold, 2004; Oehmigen et al., 2010; Van Gils, Hofmann, Boekema, Brandenburg, & Bruggeman, 2013).

0.272 (0.985) 0.168 (0.927) 0.020 (0.949)

30 °C 20 °C 10 °C 20 °C 10 °C 10 °C 10 °C

20 °C

Ascorbic acid content (Day−1) Percent Transmittance (Day−1) Sample

Table 2A Rate constant of various parameters at different storage temperatures.

30 °C

Total Titratable acidity (Day−1)

30 °C

Total color change (a.u. Day−1)

N.K. Mahnot, et al.

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Fig. 5. Arrhenius plot indicating natural logarithm of rate constant for changes in (A) transmission (B) ascorbic acid content (C) total titratable acidity and (D) total change in color of different coconut water samples versus 1/Absolute temperature i.e. ln(k) vs 1/T. (-■- Batch 1) (-●- batch 2) and (-▲- Batch 3).

Fig. 6. Microbial growth during storage at different temperatures viz. at (-◊-) 10 °C, (-○-) 20 °C, (-□-) 30 °C and (-Δ-) 5 °C. (A) and (C) show the total plate counts for Batch 1, (B) and (D) are the total fungal counts for Batch 2. (Data for Batch 3 not shown as no microbial growth was observed during the storage period).

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3.6. Influence of storage temperature on microbiological and quality parameters

Table 2B Activation energy (Jmol−1) of parameters under study. Sample

Batch 1 Batch 2 Batch 3

Percent Transmittance

Ascorbic acid content

Total Titratable acidity

Total color change

64756.08 56355.62 61995.00

71492.91 6651.94 26425.21

47009.85 56204.30 35477.50

60712.98 52088.67 36887.55

We monitored the changes in transmission, ascorbic acid content, TTA and total color change with respect to temperatures at 10 °C, 20 °C and 30 °C (Fig. 4. A, B, C, D, E, F, G, H, I, J, K and L) of the three batches of samples viz. Batch 1, Batch 2 and Batch 3. The rate of reaction for the various parameters is shown in Table 2(A) which suggests that the rate constants of the studied parameters increased with higher storage temperature. The transmission of the samples of Batch 1 and Batch 2 decreased with increase in temperature which is attributed to the increase in microbial loads with time. The increase in microbial populations, both total plate counts and total fungal counts, are shown in Fig. 6. While for the ACP treated samples in Batch 3 no microbial growth were observed during storage, the percent transmission of the samples remained considerably higher. This decreasing trend likely resulted from precipitation of some suspended matter on subsequent storage after ACP treatment. These particles might be of proteinaceous nature and reactive oxygen species have been shown to cause crosslinking of proteins leading to precipitation or settling (Davies, 2003). The degradation of ascorbic acid during storage was noted. The extent of degradation was higher at higher storage temperatures as expected. We observed that ascorbic acid degradation was also related to the change in color. Specifically, a higher decrease in ascorbic acid caused a higher change in the color of the stored samples, visually observed as a brownish tinge. The effect of ascorbic acid to cause non-enzymatic browning is also reported in literature even at relatively lower temperatures (Otsuka, Kurata, & Arakawa, 1968; Manso, Oliveira & Friar, 2001; Nursten, 2005). We figured that an unacceptable color change arises when the ascorbic acid content falls below 75% of the original concentration. This naturally becomes the primary cut-off criteria in identifying the point at which the samples may be considered unfit. The total color changes of the samples was also affected by microbial growth. An increased microbial growth increased the turbidity and the samples with microbial growth had a milky white color. The TTA of the samples showed a gradual increase during storage which could be attributed to microbial metabolism and possible oxidation of reducing sugars that happens during juice storage (Okudu & Ene-Obong, 2015). The rate of increase of TTA was higher in untreated samples and increased with rise in storage temperature. It should be noted that for the ACP treated samples, the rate of change of TTA was lower as compared to the other samples; Table 2(A). Further, it was observed that the addition of citric acid and ascorbic acid to TCW helped in reducing the reaction rates for all the parameters under study, thereby confirming that additives in juices help in maintaining the stability (DasPurkayastha et al., 2012).

3.4. Morphological characteristics of bacterial cells Fig. 3 (A, B, C and D) show the changes in morphological characteristics of E. coli and L. monocytogenes before and after ACP treatment. SEM imaging clearly showed shrinkage of the bacterial cell surface, deformation, as well as bursting of cells upon ACP treatment, as compared to untreated cells for both E. coli and L. monocytogenes. It has been reviewed that such physical changes upon ACP treatment is widely attributed to electrostatic disruption due to accumulation of charged particles on the cell membrane leading to cell bursting and eventually death (Liao et al., 2017). These morphological changes clearly confirm that ACP induced stress, and eventually caused a loss of cell functionality in bacterial cells. Similar effects of ACP on E. coli in model system was observed through SEM in a previous study (Han et al., 2016). It is suggested that reactive gas species damage microbial cells by reacting with the cell membrane as well as with their intracellular components. Reactive oxygen species have been supposed to cause lipid peroxidation of the cell membrane resulting in weaker cell membrane, causing physical damage and inactivation (Dezest et al., 2017; Han et al., 2016).

3.5. Cell membrane integrity Fig. 3(E) and (F) presents the release of UV absorbing intracellular components which includes nucleic acids absorbing at 260 nm and proteins absorbing at 280 nm, for E. coli and L. monocytogenes, respectively. The cell leakage occurs due to loss of membrane integrity and disruption of cell structure. ACP treatment in air or M65 caused an increase in absorbance for both, nucleic acids and proteins, whereas presence of citric acid in untreated samples did not cause any cellular material loss as compared to untreated inoculated TCW. The presence of citric acid combined with ACP treatment in air as well as M65 caused a significant increase in absorbance for cellular components. M65 usage for plasma treatment caused the highest loss in cell membrane integrity thereby proving its effectiveness. The results of cell leakage studies are in agreement with the visual observations from SEM study, where cell structure was found to be disrupted. The release of intracellular material in bacteria on cold plasma treatment has been widely reported in the works of Lu, Patil, Keener, Cullen, and Bourke (2014) and Hong et al. (2009).

3.7. Arrhenius equation modelling of transmission, ascorbic acid content, titratable acidity and color The order of reaction was judged based on a comparison of the coefficients of regression (R2 values), it was found that color change followed zero order reaction, while the transmittance, ascorbic acid

Table 2C Experimental and predicted rate constant values for Batch 3 stored at 5 °C along with time needed to achieve the same amount of change of parameters viz. percent transmission, ascorbic acid content, total titratable acidity and total change in color as per prediction. Quality attributes

Percent transmission Ascorbic acid content Total titratable acidity Total color change

Rate constant k (Day−1/a.u. Day−1) comparison

Storage comparison

Experimental

Arrhenius equation prediction

Experimental values of parameter at 20th day

Predicted no. of days to reach the same experimental value

0.0030 0.0330 0.0067 0.0760

0.0029 0.0300 0.0057 0.0780

85.93% 10.18 mg/100 ml 0.178% 3.090

18.24 21.94 24.27 21.15

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content and total titratable acidity followed first order reaction kinetics. The Arrhenius equation was used to describe the rate of reaction dependence on temperature. The rate of reaction for different quality parameters, k, is summarized in Table 2A Arrhenius plots were made with logarithmic values of the rates versus inverse of absolute temperature (1/T) (see Fig. 5). Activation energy (Ea) calculations serve as an indirect tool to efficiently compare the samples under study, as suggested by Ganje et al. (2016) for ASLT of tomato paste. The activation energies of the parameters under study are shown in Table 2B. From the table, it is observed that the activation energies for percent transmission, ascorbic acid content and total color change decreased upon addition of citric acid and ascorbic acid. This implies an increased temperature sensitivity of these parameters of Batch 2 and Batch 3 as compared to untreated TCW i.e. Batch 1. On the other hand, the activation energy for TTA increased on addition of additives, which points towards a relatively lower temperature sensitivity. Table 2B suggests that the percent transmission of Batch 3 samples had a marked decrease in temperature sensitivity as compared to Batch 1, but an increased temperature sensitivity as compared to Batch 2. For ascorbic acid, Batch 1 showed the least temperature sensitivity while Batch 2 had the highest temperature sensitivity, and Batch 3 occurred to be less sensitive to temperature when compared to Batch 1. The reason for such low temperature sensitivity for Batch 1 is due to less amount of ascorbic acid present initially in tender coconut water which might not be readily available for reactions. Alternatively, the number of ascorbic acid molecules cold also be low, causing lesser collision in solution and thus, an increased activation energy is noted (Ouellette & Rawn et al., 2018). On the other side, the added ascorbic acid in Batch 2 along with microbial interactions was expected to be a major cause of such high temperature sensitivity for changes in ascorbic acid content as more ascorbic acid molecules suggests greater number of collisions, leading to lower activation energy. Further, activation energy for ascorbic acid is also supposed to be dependent upon the total soluble solids as well as the mechanism of ascorbic acid degradation, as suggested in the works of Robertson and Samaniego-Esguerra (1990) in lemon juice. While comparing activation energies for total titratable acidity, we observed that Batch 2 was least temperature sensitive followed by Batch 1 and then Batch 3. The total color change sensitivity to temperature was high for Batch 3 which was followed by Batch 2 and Batch 1.

extend the shelf-life of tender coconut water. Cold plasma treatment for 120 s with air and M65 plasma followed by a post refrigeration storage of 24 h had a limited effect on reducing the bacterial populations. Addition of citric acid followed by plasma treatment with M65 as a working gas had a significant effect on inactivation of E. coli and L. monocytogenes, allowing to achieve the minimum 5 log10 reduction of microbial strains, as specified by the US-FDA. The cellular morphology also showed some interesting changes in cellular structure of Grampositive and Gram-negative bacteria showing structure collapse and shrinkage upon cold plasma treatment. The plasma treatment had minimum effect on the parameters viz. pH, total soluble solids, Hunter color values and total titratable acidity of the coconut water. Low temperature storage is important for shelf-life extension of tender coconut water. Predictions based on Arrhenius equation were found comparable to experimental results, and a shelf-life of 48 days was computed for plasma treated tender coconut water with citric acid and ascorbic acid at 5 °C storage temperature. Rapid microbial spoilage is a major concern in tender coconut water processing and effectively inactivating microorganisms using cold plasma technology provides new opportunities for juice processors. Overall, the present work proves the promising nature of cold plasma technology as a non-thermal technology for tender coconut water processing. Conflict of interest Patent applied for with the application file no. PCT/US2017/03269. Acknowledgements Nikhil is thankful to the USIEF for Fulbright-Nehru Doctoral and professional research grant to work at Purdue University together with the Institute of International Education (IIE Grantee ID 15151385). References Alleyne, T., Roache, S., Thomas, C., & Shirley, A. (2005). The control of hypertension by use of coconut water and mauby: Two tropical food drinks. West Indian Medical Journal, 54, 3–8. Almeida, F. D. L., Cavalcante, R. S., Cullen, P. J., Frias, J. M., Bourke, P., Fernandes, F. A. N., et al. (2015). Effects of atmospheric cold plasma and ozone on prebiotic orange juice. Innovative Food Science and Emerging, 32, 127–135. DasPurkayastha, M., Kalita, D., Mahnot, N. K., Mahanta, C. L., Mandal, M., & Chaudhuri, M. K. (2012). Effect of L-ascorbic acid addition on the quality attributes of microfiltered coconut water stored at 4 oC. Innovative Food Science & Emerging Technologies, 16, 69–79. Davies, M. J. (2003). Singlet oxygen-mediated damage to proteins and its consequences. Biochemical and Biophysical Research Communications, 305(3), 761–770. Dezest, M., Bulteau, A. L., Quinton, D., Chavatte, L., Béchec, M. L., Cambus, J. P., et al. (2017). Oxidative modification and electrochemical inactivation of Escherichia coli upon cold atmospheric pressure plasma exposure. PLoS One, 12(3), e0173618 ff10.1371/journal.pone.0173618ff. ffhal-01504382f. Falck, D. C., Thomas, T., Falck, T. M., Tutuo, N., & Clem, K. (2000). The intravenous use of coconut water. The American Journal of Emerging Medicine, 18, 108–111. FDA (2001). Hazard analysis and critical control point (HACCP); Procedure for the safe and sanitary processing and importing of juice. Final rule. Federal Register, 66(13), 6137–6202. Gabriel, A. A., Aba, R. P. M., Tayamora, D. J. L., Colambo, J. C. R., Siringan, A. A. T., Rosario, L. M. D., et al. (2016). Reference organism selection for microwave atmospheric pressure plasma jet treatment of young coconut liquid endosperm. Food Control, 69, 74–82. Ganje, M., Jafari, S. M., Dusti, A., Dehnad, D., Amanjani, M., & Ghanbari, V. (2016). Modelling quality changes in tomato paste containing microencapsulated olive leaf extract by accelerated shelf life testing. Food and Bioproduct processing, 97, 12–19. Han, Y., & Linton, R. H. (2004). Fate of Escherichia coli O157:H7 and Listeria monocytogenes in strawberry juice and acidified media at different pH values and temperatures. Journal of Food Protection, 67(11), 2443–2449. Han, L., Patil, S., Boehm, D., Milosavljevic, V., Cullen, P. J., & Bourke, P. (2016). Mechanisms of inactivation by high-voltage atmospheric cold plasma differ for Escherichia coli and Staphylococcus aureus. Applied and Environment Microbiology, 82(2), 450–458. Haseena, M., Kasturi, K. V., & Padmanabhan, S. (2010). Post-harvest quality and shelf-life of tender coconut. Journal of Food Science & Technology, 47(6), 686–689. Hong, Y. F., Kang, J. G., Lee, H. Y., Uhm, H. S., Moon, E., & Park, Y. H. (2009). Sterilization effect of atmospheric plasma on Escherichia coli and Bacillus subtilis endospores. Letters in Applied Microbiology, 48(1), 33–37.

3.8. Validation of ASLT model at 5 °C The result of ASLT procedure is given in Table 2C. The rate constants for the parameters for Batch 3 stored at 5 °C is compared with the calculated rate constant values predicted by Arrhenius equation at 5 °C. The results show that the values are quite close to each other. Equation (5) was applied for predicting the number of days needed to reach the experimentally determined values for light transmission, TTA and ascorbic acid, while equation (4) was employed for color. The data showed that Arrhenius equation can effectively predict the changes in parameters like light transmission, ascorbic acid content, total titratable acidity and color. Again, for predicting the shelf life of the plasma treated coconut water with additives i.e. Batch 3, we considered that a 75% degradation of ascorbic acid is a cut-off value for shelf-life of the product. It is worth recalling that at this level of ascorbic acid degradation, an unacceptable brownish color develops in the product. Consequently, considering 75% ascorbic acid degradation in product at a storage temperature of 5 °C and applying equation (5), a shelf-life of 48 days for the ACP treated product was predicted, beyond which the product can be considered not suitable for commerce. 4. Conclusion In this work, we explored the application of atmospheric cold plasma technology as a nonthermal decontamination intervention to 11

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Muhammad, A. I., Li, Y., Liao, X., Liu, D., Ye, X., Chen, S., et al. (2019). Effect of dielectric barrier discharge plasma on background microflora and physicochemical properties of tiger nut milk. Food Control, 96, 119–127. Nursten, H. (2005). The maillard reaction: Chemistry, biochemistry and implications (1st ed.). Cambridge, U.K: Royal Society of Chemistry. Oehmigen, K., Hähnel, M., Brandenburg, R., Wilke, C., Weltmann, K.-D., & von Woedtke, T. (2010). The role of acidification for antimicrobial activity of atmospheric pressure plasma in liquids. Plasma Processes and Polymers, 7(3–4), 250–257. Okudu, H. O., & Ene-Obong, H. N. (2015). Evaluation of the effect of storage time and temperature on some physicochemical properties of juice and jam developed from two varieties of monkey kola (Cola parchycarpa, Cola lepidota). African Journal of Food Science and Technology, 6(7), 194–203. Otsuka, M., Kurata, T., & Arakawa, N. (1968). Isolation and characterization of an intermediate product in the degradation of 2,3-diketo-L-gulonic acid. Agricultural & Biological Chemistry, 50, 531–533. Ouellette, R., & Rawn, J. D. (2018). Organic chemistry: Structure, mechnaism, synthesis (2nd ed.). U.S: Academic Press. Pankaj, S. K., Wan, Z., Colonna, W., & Keener, K. M. (2017). Effect of high voltage atmospheric cold plasma on white grape juice quality. Journal of the Science of Food and Agriculture, 97(12), 4016–4021. Pankaj, S. K., Wan, Z., & Keener, K. M. (2018). Effect of cold plasma on food quality: A review. Foods, 7(4), https://doi.org/10.3390/foods7010004. Polydera, A. C., Stofors, N. G., & Taokuis, P. S. (2003). Comparative shelf life study and vitamin C loss kinetics in pasteurised and high pressure processed reconstituted orange juice. Journal of Food Engineering, 60, 21–29. Prochnow, A. M., Clauson, M., Hong, J., & Murphy, A. B. (2016). Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma. Scientific Reports, 6, 38610. https://doi.org/10.1038/srep38610. Ranganna, S. (1986). Handbook of analysis and quality control for fruits and vegetable products (2nd ed.). New Delhi: Tata McGraw Hill. Robertson, G. L., & Samaniego-Esguerra, C. M. (1990). Effect of soluble solids and temperature on ascorbic acid degradation in lemon juice stored in glass bottles. Journal of Food Quality, 13, 361–374. Sadasivam, S., & Manickam, A. (2007). Biochemical methods (3rd ed.). New Delhi: New Age International Publishers. Sapie, L., & Hwa, L. (2014). Study on the kinetics of vitamin C degradation in fresh strawberry juices. Procedia Chemistry, 9, 62–68. Shi, X. M., Zhang, G. J., Wu, X. L., Li, Y. X., Ma, Y., & Shao, X. J. (2011). Effect of lowtemperature plasma on microorganism inactivation and quality of freshly squeezed orange juice. IEEE Transactions on Plasma Science, 39(7), 1591–1597. Surowsky, B., Frohling, A., Gottschalk, N., Schluter, O., & Knorr, D. (2014). Impact of cold plasma on Citrobacter freundii in apple juice: Inactivation kinetics and mechanisms. International Journal of Food Microbiology, 174, 63–71. Van Gils, C. A. J., Hofmann, S., Boekema, B. K. H. L., Brandenburg, R., & Bruggeman, P. J. (2013). Mechanisms of bacterial inactivation in the liquid phase induced by a remote RF cold atmospheric pressure plasma jet. Journal of Physics D: Applied Physics, 46(17), 175203. Vojdani, J. D., Beuchat, L. R., & Tauxe, R. V. (2008). Juice-associated outbreaks of human illness in the United States, 1995 through 2005. Journal of Food Protection, 71(2), 356–364. Weidinger, A., & Kozlov, A. (2015). Biological activities of reactive oxygen and nitrogen species: Oxidative stress versus signal transduction. Biomolecules, 5(2), 472–484. Xu, L., Garner, A. L., Tao, B., & Keener, K. M. (2017). Microbial inactivation and quality changes in orange juice treated by high voltage atmospheric cold plasma. Food and Bioprocess Technology, 10(10), 1778–1791. Yong, J. W. H., Ge, L., Ng, Y. F., & Tan, S. N. (2009). The chemical composition and biological properties of coconut (Cocos nucifera L.) water. Molecules, 14, 5144–5164.

Jackson, J. C., Gordon, A., Wizzard, G., McCook, K., & Rolle, R. (2004). Changes in chemical composition of coconut (Cocos nucifera) water during maturation of the fruit. Journal of the Science of Food and Agriculture, 84, 1049–1052. Korachi, M., Ozen, F., Aslan, N., Vannini, L., Guerzoni, M. E., Gottardi, D., et al. (2015). Biochemical changes to milk following treatment by a novel, cold atmospheric plasma system. International Dairy Journal, 42, 64–69. Kovacevi, D. B., Putnik, P., Dragovic-Uzelac, V., Pedisic, S., Rezek-Jambrak, A., & Herceg, Z. (2016). Effects of cold atmospheric gas phase plasma on anthocyanins and color in pomegranate juice. Food Chemistry, 190, 317–323. Kramida, A., Ralchenko, Y., Reader, J., & NIST ASD team (2015). NIST atomic spectra database (ver.5.3). Gaithersburf, MD: National Institute of Standards and Technology. [Online.]. Available: http://physics.nist.gov/asd 2017 January 26 . Laroussi, M., & Leipold, F. (2004). Evaluation of the roles of reactive species, heat, and UV radiation in the inactivation of bacterial cells by air plasmas at atmospheric pressure. International Journal of Mass Spectrometry, 233(1–3), 81–86. Laroussi, M., Mendis, D. A., & Rosenberg, M. (2003). Plasma interaction with microbes. New Journal of Physics, 5, 41.1–41.10. Liao, X., Li, J., Muhammad, A. I., Suo, Y., Chen, S., Ye, X., et al. (2018). Application of a dielectric barrier discharge atmospheric cold plasma (DBD-ACP) for Eshcerichia coli inactivation in apple juice. Journal of Food Science, 83(2), 401–408. Liao, X., Liu, D., Xiang, Q., Ahn, J., Chen, S., Ye, X., et al. (2017). Inactivation mechanisms of non-thermal plasma on microbes: A review. Food Control, 75, 83–91. Liu, Z. C., Liu, D. X., Chen, C., Li, D., Yang, A. J., Rong, M. Z., et al. (2015). Physicochemical processes in the indirect interaction between surface air plasma and deionized water. Journal of Physics D: Applied Physics, 48(49), 495201. https://doi. org/10.1088/0022-3727/48/49/495201. Lu, H., Patil, S., Keener, K. M., Cullen, P. J., & Bourke, P. (2014). Bacterial inactivation by high-voltage atmospheric cold plasma: Influence of process parameters and effects on cell leakage and DNA. Journal of Applied Microbiology, 116(4), 784–794. Mahnot, N. K., Kalita, D., Mahanta, C. L., & Chaudhuri, M. K. (2014). Effect of additives on the quality of tender coconut water processed by nonthermal two stage microfiltration technique. LWT-Food Science and Technology, 59(2), 1191–1195. Mahnot, N. K., Mahanta, C. L., Keener, K. M., & Misra, N. N. (2019). Strategy to achieve a 5-log Salmonella inactivation in tender coconut water using high voltage atmospheric cold plasma (HVACP). Food Chemistry, 284, 303–311. Ma, T., & Lan, W. (2015). Effects of non-thermal plasma sterilization on volatile components of tomato juice. International journal of Environmental Science and Technology, 12, 3767–3772. Manso, M. C., Oliveira, F. A. R., Oliveira, J. C., & Frias, J. M. (2001). Modelling ascorbic acid thermal degradation and browning in orange juice under aerobic conditions, International. Journal of Food Science & Technology, 36, 303–312. Misra, N. N., Keener, K. M., Bourke, P., & Cullen, P. J. (2015). Generation of in-package cold plasma and efficacy assessment using methylene blue. Plasma Chemistry and Plasma Processing, 35, 1043–1056. Misra, N. N., Martynenko, A., Chemat, F., Paniwnyk, L., Barba, F. J., & Jambrak, A. R. (2018). Thermodynamics, transport phenomena, and electrochemistry of external field-assisted nonthermal food technologies. Critical Reviews in Food Science and Nutrition, 58, 1832–1863. Misra, N. N., Sullivan, C., Pankaj, S. K., Alvarez-Jubete, L., Cama, R., Jacoby, F., et al. (2014). Enhancement of oil spreadability of biscuit surface by nonthermal barrier discharge plasma. Innovative Food Science & Emerging Technologies, 26, 456–461. Misra, N. N., Yepez, X., Xu, L., & Keener, K. (2019). In-package cold plasma technologies. Journal of Food Engineering, 244, 21–31. Mokrzycki, W., & Tatol, M. (2011). Color difference Delta E- A survey. Machine Graphics And Vision, 20(4), 383–412. Montenegro, J., Ruan, R., Ma, H., & Chen, P. (2002). Inactivation of E. coli O157: H7 using a pulsed nonthermal plasma system. Journal of Food Science, 67(2), 646–648.

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