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Effects of the photosensitizer curcumin in inactivating foodborne pathogens on chicken skin
Food Control 109 (2020) 106959
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Effects of the photosensitizer curcumin in inactivating foodborne pathogens on chicken skin
T
Jingwen Gao, Karl R. Matthews∗ Department of Food Science, Rutgers University, New Brunswick, NJ, 08901, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Photosensitizer Curcumin Chicken safety Listeria monocytogenes Salmonella
This study investigated the antimicrobial efficacy of the water-soluble photosensitizer curcumin (PSC) on liquid media and chicken skin. The water-soluble PSC showed strong absorption at 410 nm, and the light-emitting diode (LED) used to activate PSC had strong emission at 430 nm with power density of 107 W/m2. Listeria monocytogenes (3 isolates) and Salmonella (8 isolates) were evaluated in this study. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of PSC for L. monocytogenes were 10 ppm and 20 ppm, respectively. For Salmonella, 200 ppm of PSC led to maximum 3.6 log reduction. No significant differences for antimicrobial activities among different incubation (1, 2.5, and 5 min) or light dose (6.4, 32.1, and 64.2 kJ/m2) were observed. Compared with the water control, no significant color change was observed on chicken skin exposed to illumination equal to or greater than 32.1 kJ/m2. After 5-min incubation followed by 32.1 kJ/m2 of illumination, treatment with 300 ppm of PSC resulted in 2.9 and 1.5 log CFU/cm2 of L. monocytogenes and Salmonella on chicken skin, respectively. PSC showed equivalent or better efficacy in reducing the population of foodborne pathogens on chicken skin, as compared with a commercial antimicrobial containing 300 ppm of peracetic acid. This study suggests that PSC effectively inactivates pathogens on media and chicken skin without causing skin discoloration, indicating a potential application as an antimicrobial intervention in the poultry industry.
1. Introduction Poultry consumption continues to increase globally. In 2018, the global consumption of poultry was estimated to be around 93 million tons, with an expected 2% increase in the poultry production in 2019 (USDA, 2019). Although poultry is seldom consumed raw, it carries a high safety risk as it provides optimum conditions for bacterial growth: high water activity, near neutral pH, and abundant nutrients. These conditions increase the survival and growth of bacteria, perhaps exacerbating cross-contamination (Dave & Ghaly, 2011). From 1998 to 2008, foodborne outbreaks associated with poultry resulted in the greatest number of deaths (19%), which were two times higher than for leafy vegetables (Painter et al., 2013). It was reported that Listeria monocytogenes and Salmonella spp. were the main contributors, accounting for 63% and 26% of poultry-associated deaths. Chlorine and acids are the two most common chemical interventions to decontaminate the surfaces of poultry carcasses (Sohaib, Anjum, Arshad, & Rahman, 2016). However, high concentrations of chlorine and acids may result in off-flavor, discoloration, equipment corrosion, and other problems. The EU has prohibited the import of
∗
poultry products that are treated with chlorine, trisodium phosphate, and peracetic acid; leading to an estimated loss of $200 - $300 million annually (Johnson, 2015). The concept of natural antimicrobials has become more and more popular; and served as a driver of the present study to search for natural antimicrobials that can replace or augment conventional chemical interventions. Photoinactivation using photosensitizers has been widely studied in clinical medicine as a potential treatment of bacterial infections such as skin diseases and cavities (Niu, Tian, Cai, Ren, & Wei, 2015; Ricci Donato et al., 2017). Photoinactivation of microorganism is based on the activation of a photosensitizer by light exposure at a predetermined wavelength. The excited photosensitizer molecules transfer the excessive protons/electrons or energy to surrounding substances, resulting in generation of reactive oxygen species (ROS). ROS oxidize lipid membranes, proteins, and nucleic acids, leading to cell death. Since ROS have an extremely short half-life and cause non-specific damage to bacterial cells, it is difficult for bacteria to trigger defense systems or develop resistance to photoinactivation (Maisch, 2015). A number of photosensitizers have been evaluated and demonstrate photoinactivation against a variety of microorganisms on food or food-
Corresponding author. E-mail address: [email protected] (K.R. Matthews).
https://doi.org/10.1016/j.foodcont.2019.106959 Received 1 July 2019; Received in revised form 18 October 2019; Accepted 18 October 2019 Available online 22 October 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
Food Control 109 (2020) 106959
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2.4. MIC and microtiter plate assay
contact surfaces (D'Souza et al., 2015). Curcumin, which can be extracted naturally from Curcuma longa plants, is one of the most wellstudied photosensitizers. Studies showed that the photosensitizer curcumin achieved a 5-log reduction of Vibrio parahaemolyticus on oysters without promoting lipid oxidation (Liu et al., 2016; Wu et al., 2016). PSC may be part of a novel approach to improve the microbial safety of poultry. A potential limitation of curcumin is its low solubility in water, purified curcumin is typically dissolved in ethanol with or without a solubility enhancer such as dimethyl sulfoxide (DMSO), and then diluted with distilled water. However, curcumin readily precipitates as the concentration of ethanol decreases. Use of water-soluble photosensitizer curcumin (PSC) would eliminate limitations in the use of curcumin in commercial poultry processing. The goal of this study was to investigate the activity of PSC in inactivating L. monocytogenes and Salmonella on media and chicken skin. The effects of incubation time and light dose on antimicrobial activity and chicken skin color were evaluated to optimize the photoinactivation conditions.
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the microdilution broth method (CLSI, 2016). Light controls were prepared by illuminating cultures in Mueller-Hinton broth (MHB Difco, Becton Dickinson, Sparks, MD). The MIC was defined as the lowest concentration of PSC that inhibits visible growth after 16 h incubation. The MBC was determined by plating 100 μl of the suspension from each well onto TSA, and it was defined as the lowest concentration showing the absence of growth on TSA after 20 h incubation at 37 °C. Each concentration of PSC was tested in replicated wells. The bacterial population after PSC treatment was determined using a microtiter plate assay. In brief, inoculum was prepared as described and diluted to achieve 104 CFU per well. The culture was mixed with an equal volume of PSC. The culture was incubated with different concentrations of PSC for 5 min, followed by illumination (64.2 kJ/m2). A hundred microliters were removed from a well and spread plated on TSA supplemented with appropriate antibiotics. Plates were incubated at 37 °C for 18–20 h. For L. monocytogenes, experiments were conducted in a walk-in cooler (9.3 ± 0.4 °C). Triplicate wells were prepared for each sample in a single experiment.
2. Materials and methods 2.1. Chemicals and bacterial strains Water-soluble photosensitizer curcumin (PSC; uC3 Clear) was a kind gift from Sabinsa Corp. (East Windsor, NJ). It was dissolved and diluted using sterile distilled water (SDW). Peracetic acid antimicrobial (PAA; Spectrum®) was kindly provided by PeroxyChem (Philadelphia, PA). PAA was diluted using SDW, and the PAA concentration was confirmed using test strips (Micro Essential Laboratory Inc., Brooklyn, New York). Three strains of L. monocytogenes were obtained from Dr. Joshua Gurtler (Eastern Region Research Center, USDA, Wyndmoor, PA): L008 (serotype 4b), L2624 (serotype 1/2b), and L2625 (serotype 1/2a). Eight strains of Salmonella were obtained from Dr. Donald Schaffner (Rutgers University, New Brunswick, NJ): S. Thompson (S8), S. Hadar (S20), S. Typhimurium Copenhagen (S21), S. Montevideo (S24), S. Typhimurium (S25), S. Saintpaul (S26), S. Heidelberg (S40), and S. Heidelberg (107a). All L. monocytogenes and Salmonella strains were resistant to 100 μg/ml of nalidixic acid (Alfa Aesar, England) and 50 μg/ml streptomycin (MP Biomedicals), respectively. All media used to culture bacteria were supplemented with appropriate antibiotics unless otherwise noted. Stock cultures were stored at −80 °C in tryptic soy broth (TSB; Difco, Becton Dickinson, Sparks, MD) containing 20% glycerol. To prepare the inoculum, an isolated colony from tryptic soy agar (TSA; Difco, Becton Dickinson, Sparks, MD) was transferred to 30 ml of brain heart infusion (BHI; Difco, Becton Dickinson, Sparks, MD) broth for L. monocytogenes or TSB for Salmonella, followed by 20 h incubation at 37 °C. The culture was centrifuged at 4000×g for 10 min and washed with 0.1% sterile peptone water (SPW; Difco, Becton Dickinson, Sparks, MD) and suspended in SDW.
2.5. Treatment of chicken skin Chicken was purchased from a local market. Pieces of chicken skin 5 × 5 cm2 were carefully removed using a sterile scalpel and immobilized on a stainless-steel wire mesh that was then placed inside a petri dish. A 10 μl volume of inoculum, either L. monocytogenes cocktail (L008, L2624, and L2625) or Salmonella cocktail (S20 and 170a), was evenly spread on the chicken skin. Chicken skin samples were dried for 5 min with the lid of the petri dish ajar, and then kept for an additional 25 min with the lid covered. Solutions of SDW, 300 ppm PSC, or 300 ppm PAA were kept in the refrigerator for at least 30 min for cooling (12.7 ± 0.5 °C), and each treatment contained 300 ml of solution. Foil was used to protect PSC from natural light before use. Two pieces of inoculated chicken skin were dipped in 300 ml of a treatment for 5 min, followed by illumination (32.1 kJ/m2). Each skin sample was allowed to drip for 5 min before being placed in a sterile plastic bag containing 25 ml of sterile phosphate buffered saline (PBS; VWR International, LLC.). Each sample was stomached for 2 min. The homogenized sample was serial diluted (1:10) with SPW and plated (100 μl) on modified oxford agar (MOX; Difco, Becton Dickinson, Sparks, MD) or XLT-4 (Becton Dickinson, Sparks, MD) in duplicate. The plates were incubated at 37 °C for 24 h. 2.6. Incubation time and light dose The L. monocytogenes cocktail (L008, L2624, and L2625) inoculum was diluted to approximate 105 CFU/ml. The concentration of PSC was 300 ppm for studies using media and chicken skin. Incubation time (1, 2.5, and 5 min) was studied by fixing the light dose at 64.2 kJ/m2; meanwhile, light doses (0, 6.4, 32.1, and 64.2 kJ/m2) were studied using 5-min contact time. The experiment was performed in a walk-in cooler. To evaluate efficacy in media experiments were conducted in a 96well plate. A 100 μl aliquot of sample was plated in duplicate on TSA supplemented with appropriate antibiotic, followed by 20 h incubation at 37 °C. For chicken skin studies, the same method of inoculation, PSC treatment, and microbiological analysis was used as described previously.
2.2. Absorption spectrum of PSC The absorption spectrum of PSC was determined using UV–Vis spectroscopy (Cary 60 Spectrophotometer, Agilent Technologies, Santa Clara, CA). Quartz cuvettes (1 cm light path) were used. 2.3. Light source The light box unit was evaluated to have a peak wavelength near 430 nm. The light box unit was composed of six solderless exotic LED (7.2 W; LED Group Buy, Saint Louis, MO) evenly distributed across the interior top. All sides of the light box unit were made of high polished stainless steel. The power density of the light box unit was measured using a spectroradiometer (Model PS-300, Apogee Instruments, Inc., Logan, UT), based on the average of five readings. Light dose was calculated by multiplying power density by illumination time.
2.7. Color measurement Pieces of 5 × 5 cm2 chicken skin with approximate 3-mm thickness were carefully excised using a sterile scalpel. Skin samples were dipped 2
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into 300 ml of 300 ppm PSC for 5 min and subjected to predetermined light doses. Excessive blood or PSC solution on chicken skin were gently removed using tissue paper. Values of L*, a*, and b* were measured using a colorimeter (Konica Minolta CR410, Osaka, Japan). Each treatment was conducted in triplicate, and the final results were reported as the average of five readings obtained from different locations of each chicken skin sample. Color difference before and after treatment were described as the values calculated by Eq. (1) to Eq. (5). The subscript “o” in the equation refers to the color reading of the skin before treatment.
ΔL* = L* − L*o
1
Δa * = a * − a *o
2
Δb* = b* − b*o
3
Total color difference (ΔE) =
Table 1 MIC and MBC of PSC on Listeria monocytogenes. Bacterial Strain L. monocytogenes
L008 L2624 L2625
MIC (ppm)
MBC (ppm)
10 10 10
20 20 20
†Light dose: 64.2 kJ/m2.
(L* − L*o)2 + (a * − a *o)2 + (b* − b*o)2 4
Whiteness Index (WI) = 100 − (100 −
* 2 L)
+
(a *)2
+
(b*)2
5
2.8. Statistical analysis All experiments were conducted three times independently. The mean values were compared by ANOVA and Duncan's post hoc analysis using a SAS software (SAS university edition, SAS Institute Inc., USA). P < 0.05 was considered as a significant difference. 3. Results and discussion
Fig. 2. Population of L. monocytogenes on media after 5-min incubation with PSC, followed by 64.2 kJ/m2 of illumination. Dotted line represents the limit of detection.
3.1. Properties of water-soluble PSC and light source Fig. 1 shows the absorption spectrum of PSC at two concentrations. Two absorption maxima were identified: 260 nm and 410 nm. The strongest absorption maximum within visible wavelength was at 410 nm, which was consistent with published literature (Priyadarsini, 2009) The power density of the light apparatus used in this study was 107 W/m2. As a result, light doses of 6.4, 32.1, and 64.2 kJ/m2 corresponded to 1, 5 and 10 min of light exposure.
64.2 kJ/m2 of light exposure, are shown in Table 1. All three L. monocytogenes isolates shared the same MIC and MBC of 10 ppm and 20 ppm, respectively. For samples treated at cooler temperature, no colonies were observed following exposure to 30 ppm and 300 ppm of PSC (Fig. 2). Under same dose of light exposure (64.2 kJ/m2), PSC at 1/ 2 × MIC (5 ppm) achieved an approximate 3-log reduction of L. monocytogenes, a decrease which is generally considered an effective antimicrobial treatment (Anonymous, 2019). However, no inactivation was observed in the absence of light exposure, regardless of the PSC concentrations evaluated (data not shown). The results suggest that illumination was essential for PSC to induce inhibitory effects at low concentration. Previous studies showed that curcumin alone can inhibit a variety of microorganisms without light exposure, including L. monocytogenes, E. coli O157:H7, Salmonella Typhimurium, and methicillin-resistant Staphylococcus aureus (Kim et al., 2005; Niamsa & Sittiwet, 2009). The MIC of purified curcumin varied with different bacterial strains, but it was typically above 1000 ppm if no light activation was applied (Kim et al., 2005; Negi, Jayaprakasha, Jagan Mohan Rao, & Sakariah, 1999); a concentration much greater than the highest concentration evaluated in this study (300 ppm). In other words, a lower concentration of curcumin is required for bacterial inhibition/ inactivation when light of the appropriate wavelength is applied. Importantly, using a lower concentration would both reduce the cost of the antimicrobial solution and any adverse effects on chicken skin color. In addition to light exposure, 0 ppm of PSC without illumination did not reduce the bacterial population, suggesting the presence of PSC was essential to elicit an antimicrobial effect. It was reported that 430 nm light (123.3 J/cm2) resulted in significant reduction of L. monocytogenes compared with a dark (no light exposure) control (Endarko, Maclean, Timoshkin, MacGregor, & Anderson, 2012). However, the light dose used in the present study was much lower, which may in part explain the lack of photoinactivation.
3.2. Antimicrobial activity of PSC on media The MIC and MBC of the water-soluble PSC used in this study, after
Fig. 1. Absorption spectrum of PSC. 3
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Fig. 3. Population of Salmonella spp. on media after 5-min incubation with PSC, followed by 64.2 kJ/m2 of illumination. Asterisk indicates significant difference between PSC concentrations for individual strain (P < 0.5). Dotted line represents the limit of detection.
In terms of Salmonella, PSC at 200 ppm exhibited the greatest antimicrobial activity, resulting in 1.8–3.6 log reduction of Salmonella (Fig. 3). Interestingly, the antimicrobial efficacy of photoinactivation was lower at higher concentrations of PSC solution (2000 ppm). The decrease in efficacy of higher PSC concentrations may be associated with a self-shielding effect of light (Barr, MacRobert, Tralau, Boulos, & Bown, 1990). Curcumin at lower concentration is able to absorb light energy and be exited, leading to greater generation of ROS. However, in a highly concentrated solution, a large number of solutes (curcumin molecules) block the light source, interfering with the process of light activation. L. monocytogenes was completely inactivated when exposed to 300 ppm PSC which was not the case for Salmonella strains. Differences associated with antimicrobial activity of PSC against Gram-positive and Gram-negative bacteria may be related to differences in the cellular membranes. Photosensitizers bind and penetrate through the cell membrane of Gram-positive bacteria more readily since the peptidoglycan layer is more porous than the outer-inner membrane complex of Gram-negative bacteria (Dai, Huang, & Hamblin, 2009; Minnock et al., 2000). To improve the antimicrobial efficacy of PSC on gramnegative bacteria, two approaches are commonly applied – mixing with EDTA or increasing the positive charge of the photosensitizer (Malik, Ladan, & Nitzan, 1992). Further enhancement of the efficacy of PSC may be realized by applying either of the approaches to develop a foodgrade antimicrobial curcumin photosensitizer that is safe, efficacious, and cost effective. The antimicrobial activity of photoinactivation by PSC was found to be strain dependent. For example, 200 ppm of PSC resulted in a 1.8 log reduction of Salmonella S8, which was significantly (P < 0.05) less than the 3.6 log reduction of Salmonella S26 (Fig. 3). Specific reasons for differences in strain susceptibility are still debated. It was proposed that strain differences in susceptibility may be related to the amount of capsular polysaccharides and the ability of a strain to form a biofilm protecting the cell (Grinholc, Szramka, Kurlenda, Graczyk, & Bielawski, 2008); and the production of extracellular slime (Gad, Zahra, Hasan, & Hamblin, 2004). Previous studies focused on susceptibility of different strains of S. aureus, especially differences between methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) (Grinholc et al., 2008; Maisch, Bosl, Szeimies, Lehn, & Abels, 2005; Szpakowska, Lasocki, Grzybowski, & Graczyk, 2001). Greater research to elucidate strain differences in PSC sensitivity would aid in development of more
Fig. 4. Microbial count of a) L. monocytogenes and b) Salmonella on chicken skin after 5-min incubation with PSC, followed by 32.1 kJ/m2 of illumination. Dotted line represents the limit of detection.
efficacy treatments and practices.
3.3. Antimicrobial activities of PSC on chicken skin The efficacy of PSC in reducing the population of L. monocytogenes differed when evaluated on chicken skin and in liquid media (Fig. 4a). Fats and the complex surface (nooks and crannies) on chicken skin may interfere with the interaction between light and PSC, likely contributing to decreased efficacy. In addition, PSC in water has low viscosity, decreasing the ability of PSC to attach to the chicken skin (Tortik, Spaeth, & Plaetzer, 2014). Although PSC was less effective on chicken skin; nevertheless, a 2.9 log reduction of L. monocytogenes on chicken skin was achieved. The level of reduction achieved in this study is equal to or greater than that of antimicrobials presently used in processing of poultry carcasses and parts. Compared with water and PAA treatments, PSC showed the greatest reduction of L. monocytogenes and Salmonella on chicken skin (Fig. 4). PAA is one of the most common antimicrobials used by the poultry industry (McKee, 2011). PAA is effective at acid and slightly alkaline pH and retains efficacy in presence of organic matter which has permitted its use in place of chlorine (Wideman et al., 2015). In this study, the effect of PSC and PAA on reducing the population of Salmonella was not significantly different. Both treatments were significantly better than no treatment and the water alone treatment of chicken skin (Fig. 4). PSC and PAA provided similar level of control of L. monocytogenes and Salmonella, however, the advantage of PSC is that it can 4
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Fig. 6. Microbial count of L. monocytogenes after treatment with 5-min incubation time and different light doses using a) media, and b) chicken skin. Dash line indicates an estimated trend based on the obtained results. Empty circle indicates that the microbial count in the liquid media experiment was below detection limit (0.7 log CFU/ml).
Fig. 5. Microbial count of L. monocytogenes after treatment with different incubation time and 64.2 kJ/m2 of illumination using a) media, and b) chicken skin. Dash line indicates an estimated trend based on the obtained results. Empty circle indicates that the microbial count in the liquid media experiment was below detection limit (0.7 log CFU/ml).
compared to 30 min incubation, but there was not a large difference between 0 min and 5 min incubation (Hamblin et al., 2002). This is consistent with the results of the present study (Fig. 5a). Consequently, poultry carcasses can be dipped into PSC solution for as short as 1 min which would facilitate integration into current commercial poultry processing practices. In theory, light dose influences the amount of PSC being activated, and thus the production of ROS. Murdoch, Maclean, Endarko, MacGregor, and Anderson (2012) found that photoinactivation of various bacteria by 405 nm LED depended on the light dose. Similarly, Gios et al. (2010) found that applying 20 J/cm2 of light induced significantly greater reduction of S. aureus than 40 J/cm2. In this study, a 300 ppm PSC solution achieved an approximate 5-log reduction of L. monocytogenes (Fig. 6a), regardless of the light dose. Similarly, PSC exposed to 32.1 kJ/m2 of light resulted in an approximate 2-log reduction of L. monocytogenes on chicken skin, which was not affected by light dose (Fig. 6b). In other words, under the experimental conditions of this study, the antimicrobial efficacy of PSC was not influenced by the light dose. A similar phenomenon was reported where 8000 μM of curcumin combined with 24 J/cm2 and 48 J/cm2 achieved similar log reduction of Streptococcus mutans (Paschoal et al., 2013). Research is warranted to
be extracted from a natural source, which satisfies the increasing demands of sustainability and clean labeling. There are limited studies focused on the food application of photosensitizer curcumin, either a purified or modified form. Studies addressing the feasibility of using photosensitizer to improve the safety of poultry products are warranted based on the results of the present study. For example, studies may focus on application of PSC on whole poultry carcasses and parts and best approach for incorporating the technology into existing poultry processing operations.
3.4. Effects of incubation time and light dose on photoinactivation ROS have a short half-life, so it is critical to have photosensitizer and bacterial cells in close proximity. In short, the closer the proximity of the photosensitizer to the bacterial cell the more likely the ROS will negatively impact cell integrity. In this study, no significant difference was observed in the antimicrobial activity of PSC when photoinactivation between 1 min and 5 min incubation time for broth and chicken skin (Fig. 5). It was reported that survival of S. aureus and E. coli incubated with photosensitizer polylysine-ce6 for 5 min was lower 5
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Fig. 7. Effects of light dose on a) ΔL*/Δa*/Δb*, b) total color difference (ΔE), and c) whiteness index (WI) of chicken skin. Incubation time was fixed at 5 min for all the samples. Distilled water control (DW) was performed. Letters of “A, B”, “a, b”, and “x, y” represent significant difference in ΔL*, Δa*, and Δb* respectively. Asterisk indicates a significant difference of WI before and after treatment.
observed between samples treated with 32.1 kJ/m2 or greater light exposure and water controls, suggesting it is difficult for consumers to recognize the color difference after PSC treatment with longer light exposure. The mechanism of photoinactivation is based on oxidation by ROS, which can also oxidize pigment compounds and thus bleach food surfaces. Samples without illumination had significantly lower WI after treatment when compared with untreated samples (Fig. 7c) associated with an increase in yellowness on chicken skin. However, all the other samples showed no significant differences for WI, suggesting that no bleaching occurred after up to 64.2 kJ/m2 of illumination.
delineate why there is a variance in the effect of light dose on photoinactivation. In addition, it should be noted that 32.1 kJ/m2 used in the present study corresponds to 5 min of light exposure. Using a higher power density of LEDs can further reduce the illumination time, which would increase energy savings. 3.5. Effects of light dose on the color of chicken skin The greatest difference observed among L*/a*/b* was b*, since PSC exhibited yellow color (Fig. 7a). Positive Δb* indicates that a sample becomes more yellow after dip treatment. A significant increase in Δb* was observed after dipping in PSC, but chicken skin subjected to light exposure showed no significant differences on ΔL* and Δa* when compared with DW controls. Interestingly, no significant differences on Δb* were observed between water control and chicken skin samples after treatment with light dose of 32.1 and 64.2 kJ/m2, indicating that the yellow color diminished following light exposure. This phenomenon was demonstrated in a previous study, where after light exposure a loss of yellow-orange color associated with riboflavin treatment of salmon was noted (Josewin, Ghate, Kim, & Yuk, 2018). Samples exposed to 6.4 kJ/m2 of light showed a significant increase in yellowness (Δb*), indicating that such a low light dose might not be appropriate due to the color change. However, recognizing that consumers in some countries prefer chicken with yellow skin, a low light dose with shorter illumination time could be applied to poultry in those countries and actually be advantageous from a quality perspective. Apparent color change also resulted in a significant difference in ΔE between PSC and DW control after 6.4 kJ/m2 of light exposure (Fig. 7b). The larger the ΔE value, the greater likelihood that consumers would recognize a color change between samples. However, no significant differences were
4. Conclusions This study demonstrates that PSC inactivated L. monocytogenes and Salmonella in liquid media and on chicken skin. Under the experimental conditions in this study, incubation time and light dose did not influence the antimicrobial activity of PSC, suggesting that photoinactivation can be achieved in a short time. It is important to develop a rapid antimicrobial intervention to satisfy the rapid line speeds used in the poultry industry. These results are encouraging and suggest that photoinactivation can be incorporated even on a fast-moving production line. Although chicken skin treated with PSC appeared slightly more yellow, there was no recognizable difference in chicken skin color after treatment with light dose greater or equal to 32.1 kJ/m2. Therefore, upon completion of PSC photoinactivation treated chicken could not be differentiated from non-treated chicken enhancing acceptability of the method and process.
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Declaration of competing interest
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We wish to confirm that there are no known conflicts of interest associated with this publication. References Anonymous. Instructions to authors. (2019). Retrieved from https://aac.asm.org/sites/ default/files/additional-assets/AAC-ITA.pdf, Accessed date: 10 May 2019 Accessed. Barr, H., MacRobert, A. J., Tralau, C. J., Boulos, P. B., & Bown, S. G. (1990). The significance of the nature of the photosensitizer for photodynamic therapy: Quantitative and biological studies in the colon. British Journal of Cancer, 62(5), 730. CLSI (2016). Performance standards for antimicrobial susceptibility testing. CLSI supplement M100S(29th ed.). Wayne, PA: Clinical and Laboratory Standards Institute. D'Souza, C., Yuk, H. G., Khoo, G. H., & Zhou, W. (2015). Application of light-emitting diodes in food production, postharvest preservation, and microbiological food safety. Comprehensive Reviews in Food Science and Food Safety, 14(6), 719–740. Dai, T., Huang, Y. Y., & Hamblin, M. R. (2009). Photodynamic therapy for localized infections–state of the art. Photodiagnosis and Photodynamic Therapy, 6(3–4), 170–188. https://doi.org/10.1016/j.pdpdt.2009.10.008. Dave, D., & Ghaly, A. E. (2011). Meat spoilage mechanisms and preservation techniques: A critical review. American Journal of Agricultural and Biological Sciences, 6(4), 486–510. Endarko, E., Maclean, M., Timoshkin, I. V., MacGregor, S. J., & Anderson, J. G. (2012). High-intensity 405 nm light inactivation of Listeria monocytogenes. Photochemical and Photobiological Sciences, 88(5), 1280–1286. https://doi.org/10.1111/j.1751-1097. 2012.01173.x. Gad, F., Zahra, T., Hasan, T., & Hamblin, M. R. (2004). Effects of growth phase and extracellular slime on photodynamic inactivation of gram-positive pathogenic bacteria. Antimicrobial Agents and Chemotherapy, 48(6), 2173–2178. https://doi.org/10. 1128/AAC.48.6.2173-2178.2004. Gois, M. M., Kurachi, C., Santana, E. J., Mima, E. G., Spolidorio, D. M., Pelino, J. E., et al. (2010). Susceptibility of Staphylococcus aureus to porphyrin-mediated photodynamic antimicrobial chemotherapy: An in vitro study. Lasers in Medical Science, 25(3), 391–395. https://doi.org/10.1007/s10103-009-0705-0. Grinholc, M., Szramka, B., Kurlenda, J., Graczyk, A., & Bielawski, K. P. (2008). Bactericidal effect of photodynamic inactivation against methicillin-resistant and methicillin-susceptible Staphylococcus aureus is strain-dependent. Journal of Photochemistry and Photobiology B: Biology, 90(1), 57–63. Hamblin, M. R., O'Donnell, D. A., Murthy, N., Rajagopalan, K., Michaud, N., Sherwood, M. E., et al. (2002). Polycationic photosensitizer conjugates: Effects of chain length and gram classification on the photodynamic inactivation of bacteria. Journal of Antimicrobial Chemotherapy, 49(6), 941–951. Johnson, R. (2015). U.S.-EU poultry dispute on the use of pathogen reduction treatments (PRTs). Congressional Research Service. Josewin, S. W., Ghate, V., Kim, M.-J., & Yuk, H.-G. (2018). Antibacterial effect of 460 nm light-emitting diode in combination with riboflavin against Listeria monocytogenes on smoked salmon. Food Control, 84, 354–361. https://doi.org/10.1016/j.foodcont. 2017.08.017. Kim, H., Park, B.-S., Lee, K.-G., Choi, C. Y., Jang, S. S., Kim, Y.-H., et al. (2005). Effects of naturally occurring compounds on fibril formation and oxidative stress of β-amyloid. Journal of Agricultural and Food Chemistry, 53(22), 8537–8541. Liu, F., Li, Z., Cao, B., Wu, J., Wang, Y., Xue, Y., et al. (2016). The effect of a novel photodynamic activation method mediated by curcumin on oyster shelf life and quality. Food Research International, 87, 204–210. https://doi.org/10.1016/j.foodres. 2016.07.012. Maisch, T. (2015). Resistance in antimicrobial photodynamic inactivation of bacteria. Photochemical and Photobiological Sciences, 14(8), 1518–1526. https://doi.org/10. 1039/c5pp00037h.
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Effects of the photosensitizer curcumin in inactivating foodborne pathogens on chicken skin
T
Jingwen Gao, Karl R. Matthews∗ Department of Food Science, Rutgers University, New Brunswick, NJ, 08901, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Photosensitizer Curcumin Chicken safety Listeria monocytogenes Salmonella
This study investigated the antimicrobial efficacy of the water-soluble photosensitizer curcumin (PSC) on liquid media and chicken skin. The water-soluble PSC showed strong absorption at 410 nm, and the light-emitting diode (LED) used to activate PSC had strong emission at 430 nm with power density of 107 W/m2. Listeria monocytogenes (3 isolates) and Salmonella (8 isolates) were evaluated in this study. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of PSC for L. monocytogenes were 10 ppm and 20 ppm, respectively. For Salmonella, 200 ppm of PSC led to maximum 3.6 log reduction. No significant differences for antimicrobial activities among different incubation (1, 2.5, and 5 min) or light dose (6.4, 32.1, and 64.2 kJ/m2) were observed. Compared with the water control, no significant color change was observed on chicken skin exposed to illumination equal to or greater than 32.1 kJ/m2. After 5-min incubation followed by 32.1 kJ/m2 of illumination, treatment with 300 ppm of PSC resulted in 2.9 and 1.5 log CFU/cm2 of L. monocytogenes and Salmonella on chicken skin, respectively. PSC showed equivalent or better efficacy in reducing the population of foodborne pathogens on chicken skin, as compared with a commercial antimicrobial containing 300 ppm of peracetic acid. This study suggests that PSC effectively inactivates pathogens on media and chicken skin without causing skin discoloration, indicating a potential application as an antimicrobial intervention in the poultry industry.
1. Introduction Poultry consumption continues to increase globally. In 2018, the global consumption of poultry was estimated to be around 93 million tons, with an expected 2% increase in the poultry production in 2019 (USDA, 2019). Although poultry is seldom consumed raw, it carries a high safety risk as it provides optimum conditions for bacterial growth: high water activity, near neutral pH, and abundant nutrients. These conditions increase the survival and growth of bacteria, perhaps exacerbating cross-contamination (Dave & Ghaly, 2011). From 1998 to 2008, foodborne outbreaks associated with poultry resulted in the greatest number of deaths (19%), which were two times higher than for leafy vegetables (Painter et al., 2013). It was reported that Listeria monocytogenes and Salmonella spp. were the main contributors, accounting for 63% and 26% of poultry-associated deaths. Chlorine and acids are the two most common chemical interventions to decontaminate the surfaces of poultry carcasses (Sohaib, Anjum, Arshad, & Rahman, 2016). However, high concentrations of chlorine and acids may result in off-flavor, discoloration, equipment corrosion, and other problems. The EU has prohibited the import of
∗
poultry products that are treated with chlorine, trisodium phosphate, and peracetic acid; leading to an estimated loss of $200 - $300 million annually (Johnson, 2015). The concept of natural antimicrobials has become more and more popular; and served as a driver of the present study to search for natural antimicrobials that can replace or augment conventional chemical interventions. Photoinactivation using photosensitizers has been widely studied in clinical medicine as a potential treatment of bacterial infections such as skin diseases and cavities (Niu, Tian, Cai, Ren, & Wei, 2015; Ricci Donato et al., 2017). Photoinactivation of microorganism is based on the activation of a photosensitizer by light exposure at a predetermined wavelength. The excited photosensitizer molecules transfer the excessive protons/electrons or energy to surrounding substances, resulting in generation of reactive oxygen species (ROS). ROS oxidize lipid membranes, proteins, and nucleic acids, leading to cell death. Since ROS have an extremely short half-life and cause non-specific damage to bacterial cells, it is difficult for bacteria to trigger defense systems or develop resistance to photoinactivation (Maisch, 2015). A number of photosensitizers have been evaluated and demonstrate photoinactivation against a variety of microorganisms on food or food-
Corresponding author. E-mail address: [email protected] (K.R. Matthews).
https://doi.org/10.1016/j.foodcont.2019.106959 Received 1 July 2019; Received in revised form 18 October 2019; Accepted 18 October 2019 Available online 22 October 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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2.4. MIC and microtiter plate assay
contact surfaces (D'Souza et al., 2015). Curcumin, which can be extracted naturally from Curcuma longa plants, is one of the most wellstudied photosensitizers. Studies showed that the photosensitizer curcumin achieved a 5-log reduction of Vibrio parahaemolyticus on oysters without promoting lipid oxidation (Liu et al., 2016; Wu et al., 2016). PSC may be part of a novel approach to improve the microbial safety of poultry. A potential limitation of curcumin is its low solubility in water, purified curcumin is typically dissolved in ethanol with or without a solubility enhancer such as dimethyl sulfoxide (DMSO), and then diluted with distilled water. However, curcumin readily precipitates as the concentration of ethanol decreases. Use of water-soluble photosensitizer curcumin (PSC) would eliminate limitations in the use of curcumin in commercial poultry processing. The goal of this study was to investigate the activity of PSC in inactivating L. monocytogenes and Salmonella on media and chicken skin. The effects of incubation time and light dose on antimicrobial activity and chicken skin color were evaluated to optimize the photoinactivation conditions.
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the microdilution broth method (CLSI, 2016). Light controls were prepared by illuminating cultures in Mueller-Hinton broth (MHB Difco, Becton Dickinson, Sparks, MD). The MIC was defined as the lowest concentration of PSC that inhibits visible growth after 16 h incubation. The MBC was determined by plating 100 μl of the suspension from each well onto TSA, and it was defined as the lowest concentration showing the absence of growth on TSA after 20 h incubation at 37 °C. Each concentration of PSC was tested in replicated wells. The bacterial population after PSC treatment was determined using a microtiter plate assay. In brief, inoculum was prepared as described and diluted to achieve 104 CFU per well. The culture was mixed with an equal volume of PSC. The culture was incubated with different concentrations of PSC for 5 min, followed by illumination (64.2 kJ/m2). A hundred microliters were removed from a well and spread plated on TSA supplemented with appropriate antibiotics. Plates were incubated at 37 °C for 18–20 h. For L. monocytogenes, experiments were conducted in a walk-in cooler (9.3 ± 0.4 °C). Triplicate wells were prepared for each sample in a single experiment.
2. Materials and methods 2.1. Chemicals and bacterial strains Water-soluble photosensitizer curcumin (PSC; uC3 Clear) was a kind gift from Sabinsa Corp. (East Windsor, NJ). It was dissolved and diluted using sterile distilled water (SDW). Peracetic acid antimicrobial (PAA; Spectrum®) was kindly provided by PeroxyChem (Philadelphia, PA). PAA was diluted using SDW, and the PAA concentration was confirmed using test strips (Micro Essential Laboratory Inc., Brooklyn, New York). Three strains of L. monocytogenes were obtained from Dr. Joshua Gurtler (Eastern Region Research Center, USDA, Wyndmoor, PA): L008 (serotype 4b), L2624 (serotype 1/2b), and L2625 (serotype 1/2a). Eight strains of Salmonella were obtained from Dr. Donald Schaffner (Rutgers University, New Brunswick, NJ): S. Thompson (S8), S. Hadar (S20), S. Typhimurium Copenhagen (S21), S. Montevideo (S24), S. Typhimurium (S25), S. Saintpaul (S26), S. Heidelberg (S40), and S. Heidelberg (107a). All L. monocytogenes and Salmonella strains were resistant to 100 μg/ml of nalidixic acid (Alfa Aesar, England) and 50 μg/ml streptomycin (MP Biomedicals), respectively. All media used to culture bacteria were supplemented with appropriate antibiotics unless otherwise noted. Stock cultures were stored at −80 °C in tryptic soy broth (TSB; Difco, Becton Dickinson, Sparks, MD) containing 20% glycerol. To prepare the inoculum, an isolated colony from tryptic soy agar (TSA; Difco, Becton Dickinson, Sparks, MD) was transferred to 30 ml of brain heart infusion (BHI; Difco, Becton Dickinson, Sparks, MD) broth for L. monocytogenes or TSB for Salmonella, followed by 20 h incubation at 37 °C. The culture was centrifuged at 4000×g for 10 min and washed with 0.1% sterile peptone water (SPW; Difco, Becton Dickinson, Sparks, MD) and suspended in SDW.
2.5. Treatment of chicken skin Chicken was purchased from a local market. Pieces of chicken skin 5 × 5 cm2 were carefully removed using a sterile scalpel and immobilized on a stainless-steel wire mesh that was then placed inside a petri dish. A 10 μl volume of inoculum, either L. monocytogenes cocktail (L008, L2624, and L2625) or Salmonella cocktail (S20 and 170a), was evenly spread on the chicken skin. Chicken skin samples were dried for 5 min with the lid of the petri dish ajar, and then kept for an additional 25 min with the lid covered. Solutions of SDW, 300 ppm PSC, or 300 ppm PAA were kept in the refrigerator for at least 30 min for cooling (12.7 ± 0.5 °C), and each treatment contained 300 ml of solution. Foil was used to protect PSC from natural light before use. Two pieces of inoculated chicken skin were dipped in 300 ml of a treatment for 5 min, followed by illumination (32.1 kJ/m2). Each skin sample was allowed to drip for 5 min before being placed in a sterile plastic bag containing 25 ml of sterile phosphate buffered saline (PBS; VWR International, LLC.). Each sample was stomached for 2 min. The homogenized sample was serial diluted (1:10) with SPW and plated (100 μl) on modified oxford agar (MOX; Difco, Becton Dickinson, Sparks, MD) or XLT-4 (Becton Dickinson, Sparks, MD) in duplicate. The plates were incubated at 37 °C for 24 h. 2.6. Incubation time and light dose The L. monocytogenes cocktail (L008, L2624, and L2625) inoculum was diluted to approximate 105 CFU/ml. The concentration of PSC was 300 ppm for studies using media and chicken skin. Incubation time (1, 2.5, and 5 min) was studied by fixing the light dose at 64.2 kJ/m2; meanwhile, light doses (0, 6.4, 32.1, and 64.2 kJ/m2) were studied using 5-min contact time. The experiment was performed in a walk-in cooler. To evaluate efficacy in media experiments were conducted in a 96well plate. A 100 μl aliquot of sample was plated in duplicate on TSA supplemented with appropriate antibiotic, followed by 20 h incubation at 37 °C. For chicken skin studies, the same method of inoculation, PSC treatment, and microbiological analysis was used as described previously.
2.2. Absorption spectrum of PSC The absorption spectrum of PSC was determined using UV–Vis spectroscopy (Cary 60 Spectrophotometer, Agilent Technologies, Santa Clara, CA). Quartz cuvettes (1 cm light path) were used. 2.3. Light source The light box unit was evaluated to have a peak wavelength near 430 nm. The light box unit was composed of six solderless exotic LED (7.2 W; LED Group Buy, Saint Louis, MO) evenly distributed across the interior top. All sides of the light box unit were made of high polished stainless steel. The power density of the light box unit was measured using a spectroradiometer (Model PS-300, Apogee Instruments, Inc., Logan, UT), based on the average of five readings. Light dose was calculated by multiplying power density by illumination time.
2.7. Color measurement Pieces of 5 × 5 cm2 chicken skin with approximate 3-mm thickness were carefully excised using a sterile scalpel. Skin samples were dipped 2
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into 300 ml of 300 ppm PSC for 5 min and subjected to predetermined light doses. Excessive blood or PSC solution on chicken skin were gently removed using tissue paper. Values of L*, a*, and b* were measured using a colorimeter (Konica Minolta CR410, Osaka, Japan). Each treatment was conducted in triplicate, and the final results were reported as the average of five readings obtained from different locations of each chicken skin sample. Color difference before and after treatment were described as the values calculated by Eq. (1) to Eq. (5). The subscript “o” in the equation refers to the color reading of the skin before treatment.
ΔL* = L* − L*o
1
Δa * = a * − a *o
2
Δb* = b* − b*o
3
Total color difference (ΔE) =
Table 1 MIC and MBC of PSC on Listeria monocytogenes. Bacterial Strain L. monocytogenes
L008 L2624 L2625
MIC (ppm)
MBC (ppm)
10 10 10
20 20 20
†Light dose: 64.2 kJ/m2.
(L* − L*o)2 + (a * − a *o)2 + (b* − b*o)2 4
Whiteness Index (WI) = 100 − (100 −
* 2 L)
+
(a *)2
+
(b*)2
5
2.8. Statistical analysis All experiments were conducted three times independently. The mean values were compared by ANOVA and Duncan's post hoc analysis using a SAS software (SAS university edition, SAS Institute Inc., USA). P < 0.05 was considered as a significant difference. 3. Results and discussion
Fig. 2. Population of L. monocytogenes on media after 5-min incubation with PSC, followed by 64.2 kJ/m2 of illumination. Dotted line represents the limit of detection.
3.1. Properties of water-soluble PSC and light source Fig. 1 shows the absorption spectrum of PSC at two concentrations. Two absorption maxima were identified: 260 nm and 410 nm. The strongest absorption maximum within visible wavelength was at 410 nm, which was consistent with published literature (Priyadarsini, 2009) The power density of the light apparatus used in this study was 107 W/m2. As a result, light doses of 6.4, 32.1, and 64.2 kJ/m2 corresponded to 1, 5 and 10 min of light exposure.
64.2 kJ/m2 of light exposure, are shown in Table 1. All three L. monocytogenes isolates shared the same MIC and MBC of 10 ppm and 20 ppm, respectively. For samples treated at cooler temperature, no colonies were observed following exposure to 30 ppm and 300 ppm of PSC (Fig. 2). Under same dose of light exposure (64.2 kJ/m2), PSC at 1/ 2 × MIC (5 ppm) achieved an approximate 3-log reduction of L. monocytogenes, a decrease which is generally considered an effective antimicrobial treatment (Anonymous, 2019). However, no inactivation was observed in the absence of light exposure, regardless of the PSC concentrations evaluated (data not shown). The results suggest that illumination was essential for PSC to induce inhibitory effects at low concentration. Previous studies showed that curcumin alone can inhibit a variety of microorganisms without light exposure, including L. monocytogenes, E. coli O157:H7, Salmonella Typhimurium, and methicillin-resistant Staphylococcus aureus (Kim et al., 2005; Niamsa & Sittiwet, 2009). The MIC of purified curcumin varied with different bacterial strains, but it was typically above 1000 ppm if no light activation was applied (Kim et al., 2005; Negi, Jayaprakasha, Jagan Mohan Rao, & Sakariah, 1999); a concentration much greater than the highest concentration evaluated in this study (300 ppm). In other words, a lower concentration of curcumin is required for bacterial inhibition/ inactivation when light of the appropriate wavelength is applied. Importantly, using a lower concentration would both reduce the cost of the antimicrobial solution and any adverse effects on chicken skin color. In addition to light exposure, 0 ppm of PSC without illumination did not reduce the bacterial population, suggesting the presence of PSC was essential to elicit an antimicrobial effect. It was reported that 430 nm light (123.3 J/cm2) resulted in significant reduction of L. monocytogenes compared with a dark (no light exposure) control (Endarko, Maclean, Timoshkin, MacGregor, & Anderson, 2012). However, the light dose used in the present study was much lower, which may in part explain the lack of photoinactivation.
3.2. Antimicrobial activity of PSC on media The MIC and MBC of the water-soluble PSC used in this study, after
Fig. 1. Absorption spectrum of PSC. 3
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Fig. 3. Population of Salmonella spp. on media after 5-min incubation with PSC, followed by 64.2 kJ/m2 of illumination. Asterisk indicates significant difference between PSC concentrations for individual strain (P < 0.5). Dotted line represents the limit of detection.
In terms of Salmonella, PSC at 200 ppm exhibited the greatest antimicrobial activity, resulting in 1.8–3.6 log reduction of Salmonella (Fig. 3). Interestingly, the antimicrobial efficacy of photoinactivation was lower at higher concentrations of PSC solution (2000 ppm). The decrease in efficacy of higher PSC concentrations may be associated with a self-shielding effect of light (Barr, MacRobert, Tralau, Boulos, & Bown, 1990). Curcumin at lower concentration is able to absorb light energy and be exited, leading to greater generation of ROS. However, in a highly concentrated solution, a large number of solutes (curcumin molecules) block the light source, interfering with the process of light activation. L. monocytogenes was completely inactivated when exposed to 300 ppm PSC which was not the case for Salmonella strains. Differences associated with antimicrobial activity of PSC against Gram-positive and Gram-negative bacteria may be related to differences in the cellular membranes. Photosensitizers bind and penetrate through the cell membrane of Gram-positive bacteria more readily since the peptidoglycan layer is more porous than the outer-inner membrane complex of Gram-negative bacteria (Dai, Huang, & Hamblin, 2009; Minnock et al., 2000). To improve the antimicrobial efficacy of PSC on gramnegative bacteria, two approaches are commonly applied – mixing with EDTA or increasing the positive charge of the photosensitizer (Malik, Ladan, & Nitzan, 1992). Further enhancement of the efficacy of PSC may be realized by applying either of the approaches to develop a foodgrade antimicrobial curcumin photosensitizer that is safe, efficacious, and cost effective. The antimicrobial activity of photoinactivation by PSC was found to be strain dependent. For example, 200 ppm of PSC resulted in a 1.8 log reduction of Salmonella S8, which was significantly (P < 0.05) less than the 3.6 log reduction of Salmonella S26 (Fig. 3). Specific reasons for differences in strain susceptibility are still debated. It was proposed that strain differences in susceptibility may be related to the amount of capsular polysaccharides and the ability of a strain to form a biofilm protecting the cell (Grinholc, Szramka, Kurlenda, Graczyk, & Bielawski, 2008); and the production of extracellular slime (Gad, Zahra, Hasan, & Hamblin, 2004). Previous studies focused on susceptibility of different strains of S. aureus, especially differences between methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) (Grinholc et al., 2008; Maisch, Bosl, Szeimies, Lehn, & Abels, 2005; Szpakowska, Lasocki, Grzybowski, & Graczyk, 2001). Greater research to elucidate strain differences in PSC sensitivity would aid in development of more
Fig. 4. Microbial count of a) L. monocytogenes and b) Salmonella on chicken skin after 5-min incubation with PSC, followed by 32.1 kJ/m2 of illumination. Dotted line represents the limit of detection.
efficacy treatments and practices.
3.3. Antimicrobial activities of PSC on chicken skin The efficacy of PSC in reducing the population of L. monocytogenes differed when evaluated on chicken skin and in liquid media (Fig. 4a). Fats and the complex surface (nooks and crannies) on chicken skin may interfere with the interaction between light and PSC, likely contributing to decreased efficacy. In addition, PSC in water has low viscosity, decreasing the ability of PSC to attach to the chicken skin (Tortik, Spaeth, & Plaetzer, 2014). Although PSC was less effective on chicken skin; nevertheless, a 2.9 log reduction of L. monocytogenes on chicken skin was achieved. The level of reduction achieved in this study is equal to or greater than that of antimicrobials presently used in processing of poultry carcasses and parts. Compared with water and PAA treatments, PSC showed the greatest reduction of L. monocytogenes and Salmonella on chicken skin (Fig. 4). PAA is one of the most common antimicrobials used by the poultry industry (McKee, 2011). PAA is effective at acid and slightly alkaline pH and retains efficacy in presence of organic matter which has permitted its use in place of chlorine (Wideman et al., 2015). In this study, the effect of PSC and PAA on reducing the population of Salmonella was not significantly different. Both treatments were significantly better than no treatment and the water alone treatment of chicken skin (Fig. 4). PSC and PAA provided similar level of control of L. monocytogenes and Salmonella, however, the advantage of PSC is that it can 4
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Fig. 6. Microbial count of L. monocytogenes after treatment with 5-min incubation time and different light doses using a) media, and b) chicken skin. Dash line indicates an estimated trend based on the obtained results. Empty circle indicates that the microbial count in the liquid media experiment was below detection limit (0.7 log CFU/ml).
Fig. 5. Microbial count of L. monocytogenes after treatment with different incubation time and 64.2 kJ/m2 of illumination using a) media, and b) chicken skin. Dash line indicates an estimated trend based on the obtained results. Empty circle indicates that the microbial count in the liquid media experiment was below detection limit (0.7 log CFU/ml).
compared to 30 min incubation, but there was not a large difference between 0 min and 5 min incubation (Hamblin et al., 2002). This is consistent with the results of the present study (Fig. 5a). Consequently, poultry carcasses can be dipped into PSC solution for as short as 1 min which would facilitate integration into current commercial poultry processing practices. In theory, light dose influences the amount of PSC being activated, and thus the production of ROS. Murdoch, Maclean, Endarko, MacGregor, and Anderson (2012) found that photoinactivation of various bacteria by 405 nm LED depended on the light dose. Similarly, Gios et al. (2010) found that applying 20 J/cm2 of light induced significantly greater reduction of S. aureus than 40 J/cm2. In this study, a 300 ppm PSC solution achieved an approximate 5-log reduction of L. monocytogenes (Fig. 6a), regardless of the light dose. Similarly, PSC exposed to 32.1 kJ/m2 of light resulted in an approximate 2-log reduction of L. monocytogenes on chicken skin, which was not affected by light dose (Fig. 6b). In other words, under the experimental conditions of this study, the antimicrobial efficacy of PSC was not influenced by the light dose. A similar phenomenon was reported where 8000 μM of curcumin combined with 24 J/cm2 and 48 J/cm2 achieved similar log reduction of Streptococcus mutans (Paschoal et al., 2013). Research is warranted to
be extracted from a natural source, which satisfies the increasing demands of sustainability and clean labeling. There are limited studies focused on the food application of photosensitizer curcumin, either a purified or modified form. Studies addressing the feasibility of using photosensitizer to improve the safety of poultry products are warranted based on the results of the present study. For example, studies may focus on application of PSC on whole poultry carcasses and parts and best approach for incorporating the technology into existing poultry processing operations.
3.4. Effects of incubation time and light dose on photoinactivation ROS have a short half-life, so it is critical to have photosensitizer and bacterial cells in close proximity. In short, the closer the proximity of the photosensitizer to the bacterial cell the more likely the ROS will negatively impact cell integrity. In this study, no significant difference was observed in the antimicrobial activity of PSC when photoinactivation between 1 min and 5 min incubation time for broth and chicken skin (Fig. 5). It was reported that survival of S. aureus and E. coli incubated with photosensitizer polylysine-ce6 for 5 min was lower 5
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Fig. 7. Effects of light dose on a) ΔL*/Δa*/Δb*, b) total color difference (ΔE), and c) whiteness index (WI) of chicken skin. Incubation time was fixed at 5 min for all the samples. Distilled water control (DW) was performed. Letters of “A, B”, “a, b”, and “x, y” represent significant difference in ΔL*, Δa*, and Δb* respectively. Asterisk indicates a significant difference of WI before and after treatment.
observed between samples treated with 32.1 kJ/m2 or greater light exposure and water controls, suggesting it is difficult for consumers to recognize the color difference after PSC treatment with longer light exposure. The mechanism of photoinactivation is based on oxidation by ROS, which can also oxidize pigment compounds and thus bleach food surfaces. Samples without illumination had significantly lower WI after treatment when compared with untreated samples (Fig. 7c) associated with an increase in yellowness on chicken skin. However, all the other samples showed no significant differences for WI, suggesting that no bleaching occurred after up to 64.2 kJ/m2 of illumination.
delineate why there is a variance in the effect of light dose on photoinactivation. In addition, it should be noted that 32.1 kJ/m2 used in the present study corresponds to 5 min of light exposure. Using a higher power density of LEDs can further reduce the illumination time, which would increase energy savings. 3.5. Effects of light dose on the color of chicken skin The greatest difference observed among L*/a*/b* was b*, since PSC exhibited yellow color (Fig. 7a). Positive Δb* indicates that a sample becomes more yellow after dip treatment. A significant increase in Δb* was observed after dipping in PSC, but chicken skin subjected to light exposure showed no significant differences on ΔL* and Δa* when compared with DW controls. Interestingly, no significant differences on Δb* were observed between water control and chicken skin samples after treatment with light dose of 32.1 and 64.2 kJ/m2, indicating that the yellow color diminished following light exposure. This phenomenon was demonstrated in a previous study, where after light exposure a loss of yellow-orange color associated with riboflavin treatment of salmon was noted (Josewin, Ghate, Kim, & Yuk, 2018). Samples exposed to 6.4 kJ/m2 of light showed a significant increase in yellowness (Δb*), indicating that such a low light dose might not be appropriate due to the color change. However, recognizing that consumers in some countries prefer chicken with yellow skin, a low light dose with shorter illumination time could be applied to poultry in those countries and actually be advantageous from a quality perspective. Apparent color change also resulted in a significant difference in ΔE between PSC and DW control after 6.4 kJ/m2 of light exposure (Fig. 7b). The larger the ΔE value, the greater likelihood that consumers would recognize a color change between samples. However, no significant differences were
4. Conclusions This study demonstrates that PSC inactivated L. monocytogenes and Salmonella in liquid media and on chicken skin. Under the experimental conditions in this study, incubation time and light dose did not influence the antimicrobial activity of PSC, suggesting that photoinactivation can be achieved in a short time. It is important to develop a rapid antimicrobial intervention to satisfy the rapid line speeds used in the poultry industry. These results are encouraging and suggest that photoinactivation can be incorporated even on a fast-moving production line. Although chicken skin treated with PSC appeared slightly more yellow, there was no recognizable difference in chicken skin color after treatment with light dose greater or equal to 32.1 kJ/m2. Therefore, upon completion of PSC photoinactivation treated chicken could not be differentiated from non-treated chicken enhancing acceptability of the method and process.
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Declaration of competing interest
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