without brushing on broiler carcass decontamination

Food Control 77 (2017) 199e209

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Trisodium phosphate dip, hot water dip, and combination dip with/ without brushing on broiler carcass decontamination P. Singh a, H.C. Lee b, M.F. Silva a, K.B. Chin c, I. Kang b, * a

Department of Food Science & Human Nutrition, Michigan State University, East Lansing, MI 48824, USA Department of Animal Science, California Polytechnic State University, San Luis Obispo, CA 93407, USA c Department of Animal Science, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500-757, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 October 2016 Received in revised form 29 December 2016 Accepted 10 February 2017 Available online 14 February 2017

The purpose of this research was to evaluate the effects of trisodium phosphate dip (TSP), hot water dip (HWD) and their combination dip with/without brushing on broiler breast skin for bacterial reduction and structural changes. Eviscerated broiler carcasses were obtained from a local slaughter plant and immediately subjected to one of six treatments: 1) two tap water dips at 25
C (TWD/TWD), 2) TWD/ TWD with brushing (TWD/TWD/B), 3) TWD and TSP at 8%/25
C (TWD/TSP), 4) TWD and HWD at 71
C (TWD/HWD), 5) TSP and HWD (TSP/HWD), and 6) TSP/HWD with brushing (TSP/HWD/B). Each dip was conducted for 45 s with or without brushing at 5 s on/off during the second dip. Compared to the control (TWD/TWD), TSP/HWD significantly reduced mesophilic aerobic bacteria (MAB), Escherichia coli (E. coli), and total coliforms by 1.1, 0.9 and 1.0 log CFU/g, respectively, and Salmonella prevalence by 53.3% (P < 0.05), whereas TWD/TSP and TWD/HWD showed intermediate reductions (P < 0.05). Upon brushing, TSP/HWD/B reduced populations of MAB, E. coli, and total coliforms, and the prevalence of Salmonella more effectively than control of brushing (TWD/TWD/B) (P < 0.05). When two sampling methods were compared, the method of stomaching released fewer MAB and total coliforms (named loosely-associated cells) than the grinding of the stomached skin (named tightly-associated cells). Compared to controls (TWD/TWD and TWD/TWD/B), both TSP/HWD and TSP/HWD/B generally resulted in darker, less reddish, and more yellowish breast skin. Scanning electron microscope and histological images indicated that both TSP/HWD and TSP/HWD/B had deeper skin penetration than controls or TWD/HWD and TWD/TSP. Overall, TSP/HWD/B showed the most effectiveness in broiler carcass decontamination. © 2017 Published by Elsevier Ltd.

Keywords: Bacterial decontamination of broiler Salmonella Trisodium phosphate dip Hot water dip Brushing Broiler skin structure

1. Introduction Broiler chicken is the most favorite meat in the United States (U.S.) and many other countries. According to OECD/FAO (2014), poultry is expected to become the world's most consumed meat over the next 5 years. Poultry meat, however, is a leading cause of foodborne illnesses, particularly salmonellosis and campylobacteriosis, in the U.S. and the European Union (Centers for Disease Control and Prevention (CDC), 2012; Chaine, Arnaud, Kondjoyan, Collignan, & Sarter, 2013; European Food Safety Authority [EFSA], 2008; Hoffmann, Fischbeck, Krupnick, & McWilliams, 2007). Most poultry-related illnesses could be results from ineffective pathogen control strategies and inaccurate pathogen detection methods that

* Corresponding author. E-mail address: [email protected] (I. Kang). http://dx.doi.org/10.1016/j.foodcont.2017.02.015 0956-7135/© 2017 Published by Elsevier Ltd.

are currently being used at farms and/or at processing plants (Interagency Food Safety Analytics Collaboration, 2015; Singh, Silva, Ryser, Ha, & Kang, 2016). Over the last two decades, the broiler industry has adopted various intervention strategies against pathogens, which include the use of chlorine, organic acids, cetylpyridinium chloride (CPC), trisodium phosphate (TSP), etc. (Lillard, 1990; Li, Slavik, Walker, & Xiong, 1997; Sakhare, Sachindra, Yashoda, & Rao, 1999; Whyte, Collins, McGill, Monahan, & O'Mahony, 2001; Zhang, Jeong, Janardhanan, Ryser & Kang, 2011). While these strategies have been partly successful, many chemicals such as chlorine and CPC are not generally recognized as safe (GRAS), and others such as organic acids have negative organoleptic effects (Ricke, Kundinger, Miller, & Keeton, 2005). Hot water sprays or dips have been shown to reduce bacterial load on broiler carcasses with their effectiveness dependent on water temperature and exposure time (Cox, Mercuri, Thomson, &

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Abbreviations TWD/TWD Tap water dipping (TWD) (25
C, 45 s) followed by second TWD (25
C, 45 s) TWD/TWD/B TWD (25
C, 45 s) followed by second TWD (25
C, 45 s) with intermittent manual brushing (5 s on/5 s off) TWD/TSPTWD (25
C, 45 s) followed by Trisodium phosphate (TSP) dipping (8%, 25
C, 45 s) TWD/HWD TWD (25
C, 45 s) followed by hot water dipping (HWD) (71
C, 45 s) TSP/HWD TSP dipping (8%, 25
C, 45 s) followed by HWD (71
C, 45 s) TSP/HWD/B TSP dipping (25
C, 45 s) followed by HWD (71
C, 45 s) with intermittent manual brushing (5 s on/5 s off)

Gregory, 1974; Purnell, Mattick, & Humphrey, 2004). In our previous study, hot water spraying (71
C, 1 min) reduced total bacteria counts and the incidence of Salmonella on broiler carcasses but caused a partially cooked appearance (Zhang, Singh, Lee, & Kang, 2013). On-line brushing with a water spray has been primarily used to remove fecal materials from broiler carcass surfaces before scalding. Berrang and Bailey (2009) assessed on-line washing with/ without brushing of broiler carcasses between bleed-out and chilling steps in a commercial processing plant. Overall, their multiple-sequential washing decreased Campylobacter and E. coli populations as well as Salmonella prevalence, although none of single steps caused a significant difference. Trisodium phosphate received GRAS status and was approved for use in broiler processing in 1992 (Capita, Alonso-Calleja, GarciaFernandez, & Moreno, 2002b). If used properly under optimum conditions, it can cause up to 79% reduction in Salmonella prevalence and 2 log reduction in E. coli population on chicken carcasses (Capita et al., 2002b). In processing plants, most bird-to-bird contamination occurs during de-feathering and evisceration (Allen, Tinker, Hinton, & Wathes, 2003; Guerin et al., 2010; Rasschaert et al., 2008; Sarlin et al., 1998). Bacteria that are loosely associated with carcasses are likely to contribute to cross-contamination during processing, while tightly associated ones are likely to survive during chemical and physical antimicrobial exposure. Therefore, an intervention that is effective against loosely and tightly associated bacteria can reduce both cross contamination during processing and subsequent growth thereafter. During antimicrobial application, physical brushing is expected to expose hidden bacteria in crevices and ridges to chemical or physical treatments. Our recent study indicated that 10 swabbings and 10 stomachings recovered only 17 and 45% of mesophilic aerobic bacteria from broiler skin, respectively, whereas the remaining bacteria were recovered after grinding the swabbed or stomached skin (Singh, Lee, Chin, Ha, & Kang, 2015). Previously, several studies also used grinding or blending of broiler skin to recover tightly associated bacteria (Lee, Park, Kang, & Ha, 2014; Tamblyn, Conner, & Bilgili, 1997; Zhang et al., 2013). Many studies have shown that a single treatment was less effective than a combination of physical and chemical treatments such as TSP with electricity, sodium carbonate with hot water, sodium carbonate with electricity, lactic acid with steam, and sodium hypochlorite with acidic electrolyzed water (Lecompte, Kondjoyan, Sarter, Portanguen, & Collignan, 2008; Li et al., 1994; Northcutt,

Smith, Ingram, Jr, Hinton, & Musgrove, 2007; Rodriguez de Ledesma, Riemann, & Farver, 1996). Until now, no research has been conducted to evaluate the single and combined effects of three treatments (TSP, hot water dip - HWD, and brushing -B) against naturally occurring bacteria on broiler carcasses. Therefore, the objective of this study was to assess the effects of TSP, HWD, and their combination with and without brushing on broiler carcasses for the reduction of bacteria after stomaching (namely loosely-associated bacteria) and after subsequent grinding of the stomached skin (namely tightly-associated bacteria) as well as structural changes on the broiler skin, using scanning electron microscopy and histological staining. 2. Materials and methods 2.1. Broiler carcasses A total of nine visits were made to a local broiler processing plant on nine different days over three months (three visits/month) to assess the effects of chemical and/or physical treatments on microbiological quality and structural changes of broiler carcass skin (~46-day-old, HubgbardM99/ross 708), using microbiological analysis and visual imaging (scanning electron microscope and histological staining), respectively. 2.2. Experiment I: control (TWD/TWD), trisodium phosphate dip (TWD/TSP), hot water dip (TWD/HWD), and combination of TSP and HWD (TSP/HWD) In each of first three visits, 20 broiler carcasses were randomly selected from an industrial broiler processing line after evisceration. The carcasses were immediately subjected to one of four treatments (5 carcasses/treatment) as follows: 1) Control of tap water dipping (TWD) at 25
C for two times - TWD/TWD, 2) TWD followed by 8% (wt/vol) trisodium phosphate dipping at 25
C TWD/TSP, 3) TWD followed by hot water dipping at 71
C e TWD/ HWD, and 4) 8% trisodium phosphate dipping at 25
C followed by hot water dipping at 71
C - TSP/HWD. Each dip was conducted for 45 s. 2.3. Experiment II: control (TWD/TWD), control with brushing (TWD/TWD/B), combination of TSP and HWD (TSP/HWD), and combination with brushing (TSP/HWD/B) During each of second three visits, 20 broiler carcasses were similarly selected as before and subjected to one of four treatments (5 carcasses/treatment) as follows: 1) Control of tap water dipping (TWD) at 25
C for two times - TWD/TWD, 2) TWD at 25
C followed by a second TWD with brushing - TWD/TWD/B, 3) 8% trisodium phosphate dipping at 25
C followed by hot water dipping at 71
C - TSP/HWD, and 4) 8% trisodium phosphate dipping at 25
C followed by hot water dipping at 71
C with brushing - TSP/HWD/B. For carcass dipping, 18-L stainless steel buckets were filled up to ¾ of the capacity. A new bucket with fresh solution was used for every 5 birds that were dipped individually. Each dip was conducted for 45 s with/without brushing (5 s on/off) on carcass breast and neck area. For brushing, polyester brushes (Sparta® Spectrum® All Purpose Utility Scrub Brushes), having a bristle density of 38/cm2, bristle diameter of 1 mm, and bristle length of 4.5 cm, were used after purchasing from Carlisle Foodservice Products (Batavia, IL). 2.4. Skin sample preparation methods in experiment I and II After each treatment, 25 g of skin was aseptically taken from the breast and neck areas, placed in sterile WhirlPak bag (Nasco,

P. Singh et al. / Food Control 77 (2017) 199e209

201

Modesto, CA) on ice, and transported to Michigan State University in 1.5 h. Each of the sample bags was poured with 225 ml of sterile phosphate buffer saline (PBS) and stomached for 1 min (Stomacher 400 Circulator, Seward, Worthing, UK) to assess loosely-associated bacteria in Experiment I and II. For tightly-associated bacteria in Experiment II, the stomached-skin was ground using a Brinkmann Polytron Homogenizer (Plytron PT10/35, Brinkmann Instruments Co., Westbury, NY), after transferring to a new WhirlPak bag containing 225 ml of fresh PBS. Between stomaching and grinding, the skin was washed with sterile water to remove any free bacteria remaining on the surface.

critically point dried in a Leica Microsystems model EM CPD300 critical point dryer (Leica Microsystems, Vienna, Austria) using liquid carbon dioxide as the transitional fluid. The samples were then mounted on aluminum stubs using carbon suspension cement (SPI Supplies, West Chester, PA) and coated with gold (~20 nm thick) in an Emscope Sputter Coater model SC 500 (Ashford, Kent, England) purged with argon gas. Samples, mounted on the stubs, were examined at 10,000 magnification in a JEOL 6610LV SEM (tungsten hairpin emitter) scanning electron microscope (JEOL Ltd., Tokyo, Japan). Total of 30 microscopic fields (6 fields/sample) were observed for each treatment.

2.5. Muscle and skin color measurements in experiment I and II

2.7.2. Histological staining and imaging Before treatments on bled-carcasses (reference before scalding) and after treatments on eviscerated-carcasses (treatment before chilling) as explained in section 2.7, skin samples (2 cm by 2 cm) were similarly obtained from breast area and mounted on wax squares followed by fixation in 10% Neutral Buffered Formalin for 48 h. The fixed skins were processed and vacuum infiltrated with paraffin using a Sakura VIP 2000 tissue processor, followed by embedding with a ThermoFisher HistoCentre III embedding station. Once the sample-mounted blocks were cooled, excess paraffin was removed from the edges. The blocks were then placed on a Reichert Jung 2030 rotary microtome and faced to expose the tissue sample. The blocks were then finely sectioned at 4e5 mm and the sections were dried for 2e24 h at 56
C in a slide incubator to ensure adherence to the slides. The slides were removed from the incubator and stained using a standard Hematoxylin and Eosin method which included two changes of xylene for 5 min each, two changes of absolute ethanol for 2 min each, and two changes of 95% ethanol for 2 min each. Thereafter, samples were rinsed in running tap water for 2 min and Endure Hematoxylin (Cancer Diagnostics Inc., Morrisville, NC) for 1½ minute followed by a 10e15 s differentiation in 1% aqueous glacial acetic acid and running tap water for 2 min to enhance nuclear detail. Following the tap water rinse, the slides were placed in one change of 95% ethanol for 2 min, 1% Alcoholic Eosin-Phloxine B for 2 min to stain cytoplasm, one change of 95% ethanol for 2 min, four changes of 100% ethanol for 2 min each, and four changes of xylene for 2 min each followed by coverslipping with synthetic mounting media for permanent retention and visualization by light microscopy at 20 magnification. The anatomical terminologies of Kiladze (2008) and Weir and Lunam (2004) were used to classify the skin layers. Total of 48 microscopic fields (16 fields/sample) were observed for each treatment.

 Commission Internationale de l’Eclairage (CIE) color (lightnessL*, redness-a*, and yellowness-b*) was measured in triplicate after treatments on the surface of breast muscle (experiment I) and the surface of breast and scapular skin (experiment II), using a colorimeter (8-mm aperture, illuminant C; CR-400, Konica Minolta Sensing Inc., Japan) after calibration with a white plate (L*, 97.28; a*, 0.23; b*, 2.43). 2.6. Microbiological analysis in experiment I and II A serial 10-fold dilution after stomaching of the skin sample or grinding of the stomached skin was made in phosphate buffer saline and plated in duplicates on aerobic and E. coli/coliform count plates (3M Company, St. Paul, MN) to incubate at 37
C for 24e48 h for bacterial enumeration. For Salmonella presence, pre-enrichment step was conducted by adding 30 ml of stomached or ground sample to 30 mL buffered peptone water (Acumedia) followed by incubation at 37
C for 20 h. After incubation, 100 mL of the preenriched solution was transferred to 10 ml Modified Rappaport Vassiliadis broth (Sigma Aldrich, MO) and incubated again at 42
C for 24 h. An aliquote (120 mL) of this enrichment was then examined for Salmonella presence using Reveal® Salmonella test kit (Neogen Corp., Lansing, MI). All positive samples were streaked onto xylose lysine tergitol-4 agar, brilliant green sulfur agar and bismuth sulfite agar (Acumedia) plates, which were then incubated at 37
C for 24 h and inspected for typical Salmonella colonies to confirm the Reveal® Salmonella results. 2.7. Experiment III: imaging of skin using scanning electron microscopy (SEM) and histological staining Last three visits were made to prepare breast skin samples for SEM imaging (5 samples/treatment) and histological staining (3 samples/treatment) after the treatments as mentioned in Experiment I and II (section 2.2 and 2.3). Three control skin samples were additionally collected from broiler carcasses after bleeding (or before scalding) to serve as a reference for the SEM and histological images of the treated samples. 2.7.1. Scanning electron microscope (SEM) imaging Before treatments on bled-carcasses (reference before scalding) and after treatments on eviscerated-carcasses (treatment before chilling) as explained in section 2.7, skin samples (3 mm  3 mm) were removed from breast area and fixed at 4
C for 12 h in 4% glutaraldehyde buffered at pH 7.4 with 0.1 M sodium phosphate. The samples were then rinsed for 4 h in the buffer followed by postfixation for 12 h in 1% osmium tetraoxide buffered with 0.1 M sodium phosphate. After fixation, the samples were rinsed again in the buffer for 4 h followed by dehydration by exchanging with graded ethanol series (25%, 50%, 75%, 95%) for 2 h at each gradation followed by three 2 h changes in 100% ethanol. The samples were

2.8. Statistical analysis Mesophilic aerobic bacteria, E. coli and total coliforms counts per gram of skin from triplicate experiments were converted to log units for statistical analysis. In experiment I, bacterial counts and muscle color values (L*, a*, b*) were compared among treatments, using General linear model (GLM) and Duncan's multiple range test at P < 0.05 (SAS 9.4, 2013, SAS Institute Inc.). To compare prevalence of Salmonella among treatments, binary distribution in GLIMMIX procedure was used with Tukey's adjustment at P < 0.05 (SAS 9.4, 2013, SAS Institute Inc.). In experiment II, a two-factor (Treatment x Sample preparation method) analysis was conducted to compare bacterial counts and Salmonella presence. Since there was no interaction between the two factors, data were pooled together. For bacterial counts, comparisons among 4 treatments and between 2 sample preparation methods were conducted using GLM and Duncan's multiple range tests at P < 0.05. For Salmonella presence, binary distribution in GLIMMIX procedure with Tukey's adjustment at P < 0.05 was used

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as before. Skin color values on breast and scapula area were also compared among treatments, using GML and Duncan's multiple range test at P < 0.05. 3. Results and discussion 3.1. Experiment I: control (TWD/TWD), trisodium phosphate dip (TWD/TSP), hot water dip (TWD/HWD), and combination of TSP and HWD (TSP/HWD) The populations of MAB, E. coli and total coliforms on carcasses were reduced (P < 0.05) by 0.5, 0.7 and 0.8 log CFU/g, respectively, compared to the tap water dip (TWD/TWD) control, after trisodium phosphate dip (TWD/TSP) (Table 1). These results were identical with the report of Stopforth et al. (2007) who observed reductions in MAB, E. coli and coliforms of 0.5, 0.7, and 0.8 log CFU/ml, respectively, after spraying TSP (8e12%) on poultry carcasses in commercial processing plants. Spraying of 10% TSP on poultry carcasses that were previously inoculated with fecal bacteria resulted in reduction of 0.5, 0.5, and 0.2 log CFU/ml in carcass rinsate for MAB, E. coli and coliforms, respectively (Fabrizio, Sharma, Demirci, & Cutter, 2002). del Río, Panizo-Moran, Prieto, Alonso-Calleja, and Capita (2007) reported that 15 min dip of chicken legs in 12% TSP resulted in 1.7 and 0.9 log reductions in MAB and coliforms, respectively. The population reduction was presumed due to the high alkalinity (pH 12.1) of TSP solution that disrupts the bacterial cell membrane leading to intracellular leakage (USDA, 2002). It is also known that an immediate TSP-treatment yielded greater reduction (3.04 logs) than the TSP-treatment after 2 (1.76 log) and 4 h (1.26 logs) delays (Fratamico, Schultz, Benedict, Buchanan, & Cooke, 1996). Compared to the control carcasses (TWD/TWD), hot water dipping (TWD/HWD) reduced MAB, E. coli and total coliforms by 1.2, 0.7, and 0.7 log CFU/g, respectively, (P < 0.05) (Table 1). Zhang et al. (2013) reported that hot water spraying at 71
C for 1 min lowered loosely- and tightly-attached MAB on broiler carcasses by 1.1 and 1.3 log CFU/g, respectively. Hot water dipping of broiler carcasses at 70
C for 40 s and 75
C for 30 s resulted in 1.1 and 1.0 log CFU/ml reductions in MAB populations, and 1.0 and 0.9 log CFU/ ml reductions in Enterobacteriacae populations, respectively (Purnell et al., 2004). After comparing different surface decontamination strategies, Sinhamahapatra, Biswas, Das, and Bhattacharyya (2004) indicated that hot water treatment is the cheapest, most convenient and simplest approach. The combination of trisodium phosphate and hot water dip (TSP/HWD) yielded greater reduction of E. coli and total coliforms

Table 1 Mean populations1 (log cfu/g, SD) of mesophilic aerobic bacteria (MAB), Escherichia coli (E. coli) and total coliforms and prevalence (%) of Salmonella on broiler skin after treatments.

than hot water dip alone (TWD/HWD) and a greater reduction in MAB than trisodium phosphate dip alone (TWD/TSP) (P < 0.05) (Table 1). The combination (TSP/HWD) lowered the prevalence of Salmonella from 80 to 26.7% (P < 0.05), with intermediate reductions seen for TWD/TSP and TWD/HWD (Table 1). Considering the reduction in Salmonella prevalence of 40 and 20% after TWD/ TSP and TWD/HWD, respectively, the reduction of 53.5% after TSP/ HWD appeared to be an additive effect. Similar reductions in Salmonella and other pathogenic bacteria were observed when TSP (10%, 10
C/15 s) and HWD (95
C/5 s) were combined (Rodriguez de Ledesma et al., 1996). In terms of treatment order, TSP followed by hot water was more effective than the reverse (Gorman, Sofos, Morgan, Schmidt, & Smith, 1995). Trisodium phosphate acts as a detergent that can dissolve thin skin layers and expose hidden bacteria to hot water (Scientific Committee on Veterinary Measures Relating to Public Health, 1998). TSP is more bactericidal towards Gram-negative pathogens (e.g., Salmonella and E. coli) due to the lipopolysaccharide layer in the cell wall than Gram-positive bacteria (e.g., S. aureus and L. monocytogenes) (Liao & Cooke, 2001). As a result, the combination of TSP and HWD resulted in better bacterial elimination than a single treatment. With regard to color change (L*, a*, b*), no significant difference in broiler breast was observed regardless of treatment, except for more yellowness after TWD/TSP than TWD/HWD (Table 2). In the case of skin color, however, Purnell et al. (2004) reported that 60 and 5e9% of the carcasses were down-graded after HWD at 75
C for 30 s and 70
C for 40 s, respectively, due to epidermal damage or skin tears during the trussing process. Immersion or spraying of broiler carcasses with 71
C water for 1 min resulted in lower appearance scores due to partially cooked appearance (Cox et al., 1974; Zhang et al., 2013). 3.2. Experiment II: control (TWD/TWD), control with brushing (TWD/TWD/B), combination of TSP and HWD (TSP/HWD), and combination with brushing (TSP/HWD/B) In Experiment II, the best antibacterial treatment (TSP/HWD) found in Experiment I was used with and without brushing to evaluate antimicrobial efficacy on broiler carcasses. For populations of loosely- and tightly-associated bacteria, stomaching of skin samples and grinding of the stomached skin samples were used, respectively, as described by Singh et al. (2015). Data were pooled together due to no significant interaction (P > 0.05) found between treatment and sample preparation method. Compared to tap water dipping (TWD/TWD), no bacterial reduction (P > 0.05) was observed on the carcasses that received the combination of water dipping and brushing (TWD/TWD/B), regardless of bacterial species (Table 3). However, brushing with TSP and HWD (TSP/HWD/B) resulted in the best reduction of MAB population followed by TSP/ HWD, and TWD/TWD or TWD/TWD/B (P < 0.05) (Table 3). The populations of E. coli and total coliform bacteria were also

Treatments2

MAB E. coli Total coliforms Salmonella

TWD/TWD

TWD/TSP

TWD/HWD

TSP/HWD

3.5a (0.46) 1.6a (0.58) 1.7a (0.62) 80a (12/15)

3.0b (0.29) 0.9bc (0.45) 0.9bc (0.46) 40ab (6/15)

2.3c (0.23) 0.9b (0.29) 1.0b (0.32) 60ab (9/15)

2.3c (0.36) 0.7c (0.05) 0.7c (0.05) 26.7b (4/15)

Table 2 Mean color values1 (L*, a*, b*) (SD) of broiler breast muscle after treatments in Experiment I. Treatment2

a-c

Means within a row with no common superscripts are different (P < 0.05). 1 Number of observations for each mean, n ¼ 15. 2 TWD/TWD: tap water dip (TWD) (25
C, 45 s) followed by TWD (25
C, 45 s). 2 TWD/TSP: TWD (25
C, 45 s) followed by Trisodium phosphate (TSP) (8%) dip (25
C, 45 s). 2 TWD/HWD: TWD (25
C, 45 s) followed by hot water dip (HWD) (71
C, 45 s). 2 TSP/HWD: TSP (8%) dip (25
C, 45 s) followed by HWD (71
C, 45 s).

L* a* b* a-b

TWD/TWD

TWD/TSP

TWD/HWD

TSP/HWD

44.20a (2.63) 0.99a (0.66) 0.36ab (1.72)

43.40a (2.70) 1.21a (0.84) 0.73a (1.50)

45.59a (3.90) 0.77a (1.43) 0.92b (1.22)

42.55a (4.41) 0.81a (0.92) 0.27ab (1.72)

Means within a row with no common superscripts are different (P < 0.05). Number of observations for each mean, n ¼ 10. Treatments: same as in Table 1.

1 2

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Table 3 Populations (log cfu/g) of MAB, E. coli, and total coliforms and prevalence (%) of Salmonella on broiler skin obtained after four treatments using two sample preparation methods. Treatments1 TWD/TWD MAB E. Coli Total coliforms Salmonella

a

3.6 1.2a 1.5a 77a (23/30)

SEMx TWD/TWD/B a

3.5 1.1a 1.6a 50ab (15/30)

TSP/HWD b

2.4 0.7b 0.7b 23bc (7/30)

TSP/HWD/B c

2.1 0.7b 0.8b 20c (6/30)

Sampling methods2 Stomach

0.07 0.05 0.08

b

2.5 0.9a 1.1b 38.3a (23/60)

SEMy Grind 3.3a 1.0a 1.2a 46.7a (28/60)

0.05 0.04 0.05

a-c and a,b

Means within a row on each side of the divider with no common superscripts are different (P < 0.05). Standard error of mean (SEM) for treatment means pooled by sampling methods. y Standard error of mean (SEM) for sampling method means pooled by treatments. 1 Number of observations for each treatment, n ¼ 30. 1 TWD/TWD: tap water dipping (TWD) (25
C, 45 s) followed by TWD (25
C, 45 s). 1 TWD/TWD/B: TWD (25
C, 45 s) followed by TWD (25
C, 45 s) with intermittent manual brushing (5 s on/5 s off). 1 TSP/HWD: TSP (8%) dipping (25
C, 45 s) followed by HWD (71
C, 45 s). 1 TSP/HWD/B: TSP dipping (25
C, 45 s) followed by HWD (71
C, 45 s) with intermittent manual brushing (5 s on/5 s off). 2 Number of observations for each sampling method, n ¼ 60. 2 Stomaching of a skin sample and grinding of the stomached skin sample. x

significantly reduced (P < 0.05) after TSP/HWD, although no additional reduction was found after TSP/HWD/B, potentially due to low initial populations (1.2e1.5 log CFU/g) compared to MAB (3.6 log CFU/g). Similarly, a significant reduction (P < 0.05) in Salmonella prevalence was observed after TSP/HWD compared to TWD/TWD, with no additional reduction observed for brushing after TSP/HWD/ B (Table 3). In accordance with our tap water dipping results with/without brushing, Berrang and Bailey (2009) found no difference for the carcasses before and after pre-scald brushing in populations of E coli, and coliforms or prevalence of Salmonella. However, postevisceration brushing significantly reduced E. coli and coliforms by ~0.5 log CFU/ml in carcass rinsate. Shackelford, Whittemore, Papa, and Wilson (1992) showed that brushing with thicker bristles removed more total solids from broiler carcasses than thinner bristles. In our study, the combination of TSP/HWD and brushing (TSP/HWD/B) significantly reduced MAB (P < 0.05) by 1.5 and 0.3 log CFU/g over the TWD/TWD and TSP/HWD, respectively (Table 3). In case of E. coli and total coliforms, the TSP/HWD/B reduced the populations over the TWD/TWD but not over the TSP/HWD, presumably due to the low initial population (0.7 log CFU/g) after TSP/ HWD. For comparison of bacterial association to skin, skin stomaching and grinding of the stomached skin were used. Regardless of treatments, fewer MAB and total coliforms were enumerated on the broiler skin after stomaching (namely loosely-associated cells) than after grinding the stomached skin (namely tightly-associated cells) (P < 0.05), whereas no difference between association types was observed for E. coli populations or Salmonella prevalence (Table 3). Again, the result of no significant difference between the association types is expected due to low populations of E. coli and Salmonella. It is generally known that bacteria introduced on poultry migrate and find better protected niches such as hair follicles, ridges, and crevices (Kim, Frank, & Craven, 1996; McNamara, 1997). As a result, those microorganisms are more difficult to eliminate. Lee et al. (2014) found that shaking of broiler skin in a buffer solution recovered fewer MAB and coliforms than grinding of the previously shaken skins. A study using electron microscopy indicated that rinsing or stomaching of broiler skin did not recover bacteria located in deep skin crevices, but recovered after shredding of the skin (Nayak, Kenney, & Bissonnette, 2001). Using confocal microscopy, Chantarapanont, Berrang, and Frank (2003) indicated that a large number of bacteria were still on chicken skin after physical rinsing, primarily inside crevices and feather

follicles. Such bacteria are better protected by the microenvironment and are less accessible to antimicrobial treatments (McMeekin, Thomas, & McCall, 1979; Thomas & McMeekin, 1980). During scalding and picking, the epidermis can be easily removed along with the colonized bacteria. The freshly exposed dermal layer is now vulnerable to colonization, primarily by gramnegative bacteria. Many channels and crevices in the dermal layer allow bacteria to associate tightly (Thomas & McMeekin, 1980), especially when the skin is swollen during water chilling (Chantarapanont et al., 2003; Jeong, Janardhanan, Booren, Karcher, & Kang, 2011). In addition, mutational changes responsible for conformational or antigenic modifications in bacterial surface structures can help bacteria attach and colonize more effectively (Wilson et al., 2010). As the results of better decontamination after immediate TSP than delayed TSP (Fratamico et al., 1996), it is important to use chemical and/or physical interventions immediately after contamination or before tight attachment of bacteria. Our previous research indicated that 83% of MAB, once tightly attached, was detected after grinding broiler skins while 8 and 9% were detected

Table 4 Mean color values1 (L*, a*, b*) (SD) of broiler carcass scapula area skin after treatments in Experiment II. Treatments2

L* a* b*

TWD/TWD

TWD/TWD/B

TSP/HWD

TSP/HWD/B

67.73a (1.56) 3.51ab (1.51) 4.32b (1.57)

66.93a (0.71) 4.6a (1.36) 0.72c (1.03)

61.92b (2.08) 2.64b (1.33) 8.35a (0.97)

61.98b (2.15) 2.83b (0.76) 7.07a (1.37)

a-c

Means within a row with no common superscripts are different (P < 0.05). Number of observations for each mean, n ¼ 6. Treatments: same as in Table 3.

1 2

Table 5 Mean color values1 (L*, a*, b*) (SD) of broiler carcass breast area skin after treatments in Experiment II. Treatments2

L* a* b* a-c

TWD/TWD

TWD/TWD/B

TSP/HWD

TSP/HWD/B

62.70a (0.71) 1.5a (1.64) 1.94bc (1.33)

60.18ab (1.88) 1.47a (1.13) 0.04c (1.65)

58.47bc (1.80) 0.35ab (0.95) 6.15a (2.23)

56c (1.16) 1.13b (0.82) 4.81ab (1.60)

Means within a row with no common superscripts are different (P < 0.05). Number of observations for each mean, n ¼ 3. Treatments: same as in Table 3.

1 1

Fig. 1. Scanning electron microscope image (A1 - G1, 10000 magnification) of skin surface and light microscope image (A2 - G2 and G3, 20 magnification) of histologically stained skin sections for reference and treated skins. A1, A2: Reference-broiler breast skin after bleeding. B1 e G3: Treatments on broilers after scalding and evisceration: same as in Tables 1 and 3 B1, B2: TWD/TWD. C1, C2: TWD/TSP. D1, D2: TWD/HWD. E1, E2: TSP/HWD. F1, F2: TWD/TWD/B. G1, G2, G3: TSP/HWD/B. White arrows point towards red blood cells.

P. Singh et al. / Food Control 77 (2017) 199e209

Fig. 1. (continued).

205

206

P. Singh et al. / Food Control 77 (2017) 199e209

after the first swabbing and the second through 10th swabbing, respectively (Singh et al., 2015). In skin color, both scapula and breast skin became darker (L*) and more yellowish (b*) after TSP/HWD and TSP/HWD/B (P < 0.05) than after TWD/TWD and TWD/TWD/B (Tables 4 and 5). A decrease in breast skin redness (a*) was observed after TSP/HWD/B compared to TWD/TWD and TWD/TWD/B (P < 0.05) (Table 5). When pig carcasses were dipped in 8% TSP at 35
C, Morris, Lucia, Savell, and Acuff (1997) reported that decrease in redness and increase in yellowness were observed compared to the control. However, dipping of chicken in 12% TSP did not affect sensory properties of raw and fried meat as well as consumer purchase intent (Hathcox, Hwang, Resurreccion, & Beuchat, 1995). 3.3. Experiment III: imaging of skin using scanning electron microscopy and histological staining Scanning electron microscope (SEM) images showed that the surface of reference breast skin, obtained before scalding, had an intact epidermal layer with a few natural bacteria (Fig. 1-A1). Light microscope images of histologically stained reference skin also showed no apparent damage to the epidermis (Fig. 1-A2). Control skin (TWD/TWD), obtained from eviscerated carcasses, had oil/fat particles surrounded by various dense debris (Fig. 1-B1). The source of debris might be organic materials from scald tank. The skin surface was hardly visible due to the debris accumulation. These results are in accordance with the SEM images of Kim, Slavik, and Bender (1994) who showed extensive debris on chicken skin after evisceration and water rinsing. Histological images of the control skin (TWD/TWD) showed no epidermis but intact layers of dermis including stratum superficiale and stratum compactum, with no apparent damage or erosion (Fig. 1-B2). Based on these observations, the epidermis appears washed off during scalding. TSP treatment (TWD/TSP) removed most debris and exposed both clean fat particles and connective tissues (Fig. 1-C1), which were expected due to erosion of the dermal stratum superficiale layer and exposure of the dermal stratum compactum layer. The exposed connective tissues appeared swollen, presumably due to water retention at high TSP pH (pH 11e12) (Capita, Alonso-Calleja, Garcia-Fernandez, & Moreno, 2002a; 2002b; Christensen, Zimmermann, Wyatt, & Goodman, 1994). Erosion of the dermis was

supported by the eroded or damaged images at stratum superficiale layer after TSP treatment (Fig. 1-C2). In addition to the skin erosion, red blood cells (white arrows) appeared to be lysed in the histological images after TWD/TSP (Fig. 1-C2), TSP/HWD (Fig. 1-E2), and TSP/HWD/B (Fig. 1-G3) compared to the control (TWD/TWD) (Fig. 1-B2), possibly due to the high pH of TSP, again. Kasschau, Byam-Smith, Gentry, and Watson (1995) showed lysis of red blood cells at pH 7.5. Hot water dipping (TWD/HWD) completely removed surface debris, resulting in a smooth surface with few fat particles (Fig. 1D1). This smooth surface is expected from the precipitation of major dermal components such as glycoproteins, glycosaminoglycans, and proteoglycans (Haake, Scott, & Holbrook, 2001). In the SEM images, no connective tissue was visible after TWD/HWD, potentially due to dissolving of collagen at 50e71
C during hot water dipping (Fig. 1-D1) (Vaclavik & Christian, 2014). Histological images indicated that TWD/HWD caused tissue discoloration in the stratum compactum and partial erosion in the stratum superficiale (Fig. 1-D2), although not as much as the erosion observed after TSP exposure (TWD/TSP) (Fig. 1-C2). After TSP/HWD, SEM images showed a smooth skin surface that resembled the images of TWD/HWD, but with some connective tissue embedded in the dermal layer, possibly due to the trisodium phosphate effect (Fig. 1-E1). Histological images showed tissue discoloration, red blood cell lysis (indicated by arrows), and deeper textural damage in the stratum compactum layer (Fig. 1-E2). These visual appearances helped explain the greater reduction in bacterial populations using TSP/HWD combination than individual treatment (TWD/HWD or TWD/TSP). When brushing on control carcasses (TWD/TWD/B), surface debris was eliminated and connective tissue was exposed, but no dermal layer was eroded as most of the connective tissue was still embedded in the dermal layer (Fig. 1-F1). Histological images showed some structural deformation due to the physical brushing (Fig. 1-F2). However, brushing alone (TWD/TWD/B) was not sufficient to remove bacteria cells as no significant (P > 0.05) bacterial reduction was observed compared to no brush control (TWD/TWD) (Table 3). When trisodium phosphate dip, hot water dip and brushing were combined (TSP/HWD/B), SEM images showed that the skin appearance was similar to the skin after TWD/TSP, but with more

Table 6 Structural changes1 of broiler skin before and after treatments based on light microscopy of histologically stained skin sections and the corresponding percentages of the structural change based on total observations. Treatment 2

Reference TWD/TWD3 TWD/TSP3

TWD/HWD3

TSP/HWD3

TWD/TWD/B3 TSP/HWD/B3

1

Structural change

Corresponding percentage (%)

Intact epidermis and dermis layers Epidermis removed and dermis intact Epidermis removed Erosion of entire stratum superficiale of dermis Lysis of red blood cells Epidermis removed Partial erosion in stratum superficiale of dermis Tissue discoloration Epidermis removed Deep damage in stratum compactum of dermis Lysis of red blood cells Epidermis removed Disorganization in structure Appearance similar to TSP/HWD Sloughing off of epidermis, entire dermal stratum superficiale & part of dermal stratum compactum

100 100 100 90 100 100 50 80 100 100 100 100 56

Number of observations for each treatment, n ¼ 48. Reference-broiler breast skin after bleeding and before scalding. Treatments on broilers after scalding and evisceration: same as in Tables 1 and 3.

2 3

P. Singh et al. / Food Control 77 (2017) 199e209

207

Fig. 2. Scanning electron microscope images (10000 magnification) of skin crevices for reference and treated skins. A: Reference-broiler breast skin after bleeding. BeG: Treatments on broilers after scalding and evisceration: same as in Tables 1 and 3 B: TWD/TWD. C: TWD/TSP. D: TWD/HWD. E: TSP/HWD. F: TWD/TWD/B. G: TSP/HWD/B.

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P. Singh et al. / Food Control 77 (2017) 199e209

and larger fat globules and thinner connective tissue (Fig. 1-G1). The larger fat globules are expected from the deeper skin layer where fat is stored. . Brushing during TSP/HWD/B seemed to penetrate deeper areas, with exposing more fat globules as well as shrinking connective tissue upon hot water exposure (Fig. 1-G1). After TSP/HWD/ B, some microscopic fields of the histologically stained skin sections showed that major portions of the stratum compactum were sloughed off as major blood vessels were not visible, indicating deeper penetration by TSP/HWD/B (Fig. 1-G2). In other fields, TSP/ HWD/B-induced damage looked like the damage after TSP/HWD but with no sloughing off of the dermal layers (Fig. 1-G3). Like in the case of TSP/HWD, the high pH of TSP/HWD/B resulted in lysis of red blood cells (white arrows in Fig. 1-G3). The percentages of structural change described above are shown in Table 6. The thickness of broiler skin is roughly 700e1500 mm (Kondjoyan & Portanguen, 2008). Bacteria can penetrate up to 50 mm with a maximum population between 0 and 10 mm (Chantarapanont et al., 2003; Jang et al., 2007). Since TSP/HWD and TSP/HWD/B can penetrate skin up to 100 mm or more (Fig. 1-E2 and 1-G3), the treatments are expected to be more effective on bacteria in deeper skin than any single application. Despite microscopic evidence showing most severe skin damage after TSP/HWD/B, there was no difference between TSP/HWD and TSP/HWD/B for E. coli, total coliforms, and Salmonella (Table 3). These results can be explained by the low populations of E. coli and total coliforms (0.7 log CFU/g), and low prevalence of Salmonella (23%) on the skin after TSP/HWD. Another possible reason would be thermo tolerance developed by the bacteria in deeper locations during sublethal exposure of trisodium phodphate. Sampathkumar, Khachatourians, and Korber (2004) indicated that exposure of Salmonella to a sublethal dose at 1.5% TSP or pH 10.0 resulted in a significant increase in thermo tolerance. SEM images of skin crevices (Fig. 2) showed a similar pattern of structural changes as seen on the skin surface (Fig. 1), although the intensity of the change was reduced. Reference images on skin crevices, obtained from carcasses after bleeding, showed an intact epidermis with larger numbers of bacteria (Fig. 2-A) than those on smooth surface (Fig. 2-A1). The epidermis layer in crevices was removed along with bacteria during scalding and evisceration (Fig. 2-B to 2-G). The pictures, taken before and after scalding in this study, support the hypothesis that the epidermal layer is removed during scalding with gram positive bacteria colonized previously, and the freshly exposed dermis becomes vulnerable to gut pathogens (Thomas & McMeekin, 1980). Similarly, the debris in skin crevices observed after TWD/TWD (Fig. 2-B) was removed and both connective tissue and fat globules were exposed after TWD/TWD/B (Fig. 2-F). More connective tissue and fat globules were exposed after TWD/TSP (Fig. 2-C) while smoothness surface increased with no visual connective tissue after TWD/HWD (Fig. 2-D). TSP/HWD treated skin (Fig. 2-E) looked similar to TWD/HWD treated skin (Fig. 2-D) except a less smooth surface. After TSP/HWD/B (Fig. 2-G), the skin was less smooth with fewer connective tissues than after TWD/TWD/B and TWD/TSP. In conclusion, six treatments were tested for bacterial decontamination on broiler carcasses after using chemical (TSP) and physical (HW, brushing) interventions with/without combination. Generally, the triple combination (TSP/HWD/B) showed the best decontamination on broiler breast skin than any single and double applications. The superior result was supported by the images of SEM and histological samples, showing deeper skin penetration and disruption. However, the combination of TSP and HWD showed darker and more yellowish appearance that could be a visual defect. Additional research is required to reduce the visual defect using different features such as exposing temperature, time, and brushing intensity.

Acknowledgement The authors thank California Polytechnic State University (San Luis Obispo, CA), Michigan State University (East Lansing, MI), Chonnam National University (Gwangju, South Korea), and National Research Foundation of Korea (NFR) (2012R1A6A3A030411439, Seoul, South Korea) for funding for this research.

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