Effects of Salmonella bacteriophage, nisin and potassium sorbate and their combination on safety and shelf life of fresh chilled pork

Food Control xxx (2016) 1e9

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Effects of Salmonella bacteriophage, nisin and potassium sorbate and their combination on safety and shelf life of fresh chilled pork Changbao Wang a, Jie Yang a, Xiaoyu Zhu a, Yingjian Lu b, Yingying Xue a, Zhaoxin Lu a, * a b

College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 July 2016 Received in revised form 19 September 2016 Accepted 24 September 2016 Available online xxx

Salmonella is an important foodborne pathogen and a serious threat to human health worldwide. This study was to reduce Salmonella and the spoilage bacteria on fresh chilled pork using bacteriophage, nisin, and potassium sorbate (PS) along with their combinations. Microbial, chemical, and sensory qualities of the fresh chilled pork (artificially contaminated with Salmonella 3 log CFU/g) treated with bacteriophage (9 log PFU/g), nisin (5000 IU/g), PS (2 mg/g) and their combinations were evaluated. The result showed that all the samples treated with phage could significantly (P < 0.05) reduce Salmonella population on fresh chilled pork. The combination treatment of nisin, PS and phage (N-PS-P) could significantly lower total viable counts (TVC), TVB-N and TBARS of the chilled pork during the storage period. The TVC of sample treated by N-PS-P was reduced by 2.3 log CFU/g at 7th day. It was also found through the electronic nose detection that the N-PS-P treatment was able to significantly reduce odour and maintain good sensory of the chilled pork. Hence, the N-PS-P treatment extended the shelf life of fresh chilled pork up to 14 days. No adverse effect of the phage on the chilled pork was observed. In conclusion, this study suggests that the phage and its combination with nisin and PS have great potential to be used as a good preservative for fresh chilled pork. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Bacteriophage Nisin Potassium sorbate Salmonella Fresh chilled pork Chemical compounds studied in this article: Nisin (PubChem CID: 16219761) Potassium sorbate (PubChem CID: 23676745)

1. Introduction According to the National Bureau of Statistics of China (NBSC, 2016), the production of pork in China was 54,870,000 tonnes in 2015, and the consumption of chilled pork has been increasing rapidly in China for its better taste and nutrition (Wang et al., 2015). However, the spoilage microorganisms and foodborne pathogens contaminated on meat shorten the shelf-life of chilled pork and often cause economic loss (Malakar, 2013; Tang et al., 2013; Wang et al., 2015). The problems associated with cross-contamination and the resistance of pathogens to antibiotics during processing, storage and distribution, are currently one of the highest meat safety risks (Domenech et al., 2015; Li et al., 2016; Majowicz et al., 2010; Newell et al., 2010). Salmonella is one of the most common food pathogens that contaminated pork products (Rajic et al., 2007; Yang et al., 2016a). Thus, contaminated pork products become a major source of human Salmonella infections in many countries (EFSA, 2011; Scallan et al., 2011). The risk of Salmonella infection

* Corresponding author. E-mail address: [email protected] (Z. Lu).

and illness increased with the elevated amount of consumed pork products (Møller et al., 2013; Teunis et al., 2010). Constant efforts have been made to reduce Salmonella population on pork from farm to consumption (Albino, Rostagno, Húngaro, & MendoncA, 2014; Hooton, Atterbury, & Connerton, 2011; Smid, Heres, Havelaar, & Pielaat, 2012). Bacteriophage (phage) is bacterial virus that has great potential for use as biocontrol agent in foods. Previous studies have demonstrated that phages can be used to successfully reduce Salmonella spp. in foods, especially meat and poultry products (Bigwood, Hudson, Billington, Carey-Smith, & Heinemann, 2008; Guenther, Herzig, Fieseler, Klumpp, & Loessner, ^a, Vanetti, & 2012; Hooton et al., 2011; Hungaro, Mendonça, Gouve Pinto, 2013). However, the narrow antimicrobial spectrum of phage limits its application for extending the shelf life of meat. One method of extending the shelf life of fresh meat is to use natural antimicrobials such as nisin on meat. Nisin, a bacteriocin produced by Lactococcus lactis subsp. Lactis, is the first antimicrobial peptide with a “generally recognized as safe” status in the United States Food & Drug Administration (FDA, 2009), but it only could inhibit gram-positive bacteria and their spores. In addition, potassium sorbate (PS) is the potassium salt of sorbic acid and primarily used

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as a food preservative. The antimicrobial activity of PS is generally used to inhibit gram-negative bacteria or molds and yeasts. PS is effective for its applications on many foods, such as meats, drinks, and chees (FDA, 2009). To date, a few publications have revealed increased effectiveness of combinative natural antimicrobial against both Gram-positive and Gram-negative bacteria and/or pathogens on meat products (Branen & Davidson, 2004; Gill and Holley, 2003). The Salmonella lytic phage preparation (SalmoFresh™) in combination with LAE or CPC significantly reduced (0.5e1.3 log CFU/g) Salmonella counts on chicken breast fillets (Sukumaran, Nannapaneni, Kiess, & Sharma, 2015). The Listeria monocytogenes lytic phage preparation (P100) had been tested individually and in combination with LAE and other chemical antimicrobials (Soni, Desai, Oladunjoye, Skrobot, & Nannapaneni, 2012). However, the combined application of phage, nisin and PS to reduce Salmonella population on fresh chilled pork has remained elusive. This study is undertaken to investigate the possible effect of using phage, nisin and PS individually or their combination to inhibit Salmonella and total viable counts and assess the efficacy of phage on fresh chilled pork. 2. Material and methods 2.1. Salmonella strain and isolation Salmonella Typhimurium (CMCC 50115) was obtained from the National Center for Medical Culture Collections (Beijing, China). The strain was reactivated from a glycerol stock culture, in vials containing 30% (v/v) glycerol in Luria-Bertani (LB) borth kept at
70  C. The isolate was inoculated into 50 mL LB and incubated for 24 h at 37  C with constant and gentle shaking (180 r/min) to obtain ~108
9 CFU/mL concentration of cells prior to experiment. 2.2. Preparation of antimicrobial solutions The lytic Salmonella phage fmb-p1 was isolated from sewage and kept by our lab in 2014. The phage fmb-p1 could lyse seven serotypes of Salmonella such as S. Typhimurium, S. Enteritidis, S. Anatum, S. Miami, S. Agona, S. Saintpaul and S. Paratyphi-C. It was stable under different temperature (40e75  C), pH (4e10) and NaCl solutions (1e11%). High titer stocks of phage were prepared by adding 100 mL of phage (~108
9 PFU/mL) and 100 mL of Salmonella overnight culture (~107
9 CFU/mL) to 100 mL of LB broth. The LB broth mixture was incubated for 12 h at 37  C to allow amplification of the phage. The culture was centrifuged at 10,000  g for 10 min and filtered through 0.22 mm filters (Agela, USA), and the filtrate was stored at 4  C until further use. The phage concentration was determined to be 1011 PFU/mL by soft agar overlay technique (Soni & Nannapaneni, 2010). The phage was diluted by SM buffer (10 mM NaCl, 10 mM MgSO4, 50 mM TrisHCl, pH 7.5) to certain concentration before use. A stock solution of nisin (activity of 1  106 IU/g according to the manufacturer, Sigma) was prepared by dissolving 5 g product in 100 mL volumetric flask with 0.02 mol/L HCl solution and sterilized by filtration through 0.22 mm filters and stored at 4  C before use. The lower concentrations of nisin solutions were prepared by diluting with HCl solution (0.02 mol/L) from the stock solution. The stock solution of potassium sorbate (98%, aladdin) was prepared by dissolving 10.2 g product with sterile ddH2O in 100 mL volumetric flask and sterilized by filtration through 0.22 mm filters. The lower concentrations of PS solutions were prepared with sterile ddH2O from the stock solution. All antimicrobial solutions were freshly prepared before use.

2.3. Preparation of fresh chilled pork Fresh chilled pork was purchased from a local grocery store and then was aseptically cut into pieces (4 cm  4 cm) of approximately 10 g. The meat samples were stored at 4  C for 1 h, then inoculated with 400 mL of S. Typhimurium culture (to achieve the final inoculum level in meat ~ 3 log CFU/g) and kept under biosafety cabinet (room temperature) for 30 min for proper bacterial attachment. Each sample was then surface treated (treatment solutions were uniformly applied over the surface of samples using micropipette) with the assigned phage, antimicrobial, or their combinations (10 log PFU/mL phage, 5% PS, 5% nisin) as shown in Table 1. The combined antimicrobial treatments followed this turns: phage, PS, nisin. Control samples were surface treated with 400 mL of sterile distilled water. All the samples were stored at 4  C for 21 days and taken at 0, 1, 4, 7, 14 and 21 days for microbiological, chemical and sensory evaluation. 2.4. Determination of Salmonella count, total viable counts and phage titer on fresh chilled pork Salmonella count, Total viable counts (TVC, CFU/g) and phage concentrations (PFU/g) were determined immediately after 1, 4, 7, 14 and 21 days of incubation. The Salmonella count was determined as described by Sukumaran et al. (2015) with a few modifications. 10 g samples were homogenized in 90 mL of saline using a stomacher blender (Luqiao, China). To avoid phages from being plated, 10 mL homogenate was centrifuged at 10,000  g for 5 min, supernatant was discarded and pellets were resuspended in 10 mL of sterile saline. 250 mL of the homogenate was plated on to four XLT4 plates, and incubated at 37  C for 24 h. For quantitative determination of TVC, decimal dilutions of aliquots (100 mL) of the homogenates were directly placed on 90 mm Plate Count Agar plates, and incubated at 37  C for 48 h. Infective phage particles recovered from the samples were enumerated as described earlier (Carlton, Noordman, Biswas, Meester, & Loessner, 2005) with some modifications. Aliquots of 100 mL of decimal dilutions from the food samples were mixed with 100 mL host cells and 5 mL LB soft agar. The suspension was poured on solid agar plates and incubated at 37  C for 12 h until plaques could be counted. After incubation, Salmonella count, TVC or plaques were counted and results were reported as log CFU/g. The tests were performed in triplicate. 2.5. Determination of total volatile basic nitrogen (TVB-N) TVB-N content in pork was measured by a stream distillation method according to Chinese standard GB/T 5009.44 (2003). Briefly, all samples for analysis were ground individually using a Panting homogeneous bag (Luqiao, China). 10 ± 0.1 g of the ground pork was treated with 100 mL distilled water for 30 min and shook the beaker every 10 min. After filtration, 5 mL of filtrate and 5 mL of 10 g/L Magnesia (MgO) were added into a Kjeldahl distillation unit (ZLQ03, East China Glass, China). Steam distillation was distilled for 5 min. The distillate was absorbed by 10 mL of 20 g/L boric acid, and then titrated with 0.01 mol/L HCl. The amount of TVB-N was calculated using the following equation. The result stated for each sample is the mean value of three measurements:

TVB
Nðmg=100gmeatÞ ¼

ðV1
V2 Þ  c  14  100 m  5=100

Where V1 is the titration volume for the tested sample (mL), V2 is the titration volume of blank (mL), and c is the actual concentration of HCl (mol/L), m is the weight of ground pork sample (g).

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Table 1 Formulation of antimicrobial solutions used for surface treatment on fresh chilled pork. Treatments

Final concentration

Salmonella (4 logCFU/mL)

Phage (10 logPFU/mL)

PS (5%)

Nisin (5%)

CK1 CK2 P N PS N-P PS-P N-PS N-PS-P

Control Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella

0 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4

0 0 0.4 0 0 0.4 0.4 0 0.4

0 0 0 0 0.4 0.4 0 0.4 0.4

0 0 0 1.0 0 0 1.0 1.0 1.0

(103 (103 (103 (103 (103 (103 (103 (103

CFU/g) CFU/g)þPhage (109 PFU/g) CFU/g)þNisin (5000IU/g) CFU/g)þPotassium sorbate (2 mg/g) CFU/g)þNisin (5000IU/g)þPhage (109 PFU/g) CFU/g)þPotassium sorbate (2 mg/g)þPhage (109 PFU/g) CFU/g)þNisin (5000IU/g)þPotassium sorbate (2 mg/g) CFU/g)þNisin (5000IU/g)þPotassium sorbate (2 mg/g)þPhage (109 PFU/g)

mL mL mL mL mL mL mL mL

mL

mL mL mL

mL mL mL mL

mL

mL mL mL

CK1: control without Salmonella; CK2: inoculated control with Salmonella; P: treated with phage. N: treated with nisin; PS: treated with potassium sorbate; N-P: treated with phage and nisin. PS-P: treated with phage and potassium sorbate; N-PS: treated with nisin and potassium sorbate. N-PS-P: treated with phage, nisin and potassium sorbate.

2.6. Determination of thiobarbituric acid reactive sub-stances (TBARS) The TBARS value was evaluated according to the method described by Yang, Lee, Won, and Song (2016b) with slight modifications. 2.0 ± 0.1 g of pork sample was treated with 10 mL of 7.5% trichloroacetic acid using a Panting homogeneous bag (Luqiao, China). After filtration, the filtrate (5 mL) was mixed with the TBA reagent (5 mL of 0.02 M 2-thiobarbituric acid) and was boiled at 95  C for 40 min. The reactive substances were then cooled using cold water, and the absorbance of the solution was determined at 532 nm using a 96-Well Plate Reader MQX200 (uQuant, Bio-Tek). The TBARS value was expressed as mg malonaldehyde (MDA)/kg meat. The tests were performed in triplicate. 2.7. Determination of pH of fresh chilled pork

(2016). Each sample was read and recorded for six times. Meat odour was determined by the PEN3 portable E-nose (Win Muster Airsense Analytics Inc., Schwerin, Germany) contains a detector unit composed of an array of 10 different metal oxide semiconductor (MOS)-type chemical sensors: W1C (S1, aromatic), W5S (S2, broad-range), W3C (S3, aromatic), W6S (S4, hydrogen), W5C (S5, aromatic aliphatics), W1S (S6, broad-methane), W1W (S7, sulfur organic), W2S (S8, broad alcohol), W2W (S9, sulfur chlorinate), and W3S (S10, methane aliphatics). 3.0 ± 0.1 g of pork was placed into a vial (20 mL) and heated at 50  C for 5 min to allow for development of headspace before analysis. Then the headspace air was automatically injected into the e-nose by a syringe at a flow rate of 500 mL/min during the measurement, and sensor responses were recorded for 60 s. Recovery time for the sensors was 120 s, during which they were flushed with fresh air. The tests were performed in triplicate. The pork samples were tested at 0, 1, 4, 7, 14 and 21 days.

The pH of fresh chilled pork was evaluated according to Miao et al. (2015). The determination of pH was performed with a digital pH meter (Eutech pH510, Thermo, Germany). 2.9. Statistical analysis 2.8. Determination of meat color and odour Color was described as coordinates: lightness (L), redness (a) and yellowness (b). Meat color was measured using a chromatic meter (HP-2132, China) following the method of Zhang et al.

Experiment was carried out in triplicate and the results were expressed as mean ± standard deviations. All data were analyzed using One-way ANOVA (SPSS 17.0, SPSS Inc., Chicago, USA), the significance of differences was defined at P < 0.05.

Table 2 Reduction of Salmonella on fresh chilled pork by phage, nisin and potassium sorbate treatment during storage at 4  C for 21 days. Treatments

Salmonella number (log CFU/g) Time(d) 0

CK1 CK2 P N PS N-P PS-P N-PS N-PS-P

NE 3.07 3.07 3.07 3.07 3.07 3.07 3.07 3.07

1 ± ± ± ± ± ± ± ±

0.13Aa 0.13Aa 0.13Aa 0.13Aa 0.13Aa 0.13Aa 0.13Aa 0.13Aa

NE 3.06 ± <1.00 3.04 ± 2.99 ± <1.00 <1.00 2.97 ± <1.00

4 0.10Aa 0.08Aa 0.11Aa

0.07Aa

NE 3.08 ± <1.00 3.09 ± 3.01 ± <1.00 <1.00 3.02 ± <1.00

7 0.11Aa 0.08Aa 0.09Aa

0.12Aa

NE 3.03 ± <1.00 3.02 ± 2.98 ± <1.00 <1.00 3.00 ± <1.00

14 0.10Aa 0.11Aa 0.15Aa

0.20Aa

NE 3.05 ± <1.00 3.10 ± 3.07 ± <1.00 <1.00 3.02 ± <1.00

21 0.11Aa 0.09Aa 0.04Aa

0.12Aa

n/a n/a n/a n/a n/a n/a n/a 3.03 ± 0.14Aa <1.00

CK1: control without Salmonella; CK2: inoculated control with Salmonella; P: treated with phage. N: treated with nisin; PS: treated with potassium sorbate; N-P: treated with phage and nisin. PS-P: treated with phage and potassium sorbate; N-PS: treated with nisin and potassium sorbate. N-PS-P: treated with phage, nisin and potassium sorbate. Mean values in the same row with the same capital letter under the same day are not significantly different (P > 0.05) and mean values in the same column with the same lowercase letter are not significantly different (P > 0.05). NE: negative control for Salmonella. n/a: not available. Detection limit: 1.00 log PFU/g.

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3. Results and discussion 3.1. Efficacy of the phage and antimicrobial combinations in reducing Salmonella on fresh chilled pork Table 2 shows the Salmonella counts on fresh chilled pork after treatment with the phage, nisin and PS individually as well in combinations. The initial Salmonella population was 3.07 log CFU/g on fresh chilled pork. Salmonella counts were significantly (P < 0.05) reduced to be lower than detection limit by all the phage treatments (P, N-P, PS-P, N-PS-P). However, treatments of nisin (5%) and PS (5%) individually and in combination without phage did not decrease the Salmonella counts (P > 0.05) on fresh chilled pork. The results implied that the phage killed specially Salmonella, but nisin and PS were not available to inhibit Salmonella. The high efficacy of the phage to eliminate Salmonella on different meat products had also been proved in previous studies. Phage P22 (1012 PFU/mL) was found to be effective in reducing S. Typhimurium by 2 log CFU/g in sliced chicken breast and minced chicken stored at 4  C (Zinno, Devirgiliis, Ercolini, Ongeng, & Mauriello, 2014). Spray application with phage cocktail (1010 PFU/mL) was found to be more than 4 and 2 log/cm2 reduction of S. Typhimurium and S. Enteritidis in pig skin, s, & Llagostera, 2013). Phage respectively (Spricigo, Bardina, Corte preparation (6 Salmonella lytic monophages, 109 and 108 PFU/mL) in combination with cetylpyridinium chloride or lauric arginate resulted in reductions of Salmonella ranging from 0.5 to 1.3 log CFU/ g on chicken breast fillets at refrigerated storage (Sukumaran et al., 2015). In this study, phage reduced Salmonella by more than 2 log CFU/g on fresh chilled pork was very efficient compared to the previous reports. In addition, Norhana, Poole, Deeth, and Dykes (2012) reported that Nisin (500 IU/mL), EDTA (0.02 M), PS (3%, w/ v) failed to reduce Salmonella counts on shrimps at 4  C for 7 days. Tu and Mustapha (2002) also found that nisin (5000 IU/mL) or nisin-EDTA (5000 IU/mL-0.02 M) failed to reduce S. Typhimurium counts on beef treated at 4  C for 25 days. Hence, these results suggested that phage was very efficient to eliminate S. Typhimurium and improve the safety of foods. No significant synergistic activity in reducing Salmonella counts on fresh chilled pork was observed among the three antimicrobials. In addition, the stability of the phage on fresh chilled pork was monitored over the storage of 21 days at 4  C. There was no significant loss in the phage infectivity and the phage was stable during the experiment period (Table 3), suggested that phage was not affected by nisin solution and PS solution. 3.2. Efficacy of the phage and antimicrobial combinations on TVC of fresh chilled pork TVC is an important indicator for meat quality and common used in shelf-life studies (Miao et al., 2015; Rahman, Wang, & Oh, 2013). As shown in Fig. 1, the initial TVC of CK1 and CK2 was 3.7

Fig. 1. Changes in total viable counts in pork samples during storage at 4  C for 21 days. Samples were inoculated with Salmonella cells (1.1  10 3 CFU/g) at time zero. Phage (1.1  10 9 PFU/g), nisin (5000 IU/g) or potassium sorbate (2 mg/g) was applied 30 min later. CK1: control without Salmonella; CK2: inoculated control with Salmonella; P: treated with phage; N: treated with nisin; PS: treated with potassium sorbate; N-P: treated with phage and nisin; PS-P: treated with phage and potassium sorbate; N-PS: treated with nisin and potassium sorbate; N-PS-P: treated with phage, nisin and potassium sorbate.

and 3.8 log CFU/g, respectively. The TVC of combined treatments of nisin and PS were significantly (P < 0.05) decreased at 4th day, and TVC of N-PS and N-PS-P was 5.78 and 5.38 log CFU/g at 14th day, respectively, which was lower than the prescribed limits (6.0 log CFU/g) of Chinese standard GB/T9959.2 (2008). This data indicated that nisin and PS were able to inhibit other bacteria. However, TVC of CK1, CK2 and P was 6.02, 6.31 and 6.20 log CFU/g at 4th day, respectively, which indicated that the phage could not inhibit other bacteria growth except of Salmonella. TVC of N, PS, N-P were also more than 6.0 log CFU/g at 7th day, and TVC of all the treated samples were in excess of 6.0 log CFU/g at 21st day. The combined treatment N-PS-P had the lowest TVC during 21 days storage, suggested that N-PS-P treatment was the best antimicrobial combination for inhibiting the TVC on fresh chilled pork. The spoilage microorganisms and foodborne pathogens are much easier to grow on fresh meat and often cause spoilage of meat (Zhou, Xu, & Liu, 2010). The efficacy of antimicrobials is usually associated with microbial species. The main foodborne pathogens on fresh pork include gram-negative bacteria of Escherichia spp., Salmonella spp. and gram-positive bacteria of Pseudomonas spp., Listeria spp. (Tsigarida, Skandamis, & Nychas, 2000). Some of these microorganisms including Pseudomonas spp. and Listeria spp. could grow well after storage at low temperature that limited the shelf

Table 3 Changes in phage titer in pork samples during storage at 4  C for 21 days. Treatments

Phage titer (log PFU/g) Time(d) 0

P N-P PS-P N-PS-P

9.42 9.42 9.42 9.42

1 ± ± ± ±

0.33Aa 0.33Aa 0.33Aa 0.33Aa

9.25 9.28 9.33 9.47

4 ± ± ± ±

0.14Aa 0.02Aa 0.22Aa 0.25Aa

9.24 9.17 8.99 8.93

7 ± ± ± ±

0.10Aa 0.17Aa 0.58Aa 0.48Aa

9.00 9.05 9.01 9.03

14 ± ± ± ±

0.14Aa 0.08Aa 0.12Aa 0.06Aa

8.97 9.29 9.20 9.05

21 ± ± ± ±

0.07Aa 0.17Aa 0.11Aa 0.06Aa

n/a n/a n/a 9.02 ± 0.10Aa

P: treated with phage; N-P: treated with phage and nisin; PS-P: treated with phage and potassium sorbate; N-PS-P: treated with phage, nisin and potassium sorbate. Mean values in the same row with the same capital letter under the same day are not significantly different (P > 0.05) and mean values in the same column with the same lowercase letter are not significantly different (P > 0.05). n/a: not available.

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life of fresh pork (Miao et al., 2015; Zhang, Liu, Li, & Qu, 2010). The combined treatment N-PS-P could cumulatively inhibit these microorganisms and extend the shelf life of fresh chilled pork about two weeks. The concentrations (5%) of nisin and PS used in this work were higher than the permissible level for PS and nisin (FDA, 2009). Similar and even higher concentrations of PS had been used as dipping solutions on sliced pork bologna (Samelis et al., 2005), beef franks (Uhart, Ravishankar, & Maks, 2004), and chicken luncheon meat (Islam, Chen, Doyle, & Chinnan, 2002). The aim of using a higher concentration of nisin and PS was that we wanted to extend the shelf life of fresh meat to observe phage efficacy on fresh pork, but we did not expect all the antimicrobial molecules in the solution would be absorbed into the pork products. In addition, fresh chilled pork is usually washed before cooking, so the final concentration of nisin and PS in pork may be lower than the permissible level of FDA.

3.4. Efficacy of the phage and antimicrobial combinations on pH of fresh chilled pork

3.3. Efficacy of the phage and antimicrobial combinations on TVB-N of fresh chilled pork

3.5. Efficacy of phage and antimicrobial combinations on TBARS of fresh chilled pork

As shown in Fig. 2, the initial TVB-N of all the pork samples was about 7 mg/100 g and then gradually increased with storage time to 21 days at 4  C. TVB-N of CK1 and P group reached to 16.8 and 15.9 mg/100 g at 4th day, respectively, which had exceeded the limitation (15 mg/100 g of meat) of the Chinese hygienic standard (GB 2707-2005) for fresh meat of livestock. At 14th day, TVB-N of PS-P and N-PS was higher than 15 mg/100 g, respectively, whereas TVBN of N-PS-P was 12.1 mg/100 g. However, TVB-N of all the treated samples had exceeded the limited standard at 21st day. The result indicated that N-PS-P could significantly extend the shelf life of fresh chilled pork. TVB-N usually contains ammonia, trimethylamine (TMA) and dimethylamine (DMA) in spoilage pork. The results of TVB-N were in good agreement with the TVC results (Fig. 1), indicated that TVB-N was mainly produced by spoilage bacteria or from enzymatic degradation during the storage of fresh chilled pork (Cai, Chen, Wan, & Zhao, 2011; Miao et al., 2015).

TBARS is a common indicator to assess the freshness of meat and usually associated with lipid oxidation. As shown in Fig. 4, the initial TBARS of the chilled pork was 0.49 MDA mg/kg. The TBARS of all the samples increased slowly during storage at 4  C. At 7th day, the TBARS of CK1, CK2, P and N were exceeded 1.00 MDA mg/kg, indicating that a high level of lipid oxidation might happen. However, it was unimaginable that the TBARS of all the PS treatments (PS, PS-P, N-PS and N-PS-P) were increased to 1.50 MDA mg/kg at first day, and then gradually increased. But the TBARS value of NPS-P was the lowest one among the four treatments with PS during the 21 days storage. Kolsarici and Candogan (1995) reported that PS could affect the TBARS value of the meat, but sensory analysis panel members could not distinguish between PS samples and control samples, PS treatment was considered to be acceptable. Hsu and Sun (2006) reported that PS treatment resulted in decreases in bacterial counts and high TBARS values in emulsified meatball, but PS had no significant effects on TBARS values, and TBARS value might be enlarged due to the test method adopted. These data

Fig. 2. Changes in total volatile basic nitrogen (TVB-N) of pork samples during storage at 4  C for 21 days. CK1: control without Salmonella; CK2: inoculated control with Salmonella; P: treated with phage; N: treated with nisin; PS: treated with potassium sorbate; N-P: treated with phage and nisin; PS-P: treated with phage and potassium sorbate; N-PS: treated with nisin and potassium sorbate; N-PS-P: treated with phage, nisin and potassium sorbate.

As displayed in Fig. 3, the pH of fresh chilled pork was about 5.7. The pH of all the chilled pork increased gradually with increase of storage time, but the different antibacterial treatments resulted in varying pH of the samples. The combined treatment retarded the increase of pH during 21 days storage at 4  C, of which N-PS-P treatment was best efficacy for inhibiting pH in chilled pork. The reason should attribute to N-PS-P effectively inhibiting the growth of microorganisms. The higher TVC may account for the higher pH of the samples of CK1, CK2, P, N, PS, N-P and PS-P, compared to NPS-P after 14 days storage. These results were also in agreement with Tan and Shelef's (2002) and Rahman's (2013) reports. The elevating pH is mainly caused by degradation of proteins and production of amines in pork, and the higher pH and longer aging periods will result in increased microbial proliferation and decreased shelf-life (Holmer et al., 2009).

Fig. 3. Changes in pH value of pork samples during storage at 4  C for 21 days. CK1: control without Salmonella; CK2: inoculated control with Salmonella; P: treated with phage; N: treated with nisin; PS: treated with potassium sorbate; N-P: treated with phage and nisin; PS-P: treated with phage and potassium sorbate; N-PS: treated with nisin and potassium sorbate; N-PS-P: treated with phage, nisin and potassium sorbate.

Please cite this article in press as: Wang, C., et al., Effects of Salmonella bacteriophage, nisin and potassium sorbate and their combination on safety and shelf life of fresh chilled pork, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.034

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C. Wang et al. / Food Control xxx (2016) 1e9

indicated that PS might affect the tested TBARS values of pork due to the test method, but did not mean the high level of lipid oxidation happened.

3.6. Efficacy of phage and antimicrobial combinations on pork color and odour The changes in chilled pork color of all treatments during storage were given in Table 4. The L (lightness) and a (redness) values decreased slowly during storage at 4  C. The L values and a values were significantly changed for CK1, CK2, P, N, PS, N-P and PS-P at 14th day, except of N-PS-P. In addition, the b (yellowness) values increased slowly during storage at 4  C for all samples, whereas the combined treatment N-PS-P had lower b values than other treatments at 7th day and 14th day, CK1 and CK2 had the highest values at 7th day and 14th day (Table 4). Meat color was affected by the amount of myoglobin and the proportions of its forms, including bright-red oxymyoglobin (MbO2), red myoglobin (Mb) and graybrown MetMb, and the discoloration of fresh meat was mainly caused by the increase of the amount of MetMb (Zhang et al., 2016). Hence, the above results suggested that the combined treatment of N-PS-P would not cause any negative effects on color of fresh chilled pork. Fig. 5 showed the polar plots of the average response signals of each sensor at 55e59 s for fresh chilled pork by E-nose detection. The sensors provided similar signals pattern for CK2 and N-PS-P,

Fig. 4. Changes in TBARS value of pork samples during storage at 4  C for 21 days. CK1: control without Salmonella; CK2: inoculated control with Salmonella; P: treated with phage; N: treated with nisin; PS: treated with potassium sorbate; N-P: treated with phage and nisin; PS-P: treated with phage and potassium sorbate; N-PS: treated with nisin and potassium sorbate; N-PS-P: treated with phage, nisin and potassium sorbate.

Table 4 Changes in Lightness (L), Redness (a) and Yellowness (b) of pork samples during storage at 4  C for 21 days. Treatments

Time(d) 0

Lightness (L) CK1 CK2 P N PS N-P PS-P N-PS N-PS-P Redness (a) CK1 CK2 P N PS N-P PS-P N-PS N-PS-P Yellowness (b) CK1 CK2 P N PS N-P PS-P N-PS N-PS-P

1

48.7 48.7 48.7 48.7 48.7 48.7 48.7 48.7 48.7

± ± ± ± ± ± ± ± ±

0.6Aa 0.6Aa 0.6Aa 0.6Aa 0.6Aa 0.6Aa 0.6Aa 0.6Aa 0.6Aa

4

49.0 49.8 48.8 49.6 49.3 48.8 48.6 48.9 49.1

± ± ± ± ± ± ± ± ±

0.8Aa 0.7Aa 0.4Aa 1.0Aa 1.1Aa 0.8Aa 0.6Aa 0.5Aa 0.7Aa

7

48.9 48.1 48.0 47.8 48.4 48.4 49.0 48.3 47.9

± ± ± ± ± ± ± ± ±

0.4Aa 0.9Aa 0.4Aa 0.8Aa 0.4Aa 0.3Aa 1.0Aa 0.8Aa 0.7Aa

14

48.5 47.8 47.7 46.8 47.3 47.5 47.8 47.3 47.2

± ± ± ± ± ± ± ± ±

0.8Aa 1.0Aa 0.5Aa 0.8Aa 0.9Aa 1.3Aa 0.4Aa 0.4Aa 0.3Ab

21

45.9 44.6 44.7 44.3 45.0 45.6 44.9 46.4 47.0

± ± ± ± ± ± ± ± ±

0.5Ba 0.6Ba 0.8Ba 1.0Ba 0.3Ba 0.8Ba 0.8Ba 0.5Aa 0.6Ab

n/a n/a n/a n/a n/a n/a n/a 45.1 ± 0.7Ba 45.7 ± 0.7Aa

7.8 7.8 7.8 7.8 7.8 7.8 7.8 7.8 7.8

± ± ± ± ± ± ± ± ±

0.3Aa 0.3Aa 0.3Aa 0.3Aa 0.3Aa 0.3Aa 0.3Aa 0.3Aa 0.3Aa

9.1 8.6 8.1 8.1 7.9 8.3 8.7 8.7 8.5

± ± ± ± ± ± ± ± ±

0.3Ba 0.4Ba 0.7Aa 0.3Aa 0.6Aa 0.7Aa 0.4Ba 0.8Aa 0.5Aa

7.2 7.4 7.3 7.7 7.5 8.2 8.6 8.5 8.4

± ± ± ± ± ± ± ± ±

0.3Aa 0.2Aa 0.4Aa 0.5Aa 0.4Aa 0.6Aa 0.4Aa 0.5Aa 0.3Aa

6.9 6.2 5.6 7.6 6.5 7.2 7.5 8.1 8.2

± ± ± ± ± ± ± ± ±

0.5Aa 0.2Aa 0.5Aa 0.5Aa 0.4Aa 0.2Aa 0.7Aa 0.7Aa 0.1Ab

5.6 5.7 5.4 6.2 6.2 5.7 5.3 6.3 7.9

± ± ± ± ± ± ± ± ±

0.4Ba 0.2Ba 0.2Ba 0.5Ba 0.5Ba 0.4Ba 0.2Ba 0.5Ba 0.2Ab

n/a n/a n/a n/a n/a n/a n/a 5.8 ± 0.2Ba 7.1 ± 0.4Bb

2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5

± ± ± ± ± ± ± ± ±

0.2Aa 0.2Aa 0.2Aa 0.2Aa 0.2Aa 0.2Aa 0.2Aa 0.2Aa 0.2Aa

3.0 2.5 2.4 3.2 2.6 2.9 2.6 2.8 2.7

± ± ± ± ± ± ± ± ±

0.3Aa 0.3Aa 0.3Aa 0.3Aa 0.2Aa 0.2Aa 0.2Aa 0.1Aa 0.2Aa

3.5 3.9 3.8 3.6 3.3 3.1 3.2 3.0 3.1

± ± ± ± ± ± ± ± ±

0.3Ba 0.2Ba 0.3Ba 0.2Ba 0.4Ba 0.1Ba 0.3Ba 0.2Ba 0.2Ba

4.6 4.4 4.4 4.1 4.0 3.6 3.8 3.4 3.3

± ± ± ± ± ± ± ± ±

0.4Ca 0.3Ca 0.4Ca 0.1Ca 0.3Ca 0.2Cb 0.2Cb 0.3Bb 0.2Bc

5.8 5.4 4.5 4.6 4.3 4.6 5.9 4.3 3.5

± ± ± ± ± ± ± ± ±

0.4Da 0.3Da 0.5Db 0.3Db 0.2Db 0.2Db 0.4Da 0.2Cb 0.2Bc

n/a n/a n/a n/a n/a n/a n/a 5.1 ± 0.3Da 3.9 ± 0.1Cb

CK1: control without Salmonella; CK2: inoculated control with Salmonella; P: treated with phage. N: treated with nisin; PS: treated with potassium sorbate; N-P: treated with phage and nisin. PS-P: treated with phage and potassium sorbate; N-PS: treated with nisin and potassium sorbate. N-PS-P: treated with phage, nisin and potassium sorbate. Values are means ± standard deviations of three replicates experiments. Mean values in the same row with the same capital letter under the same day are not significantly different (P > 0.05) and mean values in the same column with the same lowercase letter are not significantly different (P > 0.05). n/a: not available.

Please cite this article in press as: Wang, C., et al., Effects of Salmonella bacteriophage, nisin and potassium sorbate and their combination on safety and shelf life of fresh chilled pork, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.034

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Fig. 5. Changes in odour (electronic nose) of pork samples and response of sensor W1W (S7) and W2W (S9) of pork samples during storage at 4  C for 21 days. CK2: inoculated control with Salmonella; N-PS-P: treated with phage, nisin and potassium sorbate.

but the response strengths of S4, S6, S7, S8 and S9 were different. The S4, S6 and S8 signals represented aromatic compounds, which existed in the common pork. The S7 and S9 sensor signals expressed special chemicals with sulfur atom in deterioration meat. The combined treatment N-PS-P produced spoilage odour at 21st day, whereas CK2 did spoilage odour after 4 days storage at 4  C. This result indicated that N-PS-P treatment delayed the spoiled time of fresh chilled pork owing to inhibiting bacteria growth. Fig. 5 displayed the evolution of a typical response generated by

the sensor array composed of S7 and S9 sensors. When the E-nose was exposed to the volatiles of pork samples, the conductivity ratio (G/G0) of S7 and S9 in CK2 was more than 25 and 16 at 14th day, respectively; whereas the G/G0 of S7 and S9 in N-PS-P was 1.5 and 2.5, respectively. However, the G/G0 of S7 and S9 in N-PS-P increased rapidly at 21st day, which indicated that the chilled pork began to deteriorate. This further proved that treatment N-PS-P was able to reduce the spoilage odour and kept quality of fresh chilled pork.

Please cite this article in press as: Wang, C., et al., Effects of Salmonella bacteriophage, nisin and potassium sorbate and their combination on safety and shelf life of fresh chilled pork, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.034

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C. Wang et al. / Food Control xxx (2016) 1e9

The prevalence of Salmonella in slaughtered pigs and pork products was high worldwide. It is difficult to eliminate food-borne pathogenic bacteria such as Salmonella contaminated on fresh chilled meat using chemical antimicrobials or without impact on quality and sensorial properties of the meat product (Hungaro et al., 2013). This work showed that phage was very useful to lyse Salmonella contaminated on chilled pork without any adverse effect on sensorial properties of chilled pork. Although nisin and PS could not reduce Salmonella contaminated on fresh chilled pork, they had been approved and widely used in food industry, and could inhibit the other bacteria and play a key role in extending the shelf life of fresh chilled pork. Hence, the combination of phage, nisin and potassium sorbate could improve the safety and extend shelf life of fresh chilled pork up to 14 days at 4  C. 4. Conclusion In conclusion, the combined treatment of bacteriophage, nisin and potassium sorbate on fresh chilled pork was able to significantly reduce Salmonella counts, inhibit bacterial growth, improve organoleptic properties and extend the shelf life of fresh chilled pork up to 14 days. This result could meet the demand of transportation and storage of fresh chilled pork in commerce. No adverse effect of phage on chilled pork was observed during the study. This study indicated that phage and phage in combination with nisin and potassium sorbate have potential to be used as a good preservative on fresh chilled pork. However, further studies are necessitated to determine the changes of the nutritional components of fresh chilled pork treated by phage, antimicrobials and their combinations. Acknowledgements The authors would like to thank the National Research Program of China (No. 2015BAD16B04) and the Priority Academic Program Development of the Jiangsu Higher Education Institutions (PAPD) and Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control for financial support. References Albino, L. A. A., Rostagno, M. H., Húngaro, H. M., & Mendonca, R. C. S. (2014). Isolation, characterization, and application of bacteriophages for Salmonella spp. biocontrol in pigs. Foodborne Pathogens & Disease, 11(8), 602e609. Bigwood, T., Hudson, J. A., Billington, C., Carey-Smith, G. V., & Heinemann, J. A. (2008). Phage inactivation of foodborne pathogens on cooked and raw meat. Food Microbiology, 25, 400e406. Branen, J. K., & Davidson, P. M. (2004). Enhancement of nisin, lysozyme, and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin. International Journal of Food Microbiology, 90(1), 63e74. Cai, J., Chen, Q., Wan, X., & Zhao, J. (2011). Determination of total volatile basic nitrogen (TVB-N) content and Warner-Bratzler shear force (WBSF) in pork using Fourier transform near infrared (FT-NIR) spectroscopy. Food Chemistry, 126(3), 1354e1360. Carlton, R. M., Noordman, W. H., Biswas, B., Meester, E. D. D., & Loessner, M. J. (2005). Bacteriophage p100 for control of Listeria monocytogenes, in foods: Genome sequence, bioinformatic analyses, oral toxicity study, and application. Regulatory Toxicology & Pharmacology, 43(3), 301e312. Domenech, E., Jimenez-Belenguer, A., Amoros, J. A., Ferrus, M. A., & Escriche, I. (2015). Prevalence and antimicrobial resistance of Listeria monocytogenes and Salmonella strains isolated in ready-to-eat foods in Eastern Spain. Food Control, 47, 120e125. European Food Safety AuthoritydEFSA. (2011). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2009. EFSA Journal, 9, 1e378. Available from: http://www.efsa. europa.eu/en/efsajournal/pub/2090.htm (Accessed October, 2012). Food and Drug Administration (FDA). (2009). Listing of food additives status. Available at: http://www.fda.gov/Food/FoodIngredientsPackaging/FoodAdditives/ FoodAdditiveListings/ucm091048.htm. Gill, A. O., & Holley, R. A. (2003). Interactive inhibition of meat spoilage and pathogenic bacteria by lysozyme, nisin and EDTA in the presence of nitrite and sodium chloride at 24 C. International Journal of Food Microbiology, 80(3),

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Please cite this article in press as: Wang, C., et al., Effects of Salmonella bacteriophage, nisin and potassium sorbate and their combination on safety and shelf life of fresh chilled pork, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.034