1 Use of Oriental Mustard and Allyl Isothiocyanate to Control Salmonella, Campylobacter and L. monocytogenes in Poultry Meat Products Amin Naser Olaimat PhD Candidate Department of Food Science University of Manitoba Canadian Meat Council's 94 th Annual Conference Toronto, ON, Canada, May 7-9, 2014

 Every year, 48 and 4 million cases of foodborne illnesses occur in USA and Canada, respectively.  Meat and meat products, particularly poultry meats, are the major category linked to bacterial foodborne illness outbreaks. 2

 The interest in antimicrobials derived from natural sources has increased in the past few years.  Natural compounds are used in processed food as antimicrobials  extend the shelf life of food.  reduce health hazards and economic losses. 3

4 Glucosinolate Isothiocyanate (ITC)  Mustard is the major spice used in meat manufacturing.  Mustard and other vegetables (broccoli, horseradish and wasabi) contain high levels of glucosinolates (GSLs)

 GSLs also can be converted into ITC by bacterial myrosinase-like enzyme action present in E. coli O157:H7, Campylobacter, Salmonella and L. monocytogenes and other microorganisms. 5

 Edible films or coatings provide a novel option to improve the safety and shelf-life of food.  The antimicrobials in films and coatings are gradually released at the surface of food.  Lower concentrations are required to inhibit foodborne pathogens. 6

 Chitosan is a derivative of chitin and has good-film forming, antimicrobial and biocompatibility properties.  K-carrageenan, a sulfated polysaccharide extracted from edible red seaweeds, has excellent functional properties that facilitate gelling, thickening, emulsifying and stabilizing characteristics, and it is widely used in the food industry. 7

 Determine the inhibitory and bactericidal effects of AITC against Salmonella, Campylobacter and L. monocytogenes.  Screen these pathogens for their ability to degrade sinigrin.  Study factors affecting the sinigrin degradation by these pathogens. 8

 Determine the antimicrobial effects of oriental mustard extract and AITC directly incorporated in an edible coating against:  L. monocytogenes on cooked cured chicken meat.  Salmonella and Campylobacter in fresh, refrigerated, packaged chicken. 9

10 Bacteria 21ºC37ºC MICMBCMICMBC 5 Salmonella strains L. monocytogenes strains Campylobacter strainsNT MIC: the lowest concentration at which no visible growth occurred MBC: the lowest concentration causing a 99.9% reduction of the initial inoculum NT: Not tested

11 Day 4ºC10ºC21ºC pH5pH7pH9pH5pH7pH9pH5pH7pH a d 0.46 cd 0.63 bcd 1.10 b 1.26 b 0.71 bc 3.59 a 3.67 a 0.43 cd e 0.78 e 0.07 e 2.15 d 2.58 cd 0.45 e 7.39 a 4.31 b 3.39 bc ef 1.76 e 0.07 f 3.77 cd 4.58 bc 0.16 f 8.11 a 5.70 b 2.62 de c 2.16 c 0.13 d 6.48 a 4.45 b 0.14 d 7.90 a d 2.84 c Means from each sampling time in the same row with the same letters are not significantly different (p>0.05)

12 Day 4ºC10ºC21ºC pH5pH7pH9pH5pH7pH9pH5pH7pH a c 0.73 c 0.34 c 0.01 c 2.38 b c 1.89 b 8.30 a 0.56 c ef 1.76 d 0.31 ef 0.42 e 5.76 c f 7.15 b 8.88 a 0.23 ef d 2.95 c 0.25 d 0.61 d 7.87 b e 7.47 b 8.85 a 0.10 d d 4.14 c d 3.07 c 8.45 a 0.02 d 5.93 b 8.46 a 1.30 d Means from each sampling time in the same row with the same letters are not significantly different (p>0.05)

13 Day 4ºC10ºC21ºC pH5pH7pH9pH5pH7pH9pH5pH7pH a 10.0 a a a 81.6 a ND d 67.8 ab 53.1 bc ND d 62.0 abc 35.6 c ND d a 69.6 ab ND d 50.2 bc 33.6 c ND d 35.8 c 10.2 d ND d a 52.6 a ND d 40.4 b 19.4 c ND d 7.2 d ND d a 36.5 a ND b 29.3 a ND b 2.2 d ND d ND: AITC was not detected (the detection limit was 2.5 ppm) Means from each sampling time in the same row with the same letters are not significantly different (p>0.05)

14 Bacteria% sinigrin degraded after 21 d Salmonella > 82% L. monocytogenes > 81% Campylobacter > 87%

15  Neutral pH (7.0), room temperatures (21-25ºC), ferrous and ferric iron enhanced the sinigrin degradation by pathogens.  Refrigerator temperature (4ºC), lower pH (≤5.0) and > 0.25% glucose reduced the sinigrin degradation.

16 M: 0.5% Mustard, MA: 0.2% Malic Acid, E: 0.2% EDTA ND: Not detected (detection level was ≤ 1 CFU/ml) * Cells were not detected after overnight enrichment in MH broth DayControlMMAEM+ MAM+ EE + MAM + E + MA 03.5±0.2 a 18.0±0.4 a 7.8±0.8 a 5.0±1.0 b 3.3±0.4 cd 3.7±0.2 c 3.0±0.1 cd 2.2±0.1 d 2.4±0.4 d 39.0±0.2 a 8.7±0.5 a 7.2±0.5 b 3.0±0.1 d 6.0±0.4 c 2.4±0.2 d 0.4±0.5 e 79.1±0.2 a 9.0±0.0 a 8.2±0.1 ab 1.0±0.7 c 7.4±0.7 b ND * 109.0±0.2 a 9.0±0.1 a 8.6±0.5 a ND * 8.1±0.1 b ND * Means from each sampling time in the same row with the same letters are not significantly different (p>0.05)

17 DayControlMMAEM+ MAM+ EE + MAM + E + MA 03.1±0.6 a 18.6±0.1 a 3.1±0.4 b 3.0±0.3 b 3.0±0.5 b 2.9±0.4 b 2.8±0.7 b 2.5±0.2 b 2.4±0.2 b 39.0±0.01 a 4.8±0.6 b 3.0±0.3 c 3.0±0.5 c 2.9±0.5 c 2.2±0.2 c 79.3±0.1 a 8.9±0.1 b 2.8±0.2 c 2.7±0.2 c 2.0±0.1 d 2.6±0.2 c 1.3±0.4 e 1.0±0.2 e 109.0±0.1 a 8.5±0.4 a 2.3±0.4 b 2.4±0.2 b 1.1±0.3 c 2.0±0.5 b ND * 148.7±0.1 a 8.4±0.4 a 1.3±0.5 b 1.5±0.8 b ND * 1.5±0.8 b ND * M: 0.5% Mustard, MA: 0.2% Malic Acid, E: 0.2% EDTA ND: Not detected (detection level was ≤ 1 CFU/ml) * Cells were not detected after overnight enrichment in MH broth Means from each sampling time in the same row with the same letters are not significantly different (p>0.05)

18 ND: Not detected (detection level was ≤ 1 log CFU/ml) * Cells were not detected after overnight enrichment in MH broth DayControlMustard extract (mg/g)AITC (µl/g) UncoatedCoated ±0.7 a 5 5.1±0.3 a 4.4±0.8 ab 4.4±0.3 ab 4.0±0.4 ab 3.9±0.2 ab 3.5±0.1 ab 4.1±0.9 ab 2.9±0.3 b NDND* ±0.2 a 4.5±0.4 ab 4.5±0.2 ab 3.9±0.2 ab 3.8±0.5 ab 3.1±0.2 b 3.5±0.8 ab 2.7±1.0 b NDND* ±0.3 a 4.4±0.1 ab 4.1±0.4 abc 3.8±0.4 bc 3.3±0.3 cd 2.7±0.1 de 3.2±0.1 cde 2.2±0.5 e NDND* ±0.8 a 4.3±0.6 ab 4.2±0.4 ab 3.4±0.3 abc 2.6±0.8 abc 1.5±0.5 cd 3.7±0.3 ab 2.4±0.9 bc NDND* Means from each sampling time in the same row with the same letters are not significantly different (p>0.05)

19 AITC: 50 uL/g, Mustard: 250 mg/g, EDTA: 15 mg/g Day Uncoated control Coating control AITC MustardEDTA AITC + EDTA Mustard + EDTA 0 6.1±0.1 a 5 5.9±0.2 a 5.4±0.1 ab 4.6±0.1 cd 4.8±0.5 bc 5.4±0.1 ab 3.8±0.3 de 3.7±0.6 e ±0.0 a 5.3±0.3 ab 4.3±0.2 cd 4.7±0.9 bc 5.4±0.1 ab 3.4±0.4 e 3.8±0.4 cd ±0.0 a 5.2±0.2 a 4.2±0.4 b 4.0±0.8 b 5.2±0.1 a 3.0±0.1 c 3.5±0.4 bc ±0.4 a 5.3±0.1 b 3.7±0.0 cd 3.8±0.2 c 5.2±0.1 b 3.3±0.3 de 3.1±0.0 e Means from each sampling time in the same row with the same letters are not significantly different (p>0.05)

20 Pathogens had ability to synthesize myrosinase-like activity and degraded sinigrin to form AITC EDTA and malic acid enhnced the antimicrobial activity of AITC and oriental mustard against pathogens AITC and oriental mustard are potent antimicrobials against Campylobacter, Salmonella and L. monocytogenes

 Results will be useful to:  Consumers (safe poultry meats).  Food regulatory agencies (create a new processing tool to reduce food contamination).  Meat industry (reduce recalls and increase the safety and shelf life of poultry meat products). 21

22  Natural Sciences and Engineering Research Council, Canada.  Prof. Richard A. Holley  University of Manitoba Graduate Student Travel Award University of Manitoba Graduate Fellowship James W. Barlow Graduate Fellowship International Graduate Student Scholarship International Graduate Student Entrance Scholarship

23  Canadian Meat Science Association, Associate Members of the Canadian Meat Council and Newly Weds Foods, Canada. Percy Gitelman Memorial Scholarship Percy Gitelman Memorial Scholarship

Amin Naser Olaimat PhD Candidate/Food Safety and Microbiology Department of Food Science/University of Manitoba Tel: ; 24