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1Use of Oriental Mustard and Allyl Isothiocyanate to Control Salmonella, Campylobacter and L. monocytogenes in Poultry Meat ProductsAmin Naser OlaimatPhD CandidateDepartment of Food ScienceUniversity of ManitobaCanadian Meat Council's 94th Annual Conference

Toronto, ON, Canada, May 7-9, 2014

1 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.

2Introduction2 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.

3Introduction34Introduction

GlucosinolateIsothiocyanate(ITC) Mustard is the major spice used in meat manufacturing.

Mustard and other vegetables (broccoli, horseradish and wasabi) contain high levels of glucosinolates (GSLs)

4 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.

5Introduction5 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.

6Introduction6Chitosan 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. 7Introduction7Determine 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.

8ObjectivesDetermine 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.

9Objectives 10Table 1: MIC and MBC (ppm) of AITC against Salmonella, Campylobacter or L. monocytogenes strains.Bacteria21C37CMICMBCMICMBC5 Salmonella strains10500-60060-100120-1805 L. monocytogenes strains20-40200-300100120-1604 Campylobacter strainsNT5-100.63-1.252.5MIC: the lowest concentration at which no visible growth occurredMBC: the lowest concentration causing a 99.9% reduction of the initial inoculumNT: Not tested1011Table 2: Reduction of Salmonella (log10 CFU/ml) by AIT (200ppm) at different pH and temperature combinations.Day4C10C21CpH5pH7pH9pH5pH7pH9pH5pH7pH900.00 a0.00 a0.00 a0.00 a0.00 a0.00 a0.00 a0.00 a0.00 a10.06 d0.46 cd0.63 bcd1.10 b1.26 b0.71 bc3.59 a3.67 a0.43 cd30.47 e0.78 e0.07 e2.15 d2.58 cd0.45 e7.39 a4.31 b3.39 bc61.22 ef1.76 e0.07 f3.77 cd4.58 bc0.16 f8.11 a5.70 b2.62 de102.56 c2.16 c0.13 d6.48 a4.45 b0.14 d7.90 a-0.38 d2.84 c

Means from each sampling time in the same row with the same letters are not significantly different (p>0.05)1112Table 3: Reduction of L. monocytogenes (log10 CFU/ml) by AIT (200 ppm) at different pH and temperature combinations.Day4C10C21CpH5pH7pH9pH5pH7pH9pH5pH7pH900.00 a0.00 a0.00 a0.00 a0.00 a0.00 a0.00 a0.00 a0.00 a10.12 c0.73 c0.34 c0.01 c2.38 b-0.08 c1.89 b8.30 a0.56 c30.15 ef1.76 d0.31 ef0.42 e5.76 c-0.34 f7.15 b8.88 a0.23 ef60.44 d2.95 c0.25 d0.61 d7.87 b-1.20 e7.47 b8.85 a0.10 d101.19 d4.14 c-0.18 d3.07 c8.45 a0.02 d5.93 b8.46 a1.30 d

Means from each sampling time in the same row with the same letters are not significantly different (p>0.05)1213Table 4: Stability of 200 ppm AITC (% remaining) at different pH and temperatures.Day4C10C21CpH5pH7pH9pH5pH7pH9pH5pH7pH90100.0 a100.0 a100.0 a10.0 a100.0 a100.0 a100.0 a100.0 a100.0 a188.1 a81.6 aND d67.8 ab53.1 bcND d62.0 abc35.6 cND d372.6 a69.6 abND d50.2 bc33.6 cND d35.8 c10.2 dND d654.7 a52.6 aND d40.4 b19.4 cND d7.2 dND dND d1048.3 a36.5 aND b29.3 aND bND b2.2 dND dND 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)13 14Table 5: Degradation of sinigrin by 5 L. monocytogenes strains in MHB at pH 7.0 and 25C.Bacteria% sinigrin degraded after 21 dSalmonella> 82%L. monocytogenes> 81%Campylobacter> 87%14 15Factors affecting the sinigrin degradation by pathogens Neutral pH (7.0), room temperatures (21-25C), ferrous and ferric iron enhanced the sinigrin degradation by pathogens.

Refrigerator temperature (4C), lower pH (5.0) and > 0.25% glucose reduced the sinigrin degradation.

15 16Table 6: Combined effects of oriental mustard extract with malic acid or EDTA against S. Typhimurium (log10 CFU/ml) in MHB at 21 C M: 0.5% Mustard, MA: 0.2% Malic Acid, E: 0.2% EDTAND: Not detected (detection level was 1 CFU/ml)* Cells were not detected after overnight enrichment in MH brothDayControlMMAEM+ MAM+ EE + MAM + E + MA03.50.2 a3.50.2 a3.50.2 a3.50.2 a3.50.2 a3.50.2 a3.50.2 a3.50.2 a18.00.4 a7.80.8 a5.01.0 b3.30.4 cd3.70.2 c3.00.1 cd2.20.1 d2.40.4 d39.00.2 a8.70.5 a7.20.5 b 3.00.1 d6.00.4 c2.40.2 d0.40.5 e0.40.5 e79.10.2 a9.00.0 a8.20.1 ab1.00.7 c7.40.7 bND *ND *ND *109.00.2 a9.00.1 a8.60.5 aND *8.10.1 bND *ND *ND *

Means from each sampling time in the same row with the same letters are not significantly different (p>0.05)16 17Table 7: Combined effects of oriental mustard extract with malic acid or EDTA against L. monocytogenes (log10 CFU/ml) in MHB at 21 C DayControlMMAEM+ MAM+ EE + MAM + E + MA03.10.6 a3.10.6 a3.10.6 a3.10.6 a3.10.6 a3.10.6 a3.10.6 a3.10.6 a18.60.1 a3.10.4 b3.00.3 b3.00.5 b2.90.4 b2.80.7 b2.50.2 b2.40.2 b39.00.01 a4.80.6 b3.00.3 c3.00.5 c2.90.5 c2.90.5 c2.20.2 c2.20.2 c79.30.1 a8.90.1 b2.80.2 c2.70.2 c2.00.1 d2.60.2 c1.30.4 e1.00.2 e109.00.1 a8.50.4 a2.30.4 b2.40.2 b1.10.3 c2.00.5 bND *ND *148.70.1 a8.40.4 a1.30.5 b1.50.8 bND *1.50.8 bND *ND *

M: 0.5% Mustard, MA: 0.2% Malic Acid, E: 0.2% EDTAND: Not detected (detection level was 1 CFU/ml)* Cells were not detected after overnight enrichment in MH brothMeans from each sampling time in the same row with the same letters are not significantly different (p>0.05)1718Table 8: Antimicrobial activity of K-carrageenan/chitosan coatings containing AITC or mustard extracts against C. jejuni (log10 CFU/g) on vacuum-packed chicken breasts at 4 C.ND: Not detected (detection level was 1 log CFU/ml)* Cells were not detected after overnight enrichment in MH brothDayControlMustard extract (mg/g)AITC (l/g)UncoatedCoated5010020030010255010006.20.7 a6.20.7 a6.20.7 a6.20.7 a6.20.7 a6.20.7 a6.20.7 a6.20.7 a6.20.7 a6.20.7 a55.10.3 a4.40.8 ab4.40.3 ab4.00.4 ab3.90.2 ab3.50.1 ab4.10.9 ab2.90.3 bNDND*115.40.2 a4.50.4 ab4.50.2 ab3.90.2 ab3.80.5 ab3.10.2 b3.50.8 ab2.71.0 bNDND*165.00.3 a4.40.1 ab4.10.4 abc3.80.4 bc3.30.3 cd2.70.1 de3.20.1 cde2.20.5 eNDND*214.60.8 a4.30.6 ab4.20.4 ab3.40.3 abc2.60.8 abc1.50.5 cd3.70.3 ab2.40.9 bcNDND*

Means from each sampling time in the same row with the same letters are not significantly different (p>0.05)1819AITC: 50 uL/g, Mustard: 250 mg/g, EDTA: 15 mg/gDayUncoated controlCoating controlAITC

MustardEDTAAITC + EDTAMustard + EDTA 06.10.1 a6.10.1 a6.10.1 a6.10.1 a6.10.1 a6.10.1 a6.10.1 a55.90.2 a5.40.1 ab4.60.1 cd4.80.5 bc5.40.1 ab3.80.3 de3.70.6 e116.00.0 a5.30.3 ab4.30.2 cd4.70.9 bc5.40.1 ab3.40.4 e3.80.4 cd165.70.0 a5.20.2 a4.20.4 b4.00.8 b5.20.1 a3.00.1 c3.50.4 bc215.80.4 a5.30.1 b3.70.0 cd3.80.2 c5.20.1 b3.30.3 de3.10.0 eTable 9: Antimicrobial activity of K-carrageenan/chitosan coatings containing AITC or mustard extracts against Salmonella (log10 CFU/g) on vacuum-packed chicken breasts at 4 C

Means from each sampling time in the same row with the same letters are not significantly different (p>0.05)1920Pathogens had ability to synthesize myrosinase-like activity and degraded sinigrin to form AITCEDTA and malic acid enhnced the antimicrobial activity of AITC and oriental mustard against pathogensAITC and oriental mustard are potent antimicrobials against Campylobacter, Salmonella and L. monocytogenes ConclusionsResults 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).21Benefits Relating to Meat Science2122Acknowledgments Natural Sciences and Engineering Research Council, Canada.Prof. Richard A. HolleyUniversity of ManitobaGraduate Student Travel AwardUniversity of Manitoba Graduate FellowshipJames W. Barlow Graduate FellowshipInternational Graduate Student ScholarshipInternational Graduate Student Entrance Scholarship23Acknowledgments Canadian Meat Science Association, Associate Members of the Canadian Meat Council and Newly Weds Foods, Canada.Percy Gitelman Memorial ScholarshipAmin Naser OlaimatPhD Candidate/Food Safety and MicrobiologyDepartment of Food Science/University of ManitobaTel: 204 297 8103; amin_olaimat@yahoo.com

24Thank you