1.  MC Antimicrobial Activity Test •  An aliquot of 100 µL of bacteria suspension

(4×107 CFU/mL) were added into each 4 mL MC.

•  At each contact time, 100 µL samples were diluted decimally with buffer to 3 dilutions. Each dilution was plated out on plate in triplicates and recorded.

Antimicrobial Acivity of N-Halamine Coated Materials in Brolier Chicken House

Tian Ren1, Mingyu Qiao1, Lei Zhang1, Tung-Shi Huang1, Buket Demir2 1Department of Poultry Science, Auburn University, Auburn, AL 2Department of Chemistry, Auburn University, Auburn, AL

The objectives of this study were to assess the antimicrobial activity and the antimicrobial longevity of N-halamine (1-chloro-2,2,5,5-tetramethyl-4-imidazoidinone (MC), a non-bleaching compound)-coated materials in broiler chicken houses. The antimicrobial test of MC illustrated that 0.04% MC solution was able to kill Salmonella Typhimurium and Campylobacter jejuni completely at 106 CFU/mL. Results showed that 1% MC treated samples were able to kill Salmonella Typhimurium and Campylobacter jejuni completely as long as four weeks, at the concentration of 106 CFU/cm2, where the active chlorine atoms in the treated coupons decreased from the initial 1016 atoms/cm2 to 1015 atoms/cm2. Therefore, MC is assumed to be used as a novel antimicrobial agent in chicken broiler house.

N

NH3C

H3C

NH2

CH3

H

O

Cl

Structure of MC

•  N-halamines are ideal antimicrobial agents due to their superior antimicrobial activity against a broad spectrum of microorganisms, nontoxicity, high stability, rechargeability and low cost. Antimicrobial activities of various materials that coated with N-halamines have been investigated, such as fabrics (Demir, et al., 2015) and paints (Kocer, et al., 2011).

•  MC is a type of monochlorinated N-halamine which has been reported possessing a powerful antimicrobial activity and high stability.

2. Antimicrobial Longevity Test of MC Coated Materials ---- Sample Preparation •  Stainless steel, galvanized metal, plastic, and pressure

treated woods were selected as tested materials. Slides of each material were cut into 1 inch2 coupons. Cleaned, autoclaved before use.

•  Spray with 0.1% and 1% MC in 95% EtOH, respectively. Stored in a dark, enclosed cabinet at room temperature.

3. Antimicrobial Longevity Test of MC Coated Materials ---- Antimicrobial Activity Test •  An aliquot of 25 µL of bacteria suspension was

placed onto the center of a coupon, and covered with a second identical coupon with MC coated side face down. Then, a sterile weight was placed on the top of the second coupon.

•  After 2 h contact time, coupons were transferred Na2S2O3 solution to terminate the reaction as well as wash off all the attached bacterial. Dilution and plating out as previous methods. Tests were run on day 0, 3, 6, and week 2, 3, 4.

4. Active Chlorine Content Determination •  0.25 g of KI was dissolved into 5 mL of 0.1 N

acetic acid and 45 mL of absolute ethanol. Then two coupons were added into the flask.

•  They were shaken for 2 min to mix well. Then, standardized Na2S2O3 was used to titrate. The content of active chlorine was calculated by using this following equation.

N and V are the normality (equiv/L) and volume (L) of the sodium thiosulfate, respectively, and A is the surface area (cm2) of the coupon.

Cl+ (atom/cm2 ) = 6.02×1023 ×N×V2×A

5

10

30

60

0

10

20

30

40

50

60

70

80

90

100

0 0.02

0.04 0.06

Bac

teri

a re

duct

ion

(%)

MC concentration (%)

Salmonella Typhimurium

5

10

30

60

0

10

20

30

40

50

60

70

80

90

100

0 0.02

0.04 0.08

0.16

Bac

teri

a re

duct

ion

(%)

MC concentration (%)

Campylobacter jejuni

1. Antimicrobial Activity of MC Solution •  Higher MC concentration, or longer contact time, the

antimicrobial activity of MC against S. Typhimurium and C. jejuni increased.

•  Solutions containing 0.04% or higher concentration of MC were able to completely inactivate both bacteria at 106 CFU/mL within 30 min.

2. Antimicrobial Longevity of MC Coated Materials •  Materials coated with 1% MC were able to inactivate all the

inoculated bacteria at 106 CFU/sample within 2 h contact time, and this antimicrobial activity lasted for at least four weeks.

•  Materials coated with 0.1% MC kill more than 99.8% of the S. Typhimurium and all of the inoculated C. jejuni on the initial day, and then their antimicrobial activities decrease afterwards.

3. Active Chlorine Content in the Materials •  It did not decrease in the first two weeks after materials were

treated with MC. After three weeks of treatment, it decreased significantly, from the initial 1016 atoms/cm2 to 1015 atoms/cm2 over this three-week period, and were still able to inactivate all the inoculated microbes.

•  MC is a promising antimicrobial agent, which can be applied into the chicken broiler house.

•  For the purpose of inactivating both S. Typhimurium and C. jejuni, we found that 1% MC is suitable to be used to coat on various materials.

1. Demir et al. ACS Applied Materials & Interfaces. 2015, 7(3), 1752-1757.

2. Kocer et al. ACS Applied Materials & Interfaces. 2011, 3(8), 3189-3194.

3. Scharff. Journal of Food Protection, 2011. 75(1), 9.

0

0.5

1

1.5

2

2.5

3

3.5

4

0 D 3 D 6 D 2 W 3 W 4 W

Cl+

atom

/cm

2 (×

1016

)

Storage time

0 0.5

1 1.5

2 2.5

3 3.5

4 4.5

5

0 D 3 D 6 D 2 W 3 W 4 W

Cl+

atom

/cm

2 (×

1016

)

Storage time

0

0.5

1

1.5

2

2.5

3

3.5

0 D 3 D 6 D 2 W 3 W 4 W

Cl+

ato

m/c

m2

(×10

16)

Storage time

0

0.5

1

1.5

2

2.5

3

3.5

0 D 3 D 6 D 2 W 3 W 4 W

Cl+

ato

m/c

m2

(×10

16)

Storage time

2. Antimicrobial longevity of MC coated materials and change of chlorine content Galvanized metal Plastic Pressure treated woods Stainless steel

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

DISCUSSION RESULTS

CONCLUSION

1. Antimicrobial activity of MC solution

•  The Center for Disease Control and Prevention (CDC) estimated that each year about 48 million people were infected with foodborne diseases in the US (CDC 2011), and it also led to a significant economic loss (Scharff, 2012). With the ever-growing demand in poultry products in the US (USDA ERS, 2015), incidences of pathogen contamination in poultry products can significantly affect human health. Therefore, protecting the safety of poultry products becomes a critical issue for reducing the risk of foodborne diseases.

REFERENCES

1-chloro-2,2,5,5-tetramethyl- 4-imidazolidinone

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