Food Protection Trends - March 2009 - (Page 156)

natually occuring compounds have been successfully shown to inhibit the growth of foodborne pathogens (1–3, 8,11–12, 16, 18, 19). A number of studies have found that Chinese chives possess antibacterial properties due to the presence of sulfurcontaining compounds (5–7, 10, 13–14). As a result, Chinese chives have been used to control microbial spoilage. However, their antibacterial effect against foodborne pathogens such as Salmonella has not been determined. Therefore, the objective of this study was to examine the antibacterial activity of a crude chive extract against 38 strains of Salmonella in culture media and in both beef and chicken broth as food models. To our knowledge, this is the first study to determine the antibacterial activity of a chive extract against a wide range of Salmonella strains associated with foodborne illnesses in the United States. towel, then chopped into pieces with a sterilized table knife. Chive pieces (100-g) were blended in a sterilized kitchen blender for 2–3 minutes to obtain a homogenous blend. This blend was placed in 50 ml tubes and centrifuged at 5500 × g for 45 min at 4°C. The supernatant was collected and filtered, using a 0.45 µm filter (Nagle, Rochester, NY USA), then stored overnight at 4°C. Growth over time Ten Salmonella strains were used to determine bacterial growth over time. Overnight individual strains of Salmonella (randomly selected: ED404, ED405, ED104, ED165, ED429, ED204, ED379, DT104, ED181, ED203) were serially diluted to 3 log CFU/ml, and 1 ml of each diluted strain was well mixed in a sterilized tube. This mixture was used to inoculate 10 ml batches of sterile BHI containing chive extract at eight concentrations (100 to 800 µl/ml, at 100 µl/ml intervals). Samples without chive extract were used as control. Samples were then incubated at 37°C for 24 h, and bacterial growth was monitored by measuring optical density at 610 nm, using a spectrophotometer 21 (Thermo Electron Scientific Co., Madison, WI). Antimicrobial assay A diffusion assay with some modifications was used to determine the antibacterial activity of crude chive extract. Batches of 100 ml BHI agar with 0.2% Tween 80 were prepared and sterilized at 121oC for 15 min. The agar was placed in a water bath at 49oC and allowed to cool, and each extract sample was then inoculated individually with a single strain of Salmonella to achive an inoculum level of ~5-6 log CFU/ml. The BHI agar (~45 ml) was poured into Petri dishes (100 × 15 mm) and allowed to solidify (agar thickness 12 mm). One well (7 mm in diameter) was made in the center of the agar using a sterile test tube. Chive extract (0.5 ml) was transferred into each well. Plates were incubated for 10–12 h at 35°C and then examined for a clear inhibition zone around the well. The assay was carried out in duplicate for all 38 Salmonella strains. An agar diffusion assay was also used to determine the MIV of the chive extract against all 38 strains of Salmonella. BHI agar was inoculated with a single strain of Salmonella (~5–6 log CFU/ml). Wells were made and filled with chive extracts at different concentrations (0–1000 µl/ ml with 10 µl/ml, unit concentration increase for each plate). To avoid spillage and ensure complete containment of the extract in the agar, the wells were filled gradually for complete deposit of the extract into the well cavity. Plates were incubated at 35oC and the developed clear zones were recorded. The size of the zone of inhibition was calaculated as the diameter of the zone minus the diameter of the bored test well. The MIV was defined as the lowest volume that inhibited growth. The minimum lethal volume (MLV) was defined as the lowest volume at which no growth was observed after three days of incubation. Each set of experiments was conducted three times. MATERIALS AND METHODS Bacterial strains The thirty-eight (38) strains of Salmonella were obtained from the North Carolina A&T State University food microbiology laboratory culture collection. All strains were initially obtained from a private clinical laboratory that confirmed the identification of each strain by use of several biochemical and serological methods. The identification for each strain was confirmed with API 20E and the APILAB plus the identification program 3.3/4.0 (bioMérieux, Inc., Hazelwood, MO). In addition, triple sugar iron and urea agar (Difco, Becton Dickinson) was used as a quality control test to confirm the biochemical characteristics of Salmonella isolates. Strains were kept in −80°C stock cultures in Tryptic Soy Broth with 20% glycerol. A working culture was prepared by inoculating a loopful of culture into 10 ml of TSB (TSB; Difco, Sparks, MD) with 0.8% yeast extract (Difco). The cultures were subjected to at least three successive 14 h transfers before use. Antibacterial activity of chives in food models The antibacterial activity of chives in two food models was measured in commercially canned chicken broth and beef broth samples purchased from a local store (Greensboro, NC). Each sample (beef and chicken broth) was split into two 95-ml portions. Chive extract was added at 5% (5 ml) to one portion of each food sample. Food samples without chive extract served as control samples. Samples were then incoulated with a mixture of Salmonella strains to achive a final inoculum level of 3.00 log CFU/ml. The samples were stored at 37oC for 48 h. A 0.1 ml aliquot of each sample was removed at the end of the incubation period, serially diluted in 0.1% peptone water (Bacto peptone, Becton Dickinson, Sparks, MD, USA) and spread plated, in duplicate, on prepared BHI agar. The inoculated plates were then incubated at 35oC for 24 h. The colonies were counted to determine the bacterial population in each sample. The experiments were conducted three times to determine whether addition of chive extract significantly affected bacterial growth. Preparation of crude chive extract To prepare the crude chive extract, fresh chives were obtained from a local international market in Greensboro, NC. The chives were washed under running tap water, blotted with a single-use paper Statistical analysis The Statistical Analysis System (SAS) version 6.0 computer statistical package (SAS Institute, Cary, NC) was used for 156 FOOD PROTECTION TRENDS | MARCH 2009

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Food Protection Trends - March 2009 Contents Sustaining Members Perspectives from Your President Commentary from the Executive Director Microbiological Safety of Farmstead Cheeses Made in the United States and Purchased via Online Shopping Antibacterial Activity of a Crude Chive Extract against Salmonella in Culture Medium, Beef Broth and Chicken Broth 2009–2010 Secretary Election New Members What’s Happening in Food Safety Industry Products Ivan Parkin and John H. Silliker Lectures Proposed Symposia Topics and Roundtables Event Information Networking Opportunities General Information Registration Form Coming Events Advertising Index Journal of Food Protection Table of Contents Booklet Order Form Membership Application

Food Protection Trends - March 2009

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