Silliker, Inc. Food Science Center Report RPN Part B. October 30, 2014

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1 Silliker, Inc. Food Science Center Report RPN Part B October 30, 2014 The Microbiological Shelf-life and Safety of Two Bagels Topped with two Flavors of Cheese Toppers (Tomato Mozzarella & Southwest Style Chili) when Challenged with Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, Escherichia coli O157:H7 and Bacillus cereus Prepared for: Penelope Gavriel Dunkin Donuts Dunkin' Brands, Corporate QA Manager 130 Royall Street, Mail Stop 1EB Canton, MA (781) Penelope.Gavriel@dunkinbrands.com Prepared by: Jeannie Kim, Ph.D. Research Project Manager Silliker, Inc. Food Science Center 3600 Eagle Nest Drive, South Building Crete, IL (708) Jeannie.kim@silliker.com The entire content of this REPORT is subject to copyright protection. All rights reserved. The contents of this REPORT may not be copied other than for use by non-for-profit organization, and appropriate reference with all copyright notices stated. The REPORT may not be copied, reproduced or otherwise redistributed. Except as expressly provided above, copying, displaying, downloading, distributing, modifying, reproducing, republishing or retransmitting any information, text or documents contained in this REPORT or any portion thereof in any electronic medium or in hard copy, or creating any derivative work based on such documents, is prohibited without the express written consent of Silliker, Inc. Nothing contained herein shall be construed as conferring by implication, estoppel or otherwise any license or right under any copyright of Silliker, Inc., or any party affiliated with Silliker, Inc. 1 of 10

2 BACKGROUND AND OBJECTIVE Dunkin Donuts wished to conduct an inoculated challenge study in 2 types of bagel cheese toppers (Southwest Style Chili and Tomato Mozzarella) baked on a Plain bagel. When challenging certain specialty bread and pastry filled products, the FDA has adopted NSF guidelines, and as such has specific requirements regarding which are the target microorganisms of concern (1). These guidelines are based on the ph and water activity of the product and as such, these product characteristics must be determined prior to design of these studies. Selection of challenge organisms was based on ph and water activity of products previously studied. Therefore, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, Salmonella spp. and Bacillus cereus were selected in this study. A challenge study is performed by inoculating selected microorganisms into a food to determine if the organisms are a potential health hazard risk or spoilage risk. A study can be used to determine the safest and most stable formulation, packaging, preservatives, storage conditions, product code dating, and specifications. NACMCF guidelines for conducting inoculated challenge studies recommend evaluating a minimum of two production lots, representing two separate manufacturing dates, and three samples per lot at each time point (2). The challenge study is conducted over 5 to 7 time points that cover the shelflife of the product plus 25 to 50% additional time to provide for expected consumer consumption. One lot of samples was evaluated in this study as there are no other lots available. It is prudent to analyze the product, including uninoculated control samples, at each or selected sampling points in the study to see how the background microflora is behaving during the product s shelf life (1). The absence of pathogenic growth in inoculated samples must be achieved in the absence of spoilage; otherwise, this stability may be attributed to competitive inhibition by background micro flora. Microbiological shelf-life is established by analyzing products for spoilage bacteria, yeast and mold. The number of microorganisms required to cause spoilage varies with the food item and the type of microorganism growing in it. Generally, the end of shelf-life is defined as 10,000,000 bacteria per gram, 100,000 yeast per gram or visible mold (3). The microbiological shelf-life of the product was determined by analysis of the control samples according to these criteria. The objective of this study was to assess the microbiological safety and stability of 2 types of bagel cheese toppers (Southwest Style Chili and Tomato Mozzarella) when challenged with foodborne pathogens and held at ambient temperature (73-77 F) for up to 24 hours. MATERIALS AND METHODS Test Product Samples of each tested product, labeled as Southwest Style Chili and Tomato Mozzarella" bagels were prepared at a local Dunkin Donuts store (Lansing, IL) on August 20, Products were picked up and delivered to Silliker Food Science Center, within 30 minutes post prepping (arrived at 7:15am). Products were received at ambient temperature and in good conditions in covered cartons. Basket liners were also provided by the client. Samples were separated into test and control portions. The study was initiated within 1.5 hour after receipt of samples. Inoculation was conducted at 8:35 am. Challenge Microorganisms Products were inoculated with composite cultures prepared from five strains of foodborne pathogenic microorganisms maintained in the Silliker Food Science Center Culture Collection (FSC-CC; Table 1)(1, 2). Individual strains of S. aureus, E. coli O157:H7 and Salmonella spp. were propagated in trypticase soy broth (TSB) and incubating for h at 35 C. L. monocytogenes strains were propagated in TSB plus 0.6% yeast extract (TSBYE) and incubated for h at 35 C. 2 of 10

3 Verification of S. aureus, E. coli O157:H7, Salmonella spp. and L. monocytogenes was conducted by streak plating onto Baird Parker (BP), sorbitol MacConkey agar (SMAC), xylose lysine desoxycholate agar (XLD), and modified Oxford (MOX) agar plates, respectively, and incubating at 35 C for 24 h. Typical reactions on selective agar plates were considered confirmatory. Individual strains of B. cereus were transferred to TSB and incubated for 24 ± 2 h at 35 C. The overnight culture was swabbed onto Nutrient Agar plates containing MnSO 4, and incubated for 4 d at 35 C. Spores were harvested by adding approx. 5-mL sterile deionized (DI) water to each plate and scraping the cells into a sterile test tube. The harvested culture was centrifuged at 8000 rpm for 20 min, and resuspended in sterile phosphate buffer. The spore suspension was heat shocked at 80 C for 10 min and spore levels determined by plating on trypticase soy agar (TSA). Verification of each strain was conducted by streaking B. cereus onto mannitol egg yolk polymixin (MYP). Toxin production of each strain of S. aureus and B. cereus was confirmed. A cell suspension was prepared for each strain used in an inoculum. Cell suspensions were mixed to prepare an inoculum, which contained approximately equal numbers of cells of each strain. Composite bacterial cultures were stored at 4 C during enumeration by plate count methods. Table 1. The strains for inoculation of products Staphylococcus aureus Composite FSC-CC # Source (Toxin Type) Staphylococcus aureus 2428 Whipped butter (A) Staphylococcus aureus 2431 Abscess (C) Staphylococcus aureus 2424 Ice cream (A & D) Staphylococcus aureus 3195 ATCC 27664, (E) Staphylococcus aureu 2429 Sausage (B) Escherichia coli O157:H7 Composite FSC-CC # Source Escherichia coli O157:H ATCC Escherichia coli O157:H ATCC Escherichia coli O157:H ATCC Escherichia coli O157:H ATCC Escherichia coli O157:H ATCC Salmonella spp. Composite FSC-CC # Source Salmonella Bornum 1468 Ice cream Salmonella Eastbourne 143 Food isolate Salmonella Enteritidis 1434 Ice cream Salmonella Heidelberg 539 Whole egg Salmonella Typhimurium 1443 Ice cream Listeria monocytogenes Composite FSC-CC # Source Listeria monocytogenes 2119 ATCC Listeria monocytogenes 2121 ATCC Listeria monocytogenes 525 Hot dog / food poisoning Listeria monocytogenes 518 Cheese isolate Listeria monocytogenes 2120 ATCC Bacillus cereus Composite FSC-CC # Source (type) Bacillus cereus 2116 ATCC Bacillus cereus 2117 ATCC Bacillus cereus 1668 ATCC Bacillus cereus 1401 ATCC 9592 Bacillus cereus 2637 ATCC of 10

4 Preparation of Test Samples and Storage Fifteen bagels per topping formulation were inoculated with a composite culture of each challenge microorganism at approximately 100 1,000 (3-log 10 ) colony-forming units per gram. The product was inoculated onto several locations of the product including top crust, cheese and crust-cheese interface (1, 4). Additionally, 6 uninoculated samples per formulation were prepared and serve as negative controls. After inoculation, product was allowed to rest on the bakery racks with liner provided by the client. All samples were stored at F in the bakery racks without cover for the duration of the study to mimic the display at the retail store. Three samples each per product were inoculated with B. cereus and S. aureus, respectively and stored at the same condition for toxin testing at 24 h. Sample Analyses Samples of inoculated portions were analyzed initially (time 0) and after 6, 12, 18 and 24 hours, while samples of uninoculated control portions were analyzed initially and at 24 h. Three analytical samples were taken at each interval and analyzed by plate count methods, providing a minimum of three results per data point (1, 2). The negative control was analyzed for aerobic bacteria and yeast and mold while the inoculated portions were analyzed for the challenge microorganisms. The methods of analyses are outlined in Table 2. B. cereus and S. aureus toxin testing were conducted at the end of the study in triplicate from inoculated samples. Table 2. Methods of analysis Test Medium Incubation Time/ Temperature/ Atmosphere Aerobic plate count Tryptone glucose yeast agar 2 days/35 C/aerobic Yeast/mold count Potato dextrose agar with antibiotics 5 days/25 C/aerobic Staphylococcus aureus Baird-Parker agar 48 hours/ 35 C/aerobic Escherichia coli O157:H7 Trypticase soy agar with violet red bile 48 hours/35 C/aerobic overlay Salmonella Trypticase soy agar with xylose lysine 48 hours/35 C/aerobic desoxycholate overlay Listeria monocytogenes Trypticase soy agar with yeast extract 48 hours/30 C/aerobic with modified Oxford agar overlay Bacillus cereus Mannitol egg yolk polymixin agar 24 hours/ 30 C/aerobic B. cereus toxin NA NA S. aureus toxin NA NA ph NA NA Water Activity (aw) NA NA The ph and water activity of each product was tested initially and at the end of the study in duplicate. Acceptance criteria The end of shelf-life is defined as 10,000,000 bacteria per gram, 100,000 yeast per gram or visible mold (3). Acceptance criteria for the pathogen are less than 1- log increase of E. coli O157:H7, Salmonella spp. and L. monocytogenes and less than 3- log increase of B. cereus and S. aureus or the absence of toxin over the entire shelf life (1, 2). 4 of 10

5 RESULTS AND DISCUSSION Growth of naturally occurring microflora during shelf life The taste, odor, and appearance of a food (organoleptic qualities) are the ultimate criteria used to judge a food s acceptability. These qualities begin to change as the microflora in the food (bacteria, yeast, and mold) grow and metabolize available nutrients. Organoleptic changes are generally not detectable until the microbial population is high. The number of organisms required to cause spoilage varies with the food item and the type(s) of microorganisms growing in it. Generally, the end of shelf-life is defined as 10,000,000 bacteria per gram, 100,000 yeast per gram, or visible mold (3). Shelf life results for the uninoculated control samples are summarized in Tables 4 and 5. The ph and water activity values of two bakery items did not change significantly during storage. The counts of spoilage microorganisms including aerobic bacteria, yeast and mold remained low throughout the storage period in all two bakery items (Tables 4 and 5). Therefore, the microbiological shelf-lives of the Dunkin Donuts bagel topped with Southwest Style Chili and Tomato Mozzarella cheese topper were at least 24 hours when stored at ambient temperature (73-77 F). Growth of B. cereus, E. coli O157:H7, Salmonella spp. S. aureus and L. monocytogenes in inoculated samples during shelf life A food may exhibit a long shelf-life even though the temperature, ph, water activity, and nutrient levels are permissive for microbial growth. This long shelf-life may be the result of the absence of microorganisms in the samples tested, or it may be due to the fact that the contaminating organisms will not grow in the particular product formulation. To determine the stability of a product should a chance contamination event occur, a microbiological challenge study is conducted in which the product is inoculated with the target pathogenic microorganisms of concern. If the level of the challenge microorganisms does not increase during storage, the product formulation is resistant to microbial growth and is considered microbiologically stable. Acceptance criteria for the pathogen challenge are less than 1- log increase of E. coli O157:H7, Salmonella spp. and L. monocytogenes and 3-log increase of B. cereus and S. aureus or the absence of toxin over the entire shelf life (1, 2). Test results for the inoculated samples are summarized in Tables 6 through 8 and Figure 1. The counts of B. cereus, E. coli O157:H7, Salmonella spp., S. aureus and L. monocytogenes on two bakery items did not increase when stored at ambient temperature for 24 hours. At 24 h, samples were tested for the presence of Staphylococcal enterotoxin and Bacillus diarrheal enterotoxin and found negative in both products. Therefore, the Dunkin Donuts bagel topped with Southwest Style Chili and Tomato Mozzarella cheese topper was microbiologically stable against B. cereus, E. coli O157:H7, Salmonella spp., Staph aureus and L. monocytogenes for at least 24 hours when stored at ambient temperature (73-77 F). CONCLUSION According to the FDA (1), a potentially hazardous food does not include a food for which laboratory evidence does not demonstrate rapid and progressive growth of infectious and toxigenic microorganisms. In the current study, the Dunkin Donuts bagel topped with two cheese toppings (Southwest Style Chili and Tomato Mozzarella) demonstrated no growth B. cereus, E. coli O157:H7, Salmonella spp., S. aureus and L. monocytogenes for at least 24 h days when stored at ambient temperature (73-77 F). In addition, no toxin of S. aureus and B. cereus was found in both products at 24 h. Therefore, the Dunkin Donuts bagel topped with two cheese toppings (Southwest Style Chili and Tomato Mozzarela) are considered non-potentially hazardous foods when challenged with B. cereus, E. coli O157:H7, Salmonella spp., S. aureus and L. monocytogenes and stored at ambient temperature (73-77 F) for 24 h. 5 of 10

6 References 1. Institute of Food Technologists Evaluation and definition of potentially hazardous foods. (IFT/FDA Contract No Task Order No.4. December 31.) Comp. Rev. in Food Sci. and Food Safety Vol 2. Supplement NACMCF Executive Secretariat Parameters for determining inoculated pack/challenge study protocols. J. Food Prot. 73(1): Curiale, M.S Limiting growth: microbial shelf-life testing. Food Product Design, February. 4. Scott, V.N., K.M.J. Swanson, T.A. Freier, W.P. Pruett Jr., W.H. Sveum, P.A. Hall, L.A. Smoot and D.G. Brown Guidelines for conducting Listeria monocytogenes challenge testing of foods. Food Prot. Trends, 25 (11): of 10

7 Table 3. Average weight (gram) of each product Sample number Southwest Style Chili Tomato Mozzarella Average Table 4. The microbiological counts and ph/water activity values in bagel topped with the Southwest Style Chili Cheese Topper Pull time Initial Replicate Aerobic bacteria Yeast Mold ph Aw 1 80 <10 < <10 < <10 <10 1 <10 < h 2 <10 <10 < <10 <10 <10 *CFU/g: colony forming unit per gram Table 5. The microbiological counts and ph/water activity values in bagel topped with the Tomato Mozzarella Cheese Topper Pull time Initial Replicate Aerobic bacteria Yeast Mold ph Aw 1 <10 <10 < <10 <10 < <10 <10 <10 1 <10 < h 2 <10 <10 < <10 <10 <10 *CFU/g: colony forming unit per gram 7 of 10

8 Figure 1. Changes in levels of inoculated challenge microorganisms in the products Table 6. The result of Staphylococcal enterotoxin and Bacillus diarrheal enteroxin test in samples stored at ambient temperature for 24h Product Replicate Staphylococcal enterotoxin test Bacillus diarrheal enterotoxin test Southwest Cheese Topper Margarita Cheese Topper 1 Negative/25g Negative/25g 2 Negative/25g Negative/25g 3 Negative/25g Negative/25g 1 Negative/25g Negative/25g 2 Negative/25g Negative/25g 3 Negative/25g Negative/25g 8 of 10

9 Table 7. The counts of challenge microorganisms in inoculated samples of bagel topped with Southwest Chili Cheese Topper Pull time Replicate Initial 6 h 12 h 18 h 24 h B. cereus E.coli O157:H7 Salmonella spp. S. aureus L. monocytogenes < <10 < < * CFU/g: colony forming unit per gram: Log 10 CFU/g: Logarithm base 10 colony forming unit per gram with Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, Escherichia coli O157:H7 and Bacillus cereus 9 of 10

10 Table 8. The counts of challenge microorganisms in inoculated samples of bagel topped with Tomato Mozzarella Cheese Topper Pull time Replicate Initial 6 h 12 h 18 h 24 h B. cereus E.coli O157:H7 Salmonella spp. S. aureus L. monocytogenes * CFU/g: colony forming unit per gram: Log 10 CFU/g: Logarithm base 10 colony forming unit per gram with Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, Escherichia coli O157:H7 and Bacillus cereus 10 of 10