ABSTRACT Title of Document: AN INVESTIGATION INTO THE EFFICACY OF A PH-SENSITIVE MATERIAL FOR MILK SPOILAGE DETECTION Tom Blackmon, Jonathan Chang, Amy Cheng, Tiffany Jen, Hannah Kravis, Raishay Lin, Michael Lu, Erin Ong, Tanya Pakzad, Nima Sarfaraz, Yvonne Shiau, Jacklyn Wong Directed By: Dr. Nam Sun Wang, Associate Professor, Department of Chemical Engineering Ambiguous expiration dates on milk cartons can mislead consumers into prematurely disposing unspoiled milk and potentially drinking spoiled milk. These misconceptions can lead to wastage that harms the environment, or potential discomfort and illness. The incorporation of pH-sensitive indicators into plastic milk cartons has the potential to replace stamped expiration dates as the traditional method of milk spoilage indication. We studied the correlation between bacteria count and milk pH to establish pH measurement as an effective indicator of milk quality. We then developed a method for incorporating bromothymol blue, a pH-sensitive color-changing dye, into a hydrogel made of polyacrylamide. This hydrogel can be added to existing packaging for milk or other products with detectable pH changes. Additionally, we conducted a consumer survey and analyzed current food packaging trends in the market. Our research indicates that a spoilage-indicating milk carton could have strong market potential as food industries increasingly adopt intelligent packaging designs. AN INVESTIGATION INTO THE EFFICACY OF A PH-SENSITIVE MATERIAL FOR MILK SPOILAGE DETECTION By Team Milk Tom Blackmon, Jonathan Chang, Amy Cheng, Tiffany Jen, Hannah Kravis, Raishay Lin, Michael Lu, Erin Ong, Tanya Pakzad, Nima Sarfaraz, Yvonne Shiau, Jacklyn Wong Thesis submitted in partial fulfillment of the requirements of the Gemstone Program University of Maryland, College Park 2013 Advisory Committee: Dr. Nam Sun Wang, Chair ___________ ____________ © Copyright by Tom Blackmon, Jonathan Chang, Amy Cheng, Tiffany Jen, Hannah Kravis, Raishay Lin, Michael Lu, Erin Ong, Tanya Pakzad, Nima Sarfaraz, Yvonne Shiau, Jacklyn Wong 2013 Acknowledgements We would like to thank all those who have supported and advised us throughout this project; in particular: our terrific mentor, Dr. Nam Sun Wang, who was not only a wise teacher, but also a demanding coach and an unflagging cheerleader; Dr. Bigio from the Department of Mechanical Engineering and Dr. Lo from the Department of Nutrition and Food Science for providing invaluable expertise and resources; and last but certainly not least the staff of the Gemstone Program for their continuing support these past four years. Our Gemstone experience has been extraordinary, and we look forward to seeing more exciting research from future teams. ii Table of Contents Acknowledgements………………………………………………………………………..ii Table of Contents…………………………………………………………………………iii 1. Introduction……………………………………………………………………………..1 1.1 Why is Milk Spoilage Important?..……………………………………………1 1.2 The Problem with Current Milk Spoilage Detection Methods………………..3 1.3 Why is More Milk Spoilage Detection Research Needed?...............................4 1.4 Outline of Study……………………………………………………………….5 1.5 General Study Hypotheses…………………………………………………….6 2. Literature Review……………………………………………………………………….8 2.1 Science Literature Review…………………………………………………….8 2.1.1 Milk and Bacteria……………………………………………………8 2.1.2 Detecting Spoiled Milk…………………………………………….10 2.1.2.1 Utilizing pH Indicators as a Measure of Spoilage……….10 2.1.2.2 Electrical methods for the detection of bacteria…………14 2.1.2.3 Wireless detection and monitoring of milk spoilage…….16 2.1.2.4 Utilizing Gas-Sensor Arrays as a Measure of Spoilage….17 2.1.2.5 Infrared Spectroscopy as Spoilage Indicator…………….20 2.1.2.6 Utilizing Protein or Fat Count as a Measure of Spoilage..22 2.1.2.7 Smell and Taste Sensors in Detection of Milk Spoilage…25 2.1.3 Creating the Milk Spoilage Detector………………………………26 2.2 Business Literature Review………………………………………………….27 2.2.1 Consumer Interest………………………………………………….27 2.2.2 Packaging as a Tool for Communication………………………..…27 2.2.3 Innovation in the Dairy Industry…………………………………...29 2.2.4 Challenges in the Milk Industry……………………………………34 2.2.5 Trends in the Market……………………………………………………….34 2.2.6 Contributions to the Research Field………………………………………..34 3. Methodology…………………………………………………………………………..35 3.1 Lab Methodology…………………………………………………………….35 3.1.1. Bacterial/pH Correlation…………………………………………..35 3.1.1.1 Preparing the LB Broth…………………………………..35 3.1.1.2 Preparing LB Agar……………………………………….35 3.1.1.3 Preparing Samples……………………………………….36 3.1.1.4 Testing pH………………………………………………..36 3.1.1.5 Bacterial Count Testing………………………………….37 3.1.1.6 Correlating Bacterial Count and pH……………………..38 3.1.2 Heat Treatment……………………………………………………..38 3.1.2.1 Open Flame Variation……………………………………38 3.1.2.2 Hot Plate Variation………………………………………39 3.1.2.3 Precision Plastic Melter Variation……………………….40 3.1.2.4 Oven Test Variation……………………………………...41 3.1.3 Effectiveness of Microfiltration Membranes………………………42 3.1.4 Nafion Testing……………………………………………………..43 3.1.4.1 Leakage Test……………………………………………..43 iii 3.1.4.2 Proton Permeability Test…………………………………45 3.1.4.3 Control Test……………………………………………...46 3.1.5 Hydrogel Experiments……………………………………………..46 3.1.5.1 Creation of Gelatin-Based Hydrogel…………………….46 3.1.5.2 Gelatin-based Hydrogel Leakage Test…………………...47 3.1.5.3 Polyacrylamide-Based Hydrogel Composition Test……..47 3.1.5.4 Polyacrylamide-based Hydrogel Leakage Test………….48 3.1.5.5 pH Indicator Comparison………………………………...49 3.2 Market Research Methodology………………………………………………50 3.2.1 Writing the Survey…………………………………………………50 3.2.2 Determining Survey Locations…………………………………….52 3.2.3 Marketing Assumptions……………………………………………53 3.2.4 Best Product Design………………………………………………..53 4. Results and Analysis…………………………………………………………………..56 4.1 Science Results………………………………………………………………56 4.1.1. Bacterial/pH Correlation…………………………………………..56 4.1.2 Heat Treatment……………………………………………………..57 4.1.2.1 Open Flame Variation……………………………………57 4.1.2.2 Hot Plate Variation………………………………………58 4.1.2.3 Precision Plastic Melter Variation……………………….58 4.1.2.4 Oven Test Variation……………………………………...59 4.1.3 Effectiveness of Microfiltration Membranes………………………60 4.1.4 Nafion Testing……………………………………………………..60 4.1.4.1 Leakage Test……………………………………………..60 4.1.4.2 Proton Permeability Test…………………………………62 4.1.4.3 Control Test……………………………………………...63 4.1.5.Hydrogel Experiments……………………………………………..63 4.1.5.1 Creation of Gelatin-Based Hydrogel…………………….63 4.1.5.2 Gelatin-based Hydrogel Leakage Test……………….......64 4.1.5.3 Polyacrylamide-Based Hydrogel Composition Test……..65 4.1.5.4 Polyacrylamide-based Hydrogel Leakage Test………….65 4.1.5.5 pH Indicator Comparison………………………………...66 4.2 Science Analysis……………………………………………………………..68 4.2.1 Bacterial/pH Correlation…………………………………………...68 4.2.2 Heat Treatment……………………………………………………..70 4.2.2.1 Open Flame Variation……………………………………70 4.2.2.2 Hot Plate Variation………………………………………70 4.2.2.3 Precision Plastic Melter Variation……………………….71 4.2.2.4 Oven Test Variation……………………………………...71 4.2.3 Effectiveness of Microfiltration Membranes………………………72 4.2.4 Nafion Testing……………………………………………………..72 4.2.4.1 Leakage Test……………………………………………..72 4.2.4.2 Proton Permeability Test…………………………………73 4.2.4.3 Control Test……………………………………………...74 4.2.5.Hydrogel Experiments……………………………………………..74 4.2.5.1 Gelatin-Based Hydrogel………………………………….74 iv 4.2.5.2 Polyacrylamide-Based Hydrogels …………………..…...75 4.2.5.3 pH Indicator Comparison………………………………...76 4.3 Business Results……………………………………………………………..77 4.3.1 Survey Results……………………………………………………..78 4.3.2 Results and Application of Market Research………………………82 4.3.2.1 Target Market…………………………………………….82 4.3.2.2 Household Consumer Segment Analysis………………...83 4.3.2.3 High-Volume Consumer Segment Analysis……………..83 4.3.3 Industry Analysis…………………………………………………..84 4.3.4 Competitive Advantage……………………………………………85 4.3.5 Competitor Matrix…………………………………………………86 4.3.6 Marketing Strategy and Sales Plan………………………………...87 4.3.6.1 Strategy for Household Consumers……………………...87 4.3.6.2 Strategy for High-Volume Consumers…………………..87 4.3.6.3 Target Distributers……………………………………….88 4.3.6.4 Pricing……………………………………………………89 4.3.7 Financial Plan………………………………………………………89 4.3.7.1 Costs……………………………………………………...89 4.3.7.2 Revenues…………………………………………………90 4.3.7.3 Pricing……………………………………………………90 4.3.7.4 Burn Rate and Break Even Rate…………………………90 4.3.7.5 Financial Projections……………………………………..91 4.3.8 Feasibility…………………………………………………………..91 4.3.9 Key Risks…………………………………………………………..92 5. Conclusion…………………………………………………………………………….94 6. Future Directions……………………………………………………………………...95 Appendix I: Survey Questionnaire………………………………………………………99 Appendix II: Survey Data………………………………………………………………100 Appendix III: Balance Sheet and Income Statement…………………………………...105 Appendix IV: The Milk Industry Report……………………………………………….107 Appendix V: Cost of the ColorCarton with Non-Bulk Pricing…………………………113 v List of Tables and Figures Tables: Table 1: Samples of Milk for Bacteria/pH Correlation 3.1.1.3 Table 2: Literature pH values 3.1.5.5 Table 3: pH readings of “Off the Shelf” (OTS) and “Sterile” (ST) milk samples over time 4.1.1 Table 4: Absorbance readings of filtered dye infused deionized water. Pore size of membrane was 0.45µm 4.1.3 Table 5: Absorbance readings of the deionized water was taken at 650 nm in order to quantify leakage levels. 4.1.4.1 Table 6: Absorbance Readings of Leakage from Gelatin-based Hydrogel 4.1.5.2 Table 7: Polyacrylamide-based Hydrogel Observations after the Addition of pH Buffers 4.1.5.3 Table 8: pH Indicator Comparison