Materials and Methods (Continuation) Results (Continuation) Antibacterial properties of carvacrol and cinnamaldehyde released from solution blow spun fish skin gelatin nanofibers Fei Liu1, Furkan Türker Saricaoglu1, David Bridges2, Roberto J. Avena-Bustillos1, Gary Takeoka1, Vivian Wu2, and Tara H. McHugh1 1Healthy Processed Foods Research, and 2Produce Safety and Microbiology Western Regional Research Center, Agricultural Research Service, USDA, Albany, CA 94710 Introduction Materials and Methods (Continuation) Results (Continuation) Nanofibers have been examined for controlled release of drugs and bioactive agents. The primary advantage of nanofibers over larger diameter fibers is larger surface area to volume ratios. Nanofibers can be produced from natural polymers derived from agriculture products and food-grade solvents. Solution blow spinning (SBS) is a novel and practical technique to produce nano- and microfibers. Gelatin nanofibers can be produced by SBS and used as carriers for controlled release of antioxidants and antimicrobials. Chemical analysis of nanofibers and films Nanofibers sealed in Petri dishes were stored at refrigeration until testing. Carvacrol and cinnamaldehyde were extracted from HMWFG nanofibers with ethanol and dichloromethane respectively and their concentrations determined with an Agilent 6890 GC with a flame ionization detector and a DB-5 bonded-phase fused-silica capillary column. The injector and detector temperatures were 220 °C and 250 °C, respectively. The oven temperature was programmed from 80 °C to 165 °C at 6 °C/min. The linear velocity of the helium carrier gas was 38.2 cm/s (80 °C). Antibacterial analysis of nanofibers and films Frozen stock cultures of E. coli O157:H7, S. enterica, and L. monocytogenes were activated in Trypticase Soy Broth (TSB) and incubated at 37 °C for 24 h. 70 mL of sterilized Trypticase Soy Agar (TSA) media firstly poured into Petri dishes and dried for 5 min in a biosafety hood. After drying, 10-20 mg of fibers were settled on the agar applying slight pressure to avoid breaking the agar gel. Activated cultures were diluted 2 times with peptone water and 0.1 mL of second dilution was mixed with 10 mL of sterilized TSA and poured immediately on the first agar layer, and incubated at 37 °C for 24 h. Each fiber was analyzed in triplicate for each strain. For vapor phase analysis, Petri dish lids were covered with 30-40 mg fibers, and fibers were slightly pressed with a spatula in order to stick them to lids. 14 mL of sterilized TSA were poured into dishes and dried in hood without closing the lids. After drying 0.1 mL of second dilution were spread on to agar and then with lids down were closed. All dishes were covered with Parafilm in order to inhibit vapors leaking out. Each fiber was analyzed in duplicate for each strain. Antibacterial analysis of nanofibers and films (continuation) Antibacterial diffusion activity of fish gelatin nanofibers and films Cinnamaldehyde nanofibers Cinnamaldehyde films   Mean Dia., mm Std. Dev. E. coli C 0.00 E-5 7.38 1.05 E-10 8.46 1.80 E-15 20.46 1.67 E-20 23.62 2.28 E-25 27.38 2.26 E-30 29.08 3.21 Salmonella typhimurium 6.33 0.30 10.73 0.53 13.20 0.71 18.20 1.24 19.67 0.65 2.11 22.46 2.49 Listeria monocytogenes 4.66 0.37 9.00 1.57 18.79 1.09 33.92 4.44 37.28 2.47 45.61 2.64 46.91 3.83   Mean Dia., mm Std. Dev. E. coli C 0.00 E-5 5.16 0.08 E-10 15.48 0.37 E-15 21.33 3.65 E-20 28.44 2.45 E-25 34.04 2.32 E-30 34.31 1.71 Salmonella typhimurium 9.76 0.70 17.95 2.31 19.93 1.03 21.50 1.76 25.26 1.19 29.24 0.84 32.25 1.31 Listeria monocytogenes 7.05 0.33 16.24 0.60 20.21 0.40 26.76 1.04 31.21 0.74 32.51 0.54 44.38 1.94 Objective This study evaluated antibacterial properties of carvacrol and cinnamaldehyde against three pathogenic bacteria E. coli O157:H7, S. enterica, and L. monocytogenes as released by diffusion and vapors from SBS nanofibers. Materials and Methods High molecular weight fish skin gelatin (HMWFG) at 16.5% (w/v) was solubilized in 20% (v/v) acetic acid solution. Carvacrol and cinammaldehyde was added at 5-30 % concentrations respectively, based on HMWFG weight, with lecithin at 5% in emulsions produced by microfluidization. SBS was done at a flow rate of 0.1 mL/min and 0.38 MPa air pressure. For film casting, solutions were degassed and 60-65 mL of film forming solution was cast onto glass plates lined with polyester film (Mylar) using a metallic draw down bar at a thickness of 45 mils. The films were allowed to dry for 15 h at room temperature, and then stored in a plastic bag at refrigerator until they were used for testing. Cinnamaldehyde nanofibers, vapor phase Carvacrol nanofibers, vapor phase Solution blow spinning experimental set-up Results Chemical analysis of nanofibers and films Cinnamaldehyde films, vapor phase Carvacrol films, vapor phase Antibacterial activity by vapor phase release from fish gelatin nanofibers and films Conclusions SBS of HMWFG nanofibers resulted in higher losses of carvacrol and cinnamaldehyde than by film casting. All nanofibers and films showed antibacterial activity by diffusion and vapor release at all concentrations of carvacrol and cinnamaldehyde for the three pathogenic bacteria. Bacterial inhibition area increased as concentrations of carvacrol and cinnamaldehyde were increased in the HMWFG formulations. Cinnamaldehyde was more volatile and potent antibacterial released as vapor than carvacrol from the HMWFG nanofibers and films. Gelatin nanofibers produced by solution blow spinning and casted films proved to be effective carriers of antibacterials with promising applications in food systems. Antibacterial analysis of nanofibers and films Antibacterial diffusion activity of fish gelatin nanofibers and films Carvacrol nanofibers Carvacrol films   Mean Dia., mm Std. Dev. E. coli C 0.00 E-15 19.47 2.92 E-20 15.36 2.20 E-25 14.37 0.11 E-30 12.15 2.05 Salmonella typhimurium 6.33 0.30 11.34 1.38 13.91 0.64 12.73 1.37 11.91 2.67 Listeria monocytogenes 8.37 0.44 19.79 1.58 16.00 3.95 15.25 0.31 11.31 0.95   Mean Dia., mm Std. Dev. E. coli C 4.73 6.68 E-15 21.03 0.47 E-20 30.99 5.38 E-25 35.42 2.29 E-30 40.30 0.40 Salmonella typhimurium 9.29 1.53 12.25 0.25 13.59 0.42 14.67 0.68 13.92 1.54 Listeria monocytogenes 15.70 1.70 25.77 0.23 26.77 0.36 36.97 0.61 27.84 20.81 Acknowledgment This project is supported by Agriculture and Food Research Initiative Nanotechnology for Agricultural and Food Systems Grant Program 2013-01598 from the USDA National Institute of Food and Agriculture.