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Title:   Author  item
                                            Lacombe, Alisonitem
                                            Niemira, Brendanitem
                                            Gurtler, Joshuaitem
                                            Sites, Josephitem
                                            Boyd, Glennitem
                                            Kingsley, Daviditem
                                            LI, XINHUI - University Of Delawareitem
                                            CHEN, HAIQIANG - University Of Delaware
  Submitted to: Food Microbiology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 10/13/2016
                                    Publication  Date: 5/3/2017
                                    Citation: Lacombe, A.C., Niemira, B.A., Gurtler, J., Sites, J.E., Boyd, G., Kingsley, D.H., Li, X., Chen, H. 2017. Nonthermal inactivation of norovirus surrogates on blueberries using atmospheric cold plasma. Food Microbiology. 63:1-5.Interpretive Summary: Viruses, including human norovirus, are currently the leading cause of foodborne outbreaks, most of which are associated with foods consumed raw. Because human norovirus cannot be cultured in a laboratory, non-pathogenic surrogate viruses are often used for evaluating sanitizing technologies. Cold plasma (a form of highly reactive, energized gas) is an emerging novel nonthermal technology that can be used for surface decontamination of foods. This study investigated cold plasma for the inactivation of two surrogates for human norovirus:  Tulane virus and murine norovirus. Blueberries (5g) were weighed into sterile glass jars and inoculated with either Tulane virus or murine norovirus. Samples were treated with atmospheric cold plasma for 0, 15, 30, 45, and 60 seconds. The berries were 7.5 cm from the cold plasma emitter. Temperature readings were taken with an infrared camera prior to and immediately following cold plasma treatments. In order to establish the impact of air flow during cold plasma treatment, an additional jet of room temperature air was introduced from a separate nozzle. The experiment was repeated with 90 and 120 seconds as additional treatment time points. The addition of ambient air ensured that heating was not a factor in the inactivation of virus. The amount of Tulane virus or murine norovirus on the berries was measured immediately after each treatment, using cultured cells appropriate for each surrogate. The minimum effective treatment for Tulane virus was 45 seconds, with a 96% reduction compared to the untreated control. The maximum reduction for Tulane virus was 99.97%, after a treatment of 120 seconds. Murine norovirus showed a statistically significant reduction of 68% after only 15 seconds, with maximal reduction of >99.999% after 90 seconds of treatment. These results demonstrate that cold plasma can inactivate viruses without heat as a mechanism, and is therefore a nonthermal process. With further optimization, cold plasma may be used by food processors as a new tool to improve food safety.
                                Technical Abstract: Viruses are currently the leading cause of foodborne outbreaks, most of which are associated with foods consumed raw. Cold plasma (CP) is an emerging novel nonthermal technology that can be used to surface decontaminate foods. This study investigated CP technology for the nonthermal inactivation of human norovirus surrogates, Tulane virus (TV) and murine norovirus (MNV), on the surface of blueberries. Blueberries (5g) were weighed into sterile 4 oz. glass jars and inoculated with TV, 5 log PFU/g. Samples were treated with atmospheric CP for 0, 15, 30, 45, and 60s at a working distance of 7.5 cm with 4 cubic feet/minute (cfm) of CP jet. Temperature readings were taken with an infrared camera prior to and immediately following CP treatments. In order to establish the impact of air flow during CP treatment (4 cfm), an additional 7 cfm jet of room temperature air was introduced from a separate nozzle. The experiment was repeated with 90 and 120 seconds as additional treatment time points.  Viral titers were measured immediately after each treatment with a plaque assay using LLC-MK2 cells (TV) or Raw 264.7 cells (MNV). TV was significantly reduced 1.5 PFU/g compared to the control after treatment time of 45s, which was achieved regardless of temperature conditions. With the addition of 7 cfm of ambient air, the maximum log reduction for TV was 3.5 log PFU/g after 120s of treatment. MNV was significantly reduced by 0.5 log PFU/g compared to the control at 15s, and further treatment of MNV with ambient air brought the log reduction to greater than 5 log PFU/g at 90 s of treatment (Fig 3). These results demonstrate that CP viral inactivation does not rely on thermal inactivation, and is therefore nonthermal in nature. With further optimization, CP may be used by food processors as a means of nonthermal inactivation of foodborne viruses.


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