Published on June 6, 2016
slide 1: International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-2 Issue-5 May- 2016 Page | 212 Antimicrobial efficiency of non-thermal atmospheric pressure plasma processed water PPW against agricultural relevant bacteria suspensions Uta Schnabel 1 Rijana Niquet 2 Christian Schmidt 3 Jörg Stachowiak 4 Oliver Schlüter 5 Mathias Andrasch 6 Jörg Ehlbeck 7 123467 Leibniz Institute for Plasma Science and Technology Felix-Hausdorff-Str. 2 17489 Greifswald Germany 5 Leibniz Institute for Agricultural Engineering Potsdam-Bornim Max-Eyth-Allee 100 14469 Potsdam Germany Abstract — Currently used methods for decontamination and sanitation are antimicrobial ineffective generate high costs with a high consumption of water and chemicals additionally. As an alternative non-thermal plasma at atmospheric pressure could be a versatile tool. Therefore an experimental set-up based on a microwave-plasma source which generates plasma processed air PPA containing manifold RNS-based chemical and antimicrobial compounds was used. The PPA was introduced into distilled water phosphate buffered saline PBS or nutrient broth to generate plasma processed water PPW plasma processed PBS PPP or plasma processed broth PPB which can be applied for the decontamination of packaging material fresh produce and processing equipment. This is a new and innovative method for the generation of antimicrobial active plasma processed liquids PPL. In our experiments bacterial suspensions contaminated with six different bacteria Escherichia coli K12 DSM 11250 Pseudomonas fluorescens DSM 50090 Pseudomonas fluorescens RIPAC Pseudomonas marginalis DSM 13124 Pectobacterium carotovorum DSM 30168 and Listeria innocua DSM 20649 in a concentration of 10 6 cfu . ml -1 and subsequently treated with PPW PPP PPB and HNO 3 were investigated. For PPL production the plasma was ignited for 5 15 or 50 s. After a post-plasma treatment with PPL of maximum 5 minutes a decrease of bacterial load up to 6 log steps were detected for examined bacteria. Furthermore an exclusive inactivation by acidification of PPL was excluded. The characteristics of plasma and its generated cocktail of long living chemical compounds in air and in water leading to a high bacterial inactivation and offering a wide range of possible applications. Keywords — fresh food microbial inactivation non-thermal atmospheric pressure plasma plasma processed water. Highlights: microwave plasma processed liquids used successfully for bacterial inactivation 3 different plasma processed liquids distilled water PBS and nutrient broth were investigated and their antimicrobial efficacy compared among each other and to HNO 3 solution decontamination by plasma up to 6.0 log steps for potential human and phytopathogens were achieved. I. INTRODUCTION The consumption of about 400 g up to 800 g per day of fresh fruits and vegetables is recommended by many organizations like the World Health organization WHO 1 the World Cancer Research Fund WCRF and the American Institute for Cancer Research AICR 2 as well as the German Nutrition Society e. V. DGE. Fruits and vegetables are the supplier of vitamins and minerals of dietary fiber and phytochemicals with a low energy density 3. Investigations lead to the conclusion that the more fruits and vegetables eaten the lower the risk is not only for certain types of cancer but also for obesity hypertension and coronary heart disease 4-10. However fresh and fresh-cut produce have a limited shelf life of several days which allows only a regional distribution of that produce. The limited shelf life and the associated losses of fresh produce have various causes but especially depend on microbial contamination at all stages in the value chain. The microbial contamination may also cause foodborne illnesses which occur annually and worldwide. The U.S. Food and Drug Administration FDA listed under the ten riskiest foods in their Center for Science in the Public Interest CSPI Report 2009 5 times fruits and vegetables. Whereby leafy greens are on the top 11. European institutions and customer organizations like the German Institute for Risk Assessment BfR are aware of the risk of food borne illnesses caused by fresh fruits and vegetables too 12 13. slide 2: International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-2 Issue-5 May- 2016 Page | 213 The European Food Safety Authority EFSA described in their zoonoses report of 2011 14 5648 reported food-borne outbreaks for 2011 with more than 200000 confirmed human cases. The outbreaks were caused by Bacillus toxins Campylobacter Clostridium E. coli mainly Verotoxin-producing Escherichia coli VTEC Listeria Yersinia and some others. Initial microbiological load of fresh vegetables ranges between 10 2 and 10 7 cfu g -1 whereas most germs are harmless Gram- negative bacteria like Pseudomonas spp. and Pectobacterium spp. For food safety relevant foodborne pathogens are in particular bacteria like Enterobacteriaceae Escherichia spp. Samonella spp. and Listeria spp. 15 16. Due to the low infection dose of E. coli the guidance level for it can be reduced to 100 cfu g -1 17 18. In case of fresh fruits and vegetables preservation methods such as heat treatment and freezing are not applicable because of the resulting loss of freshness properties. Conventional methods of decontamination and cleaning of fresh food are based on rinsing with water which may contain high amounts of chemicals e.g. chlorine 50-200 ppm chlorine dioxide or ozone. Although the poor stability of chlorine and the association of chlorine with a possible formation of carcinogenic chlorinated compounds in water have called the use of chlorine in food processing applications into question 19 20. Water containing disinfectant eliminates 3 to 4 log of microorganisms in solution and prevents them from attaching to the product surface. However once bacteria are attached or internalized no effective method exists to remove or destroy the contamination 21 22. Therefore the development of environmentally friendly alternative disinfection and cleaning methods is important but also the product compatibility costs environmental impact impact on product quality and regulatory provisions have to be taken into account 23. Alternative methods for both effective and safe disinfection of fresh food especially fruits and vegetables are needed to guarantee safe consumption of high-quality products. One possible alternative method could be the application of non-thermal atmospheric pressure plasma. Plasma is generated by supply of energy to a gas leading to an excitation as well as ionization of gas atoms or molecules giving the opportunity to a direct absorption of electrical power. A wide range of different plasma types are known. One type is the non-thermal atmospheric pressure plasma 24 25. Plasma is always a cocktail of a variety of species including excited and reactive atoms molecules ions and radicals but also radiation VUV UV 26-28. Plasma is currently used in various industrial fields such as electrical engineering textile and packaging industry optics automotive industry printing as well as environmental technology and much more 25 29 30. The application of non-thermal atmospheric pressure plasma is a discipline with increasing attention in the field of food processing and an emerging non-thermal technology for reducing microbial load on the surface of fresh and processed foods 31. Thus the potential applications of non-thermal atmospheric pressure plasma for the food industry are manifold and it has specific potential for the treatment of foods. Recent reports include special applications like modification of seed germination or active packaging of fruits 32 33 but also plasma applications for decontamination of different food products in most cases with the objective of further shelf-life or storage-time extension 34-39. However independent of the application a humid or wet environment is given by microbial suspensions biofilms and cell tissue fresh or liquid food. Therefore the presence of a gas-liquid environment and a gas-liquid interaction is always given. The aim of the presented work was to investigate the antibacterial efficacy of plasma processed water PPW against food- related microorganisms in suspension. Investigations of buffering effects by phosphate buffer and nutrient broth should give an insight for the capability to use PPW in food washing plants. To exclude the pH value as single responsible antimicrobial component HNO 3 solution was examined separately. II. METHODS 2.1 Generation of plasma processed liquids by microwave discharge The generation of all plasma processed liquids was realized by microwave driven discharge processed gas in contact with sterile distilled water phosphate buffered saline PBS after Sörensen pH 7.2 or nutrient broth. The used microwave driven discharge set-up is shown in Fig. 1. The microwaves had a frequency of 2.45 GHz and the supply power was in the range of slide 3: International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-2 Issue-5 May- 2016 Page | 214 1.1 kW. Accordingly the gas temperature was about 4000 K at a gas flux of 18 slm air. The so called generated plasma processed air PPA was introduced into distilled water PBS or nutrient broth and the resulting plasma processed water PPW plasma processed PBS PPP or plasma processed broth PPB were then used to inactivate the bacterial suspensions. The discharge was ignited for 5 15 or 50 s. The suspensions were treated for 1 3 and 5 minutes with the PPW PPP or PPB post-treatment time. The observed inactivation of microorganisms depended on the storage with long-lived reactive chemical species in the liquids and acidification during post-plasma treatment time. For comparability and to investigate if only the acidification is responsible for the inactivation of bacterial suspensions HNO 3 solutions with 3 different pH values were used the same way like the other liquids but without PPA treatment. The chosen pH values for HNO 3 solutions correspond to pH values received for PPW after PPA treatment. FIGURE 1. SCHEME OF THE MICROWAVE-SETUP FOR THE GENERATION OF A: PPW PLASMA PROCESSED WATER B: PPP PLASMA PROCESSED PBS AND OF C: PPB PLASMA PROCESSED BROTH 40. Measurements of the pH value were analyzed with a pH-meter Multi 3420 – WTW Wissenschaftlich-Technische Werkstätten GmbH Weilheim Germany and the pH electrode SenTix® Mic pH 0-14/ 0-100 °C - WTW Wissenschaftlich- Technische Werkstätten GmbH Weilheim Germany directly after PPW PPP or PPB generation. The observed pH values in dependency of the plasma-on time are shown in Table 2. The pH value for the used HNO 3 solutions is also shown in Table 1. TABLE 1 PH VALUES OF PPW PLASMA PROCESSED WATER PPP PLASMA PROCESSED PBS PPB PLASMA PROCESSED BROTH AND HNO 3 pre-treatment time s PPW PPP PPB HNO 3 0 6.1 7.2 7.1 - 5 1.7 7.0 6.6 35 15 1.5 6.6 5.3 25 50 1.1 2.8 3.0 15 2.2 Investigated microorganism suspensions For microbiological experiments Escherichia coli K12 DSM 11250 Pseudomonas fluorescens DSM 50090 Pseudomonas fluorescens RIPAC Pseudomonas marginalis DSM 13124 Pectobacterium carotovorum DSM 30168 and Listeria innocua DSM 20649 were used in concentrations of 10 6 cfu . ml -1 suspended in sterile distilled water see also Table 2. E. coli K12 DSM 11250 and L. innocua DSM 20649 were chosen due to their relationship to enterohemorrhagic E. coli EHEC O157:H7 and L. monocytogenes both human pathogens which could occur on food. However the chosen strains are classified as risk level 1 and therefore easy to handle. P. fluorescens DSM 50090 P. fluorescens RIPAC P. marginalis DSM 13124 and P. carotovorum DSM 30168 occur in soil or on plants and can cause spoilage of food or storage losses e.g. by soft rot. slide 4: International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-2 Issue-5 May- 2016 Page | 215 TABLE 2 BACTERIA STRAINS USED IN THIS WORK Microorganism DSM number ATCC/ NCTC number Escherichia coli DSM 11250 NCTC 10538 Pseudomonas fluorescens DSM 50090 ATCC 13525 Pseudomonas marginalis DSM 13124 ATCC 10844 Pectobacterium carotovorum DSM 30168 ATCC 15713 Listeria innocua DSM 20649 ATCC 33090 Pseudomonas fluorescens RIPAC directly isolated from cantaloupe RIPAC number: D13-0092-1-1-13 2.3 Treatment of bacterial suspensions and recovery of bacterial load The treatment of bacterial suspensions was realized by transferring 2.5 ml PPW PPP PPB or HNO 3 to 2.5 ml bacterial suspension for a specific treatment time. These treatment times were 1 3 and 5 minutes. Afterwards the antibacterial reaction was stopped with 5 ml nutrient broth tryptic soy broth from Merck KGaA Darmstadt Germany or standard nutrient broth I from Carl Roth GmbH+Co.KG Karlsruhe Germany for plasma processed liquids with 5 and 15 seconds plasma on time as well as HNO 3 solution with pH 3.5 and 2.5. In the case of 50 seconds plasma on time and pH 1.5 of HNO 3 20 ml nutrient broth was used to stop the antibacterial reaction. By using the surface-spread-plate count method with tryptic soy agar Merck KGaA Darmstadt Germany or standard nutrient agar I Carl Roth GmbH+Co.KG Karlsruhe Germany plates the recovery was realized and completed with an overnight cultivation in an incubator. The surface-spread-plate count method is a surface counting method employed for aerobic bacteria. 100 µl of all serial dilutions of the broth were plated out on the whole surface-area of the petri dish. Serial dilutions were performed as a 1 in 10 dilution. The detection limit of this procedure was 1 cfu . ml -1 . If the number of microorganisms fell below the detection limit i. e. no viable microorganisms have been found the values were set at detection limit in the graphical representations. 2.4 Statistical Analysis Data presented were mean of the logarithmic values of replicated experiments. Significant differences among non-treated references and countable plasma-treated samples were determined by the independent two-sample t-test for unequal variances also known as Welchs t-test. For calculation the T.Test function implemented in Microsoft® Excel was used. III. RESULTS The investigation of antibacterial effects of PPW PPP PPB and HNO 3 on different bacterial suspensions was based on a previous work with the use of PPA and PPW 41-43. The optimized plasma parameters for PPA production and the subsequent preparation of PPW were taken from the latter publications. 3.1 Inactivation of bacterial suspensions of phytopathogen P. fluorescens The investigations with bacterial suspensions of P. fluorescens DSMZ and RIPAC strain in combination with PPW PPP PPB and HNO 3 were done under the aspect to compare a non-buffered plasma processed liquid which is commonly used in food industry and elsewhere with weak and strong buffered plasma processed liquids as well as a non-plasma processed liquid with comparable pH values and containing RNS reactive nitrogen species. These possible RNS are also produced within the used microwave generated PPA and could dissolve in distilled water. slide 5: International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-2 Issue-5 May- 2016 Page | 216 By pipetting the plasma processed liquids PPL and HNO 3 were added to the bacterial suspensions of P. fluorescens in concentrations of 10 6 cfu . ml -1 . The PPA used for the generation of PPW PPP and PPB was generated in three different concentrations achieved by a 5 15 and 50 second microwave plasma ignition pre-treatment time. The post-treatment times of the plasma processed liquids were 1 3 and 5 minutes. The timescales reflected the time of contact between bacterial suspensions and PPL before nutrient broth was used to stop the possible reactions. The analysis with plasma processed PBS was done due to the fact that this solution would have a mild buffering effect. The dissolved salts of sodium chloride potassium chloride and phosphates should react with chemical reactive compounds of PPA and therefore less antibacterial species may be available to inactivate the bacteria in the suspension. To investigate the buffering effect of non-organic and organic components to the antimicrobial efficacy of PPW is important due to its possible applications in food industry on organic matter. Commonly microorganisms which occur in food industry and processing are in contact with complex media like the product itself or washing and rinsing liquids. Within this environment biofilm forming can occur easily. Therefore the agents used for cleaning should be compatible to the buffering capacity of these complex media. This means the antibacterial components should not be decomposed or a sufficient amount of antibacterial species should remain. All bacterial suspensions were treated with PPB generated by microwave PPA treatment of nutrient broth. The used nutrient broth was dependent on the used bacterial strain. The PPA was generated in three different concentrations by a 5 15 and 50 second microwave plasma ignition pre-treatment time whereas the post-treatment times of the PPB were 1 3 and 5 minutes. This was the time of contact between the bacterial suspension and the PPB. The experiments with PPL made from distilled water PBS and nutrient broth showed a dependency of bacterial inactivation between pre-treatment time post-treatment time and the pH-value. However a complex chemistry also happens when PPA gets into contact with the PPLs. To exclude that the pH-value change is not the only cause for the observed inactivation kinetics a HNO 3 solution in different antimicrobial effective pH-values was investigated. Therefore pH-values comparable to the lowest ones of each PPL pH 3.5 of HNO 3 compared to pH 3.0 of PPB after 50 s pre-treatment time pH 2.5 of HNO 3 compared to pH 2.8 of PPP after 50 s pre-treatment time and pH 1.5 of HNO 3 compared to pH 1.1 of PPW after 50 s pre- treatment time were examined. Experimental results Fig. 2 showed an antibacterial reduction of 6.6 log-steps for P. fluorescens DSMZ strain and 7.0 log- steps for P. fluorescens RIPAC strain maximum. Inactivation kinetics observed for 5 seconds/pH 3.5 and 50 seconds/pH 1.5 pre-treatment are comparable for both P. fluorescencs strains. Five seconds plamsa treated PBS and nutrient broth as well as HNO 3 solution with a pH value of 3.5 resulted in 0.6 log-step reduction for DSMZ strain and in 2.4 log-steps reduction for RIPAC strain maximum. Only the PPW treatment led to higher reduction rates. For P. fluorescens DSMZ strain 4.4 log-steps and for RIPAC strain 5.0 log-steps inactivation was received. In nearly all cases for this treatment parameters a tailing after 1-minute post-treatment time was gained. In the case of 50 seconds plasma treated liquids or a HNO 3 solution with a pH value of 1.5 the lowest decrease for P. fluorescens was 0.9 and 1.3 log-steps respectively. The worst inactivation kinetics with a maximal decrease of 4.5 and 3.3 log-steps was seen for HNO 3 solution. The detection limit was reached for both strains with PPW after 1-minute post-treatment time with PPP after 3 minutes’ post-treatment and with PPB within 1 up to 3 minutes. In between these results the ones for 15 seconds pre-treatment of PPL and HNO 3 with pH value of 2.5 are located. Here the strains showed different behavior. However PPW had the strongest 5.4 and 7.0 log-steps inactivation capacity and PPP the lowest 0.6 log-steps. The combination of P. fluorescens DSMZ strain and PPB led to no inactivation with HNO 3 this strain had up to 3.1 log-steps reduction. In the case of P. fluorescens RIPAC strain the behavior was slightly different. Here the treatment with 15 seconds pre-treated PPB led to 2.4 log-steps decrease and HNO 3 with pH 2.5 to 2.8 log-steps inactivation maximum. slide 6: International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-2 Issue-5 May- 2016 Page | 217 0 2 4 6 A.1 A.2 log average of survivors / cfu·ml -1 Treatment: PPW PPP PPB HNO3 B.1 Pre. time 5 s pH 3.5 0 2 4 6 B.3 B.2 Pre. time 15 s pH 2.5 0 1 2 3 4 5 0 2 4 6 A.3 post-treatment time / min 0 1 2 3 4 5 Pre. time 50 s pH 1.5 FIGURE 2. RESULTS OF THE PPL AND HNO 3 TREATMENT OF BACTERIAL SUSPENSIONS 2.5 ml. P. FLUORESCENS DSM 50090 A AND P. FLUORESCENS RIPAC B IN CONCENTRATIONS OF 10 6 cfu . ml -1 WERE INVESTIGATED. AFTER A PLASMA IGNITION FOR 5 SECONDS PRE-TREATMENT TIME OR PH 3.5 RESPECTIVELY A1/B1 FOR 15 SECONDS OR PH 2.5 RESPECTIVELY A2/B2 AND FOR 50 SECONDS OR PH 1.5 RESPECTIVELY A3/B3 THE SUSPENSIONS WERE INCUBATED WITH PLASMA PROCESSED LIQUIDS PPL OR HNO 3 IN DURATIONS OF 1 3 AND 5 MINUTES POST-TREATMENT TIME. THE AVERAGE OF THREE EXPERIMENTS IS SHOWN. EXPERIMENTS WERE DONE WITH N3. 3.2 Inactivation of bacterial suspensions of phytopathogens P. marginalis and P. carotovorum Experimental results Fig. 3 showed an antibacterial reduction of 6.2 log-steps for P. marginalis and 6.3 log-steps for P. carotovorum maximum. Inactivation kinetics observed for 5 seconds/pH 3.5 and 50 seconds/pH 1.5 pre-treatment are comparable for both investigated strains furthermore the gained kinetics for P. marginalis are very close to the ones received for P. fluorecens strains Fig. 2 A and B. Five seconds plasma treated PBS and nutrient broth as well as HNO 3 solution with slide 7: International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-2 Issue-5 May- 2016 Page | 218 a pH value of 3.5 resulted in 0.1 log-step reduction for P. marginalis and in 0.3 log-steps reduction for Pectobacterium strain maximum. Only the PPW treatment led to higher reduction rates. For P. marginalis 6.2 log-steps and for P. carotovorum 6.3 log-steps inactivation was received. In all cases of PPP PPB and HNO 3 for this treatment parameters a tailing after 1-minute post-treatment time was gained. In the case of 50 seconds plasma treated liquids or a HNO 3 solution with a pH value of 1.5 the lowest decrease for P. marginalis was 1.7 and for P. carotovorum 4.3 log-steps respectively. The worst inactivation kinetics was seen for PPB and HNO 3 solution within P. marginalis treatment and for PPP in P. carotovorum treatment. The detection limit was reached for both strains with PPW after 1-minute post-treatment time with PPP after 1-minute and 5 minutes’ post-treatment with PPB and HNO 3 within 5 minutes’ post-treatment time P. marginalis and 1-minute post- treatment for P. carotovorum. In between these results the ones for 15 seconds pre-treatment of PPL and HNO 3 with pH value of 2.5 are located C2/D2. Here the strains showed different behavior. However PPW had the strongest 6.2 and 6.3 log-steps inactivation capacity and PPP as well as PPB the lowest 0.0 to 0.4 log-steps. The combination of P. marginalis and HNO 3 led to 2.2 log-steps inactivation. In the case of P. carotovorum the behavior was different. Here the treatment with HNO 3 with pH 2.5 resulted in 0.8 log-steps inactivation maximum. 0 2 4 6 C.1 C.2 log average of survivors / cfu·ml -1 Treatment: PPW PPP PPB HNO3 D.1 Pre. time 5 s pH 3.5 0 2 4 6 D.3 D.2 Pre. time 15 s pH 2.5 0 1 2 3 4 5 0 2 4 6 C.3 post-treatment time / min 0 1 2 3 4 5 Pre. time 50 s pH 1.5 FIGURE 3. RESULTS OF THE PPL AND HNO 3 TREATMENT OF BACTERIAL SUSPENSIONS 2.5 ml. P. MARGINALIS C AND P. CAROTOVORUM D IN CONCENTRATIONS OF 10 6 cfu . ml -1 WERE INVESTIGATED. AFTER A PLASMA IGNITION FOR 5 SECONDS PRE-TREATMENT TIME OR PH 3.5 RESPECTIVELY C1/D1 FOR 15 SECONDS OR PH 2.5 RESPECTIVELY C2/D2 AND FOR 50 SECONDS OR PH 1.5 RESPECTIVELY C3/D3 THE SUSPENSIONS WERE INCUBATED WITH PLASMA PROCESSED LIQUIDS PPL OR HNO 3 IN DURATIONS OF 1 3 AND 5 MINUTES POST- TREATMENT TIME. THE AVERAGE OF THREE EXPERIMENTS IS SHOWN. EXPERIMENTS WERE DONE WITH N3. slide 8: International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-2 Issue-5 May- 2016 Page | 219 3.3 Inactivation of bacteria suspensions of human pathogens E. coli and L. innocua Experimental results Fig. 4 showed an antibacterial reduction of 6.5 log-steps for E. coli and 6.2 log-steps for L. innocua maximum. Inactivation kinetics observed for 5 seconds/pH 3.5 and 15 seconds/pH 2.5 pre-treatment are comparable for both investigated strains. The inactivation kinetics in all used parameter combinations are different from the ones gained for the phytopathogens Fig. 2 and Fig. 3. Five seconds plasma treated PBS and nutrient broth as well as HNO 3 solution with a pH value of 3.5 resulted in 0.5 log-step reduction for E. coli and in 0.0 log-steps reduction for L. innocua maximum. Only the PPW treatment led to higher reduction rates. For E. coli 2.0 log-steps and for L. innocua 0.8 log-steps inactivation was received. In the case of 15 seconds plasma treated liquids or a HNO 3 solution with a pH value of 2.5 the lowest decrease for E. coli was 0.0 and for L. innocua 0.0 log-steps respectively. The worst inactivation kinetics was seen for PPP PPB and HNO 3 solution within both strains. The detection limit was not reached for both strains with PPW after 5-minutes’ post- treatment time. However a maximum reduction of 3.0 log-steps for E. coli and 2.4 log-steps of L. innocua was seen. The results for 50 seconds pre-treatment of PPL and HNO 3 with pH value of 1.5 are shown in Fig. E3/F3. Here the strains showed a little different behavior. However PPW and PPP had the strongest 6.5/ 59 and 6.2/ 58 log-steps inactivation capacity and HNO 3 as well as PPB the lowest 1.6 to 0.6 log-steps. 0 2 4 6 E.1 E.2 log average of survivors / cfu·ml -1 Treatment: PPW PPP PPB HNO3 F.1 Pre. time 5 s pH 3.5 0 2 4 6 F.3 F.2 Pre. time 15 s pH 2.5 0 1 2 3 4 5 0 2 4 6 E.3 post-treatment time / min 0 1 2 3 4 5 Pre. time 50 s pH 1.5 FIGURE 4. RESULTS OF THE PPL AND HNO 3 TREATMENT OF BACTERIAL SUSPENSIONS 2.5 ml. E. COLI E AND L. INNOCUA F IN CONCENTRATIONS OF 10 6 cfu . ml -1 WERE INVESTIGATED. AFTER A PLASMA IGNITION FOR 5 SECONDS PRE-TREATMENT TIME OR PH 3.5 RESPECTIVELY E1/F1 FOR 15 SECONDS OR PH 2.5 RESPECTIVELY E2/F2 AND FOR 50 SECONDS OR PH 1.5 RESPECTIVELY E3/F3 THE SUSPENSIONS WERE INCUBATED WITH PLASMA PROCESSED LIQUIDS PPL OR HNO 3 IN DURATIONS OF 1 3 AND 5 MINUTES POST-TREATMENT TIME. THE AVERAGE OF THREE EXPERIMENTS IS SHOWN. EXPERIMENTS WERE DONE WITH N3. slide 9: International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-2 Issue-5 May- 2016 Page | 220 The gained results within these experiments clearly showed that not only the pH-value is responsible for the observed inactivation of bacteria by PPW. A deeper insight into the gas composition of PPA and more chemical analysis of PPW may offer new aspects for the responsible inactivation mechanisms of PPW. This will be investigated in future studies. 3.4 Statistical Analysis The results of statistical analysis of the experimental data presented in Fig. 2 to 4 are shown in the Tables 3 to 6. It is obvious that for all reduction above one order of magnitude the experimental data are significant different to the control. TABLE 3 RESULTS OF THE STATISTICAL ANALYSIS DONE WITH THE T-TEST FOR PPW PLASMA PROCESSED WATER. THE SHOWN VALUES ARE THE P-VALUES MULTIPLIED BY 1000 FOR BETTER READABILITY OF THE TABLE. PPW plasma processed water p-values x 1000 pre-reatment time s 5 15 50 post-treatment time min 1 3 5 1 3 5 1 3 5 E. coli 2 2 2 2 2 2 2 2 2 P. fluorescens DSM 25 25 25 25 25 25 25 25 25 P. fluorescens RIPAC 78 78 78 78 78 78 78 78 78 P. marginalis 15 15 15 15 15 15 15 15 15 P. carotovorum 3 3 3 3 3 3 3 3 3 L. innocua 1 0 0 0 1 1 1 1 1 TABLE 4 RESULTS OF STATISTICAL ANALYSIS DONE WITH THE T-TEST FOR PPP PLASMA PROCESSED PBS. THE SHOWN VALUES ARE THE P-VALUES MULTIPLIED BY 1000 FOR BETTER READABILITY OF THE TABLE. PPP plasma processed PBS p-values x 1000 pre-reatment time s 5 15 50 post-treatment time min 1 3 5 1 3 5 1 3 5 E. coli 896 896 896 896 522 272 896 89 86 P. fluorescens DSM 5 5 5 5 5 5 0 3 3 P. fluorescens RIPAC 212 46 62 33 67 44 25 22 22 P. marginalis 422 623 239 123 40 70 0 0 0 P. carotovorum 187 679 36 17 21 22 12 12 12 L. innocua 641 211 836 114 124 584 3 3 3 TABLE 5 RESULTS OF STATISTICAL ANALYSIS DONE WITH THE T-TEST FOR PPB PLASMA PROCESSED BROTH. THE SHOWN VALUES ARE THE P-VALUES MULTIPLIED BY 1000 FOR BETTER READABILITY OF THE TABLE. PPB plasma processed broth p-values x 1000 pre-reatment time s 5 15 50 post-treatment time min 1 3 5 1 3 5 1 3 5 E. coli 750 750 750 750 750 750 182 182 182 P. fluorescens DSM 469 417 676 117 170 499 1 1 1 P. fluorescens RIPAC 237 237 225 225 225 225 224 224 224 P. marginalis 27 9 44 94 425 235 0 0 0 P. carotovorum 359 342 410 302 846 556 226 226 226 L. innocua 50 299 582 136 244 271 0 1 2 slide 10: International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-2 Issue-5 May- 2016 Page | 221 TABLE 6 RESULTS OF STATISTICAL ANALYSIS DONE WITH THE T-TEST FOR HNO 3 . THE SHOWN VALUES ARE THE P- VALUES MULTIPLIED BY 1000 FOR BETTER READABILITY OF THE TABLE. HNO 3 p-values x 1000 pH value 3.5 2.5 1.5 post-treatment time min 1 3 5 1 3 5 1 3 5 E. coli 112 0 0 0 0 0 0 0 0 P. fluorescens DSM 139 0 0 0 0 0 0 0 0 P. fluorescens RIPAC 8 0 0 0 0 0 0 0 0 P. marginalis 921 460 478 7 6 6 6 6 6 P. carotovorum 150 303 528 2 6 0 2 2 2 L. innocua 637 846 542 982 806 938 101 85 74 IV. DISCUSSION In the manufacture of food good hygiene is a key part of the quality assurance i.e. ensuring that the product is within the microbial specifications appropriate to its use. Poor hygienic conditions and inadequate sanitation will result in healthcare- associated infections and foodborne diseases as well as high production losses in food industry. Therefore the inactivation of human- and phytopathogens is of great interest in many social and economic fields. Some typical human pathogens which can be found in food are E. coli L. monocytogenes Salmonella Yersinia S. aureus – even MRSA methicillin-resistant S. aureus and ORSA oxacillin-resistant S. aureus Clostridium and Aspergillus. In a selection of phytopathogens many molds e.g. Fusarium oomycetes Xanthomonas Erwinia including the new group of Pectobacteria and Pseudomonas can be found 4 20. The investigated bacteria represent possible food contaminations gram-positive and gram-negative which are often responsible for human or plant diseases. Non-thermal plasma treatment of foods is a promising technology in that it acts rapidly does not leave toxic residuals on processed parts or in the exhaust gas and the temperature rise can be kept at an acceptable level 15. The combination of plasma species with a non-thermal treatment mode makes non-thermal plasmas particularly suited for decontamination in food processing settings 21-25. This process is practical inexpensive and suitable for decontamination of products where heat is not desirable 26. For the inactivation of E. coli Pseudomonas and other microorganisms by microwave generated PPW PPP and PPB described within only physical stresses by chemical acidic and biocidal agents are important. Other stresses such as temperature pressure or radiation can be excluded due to the experimental set-up. The observed kinetics of antimicrobial inactivation of the investigated microorganisms with PPW PPP PPB and HNO 3 are very different. The buffering effect of different solutions was clearly demonstrated. If there was an inactivation by the plasma processed liquid its best result was gained for a 50 seconds pre-treatment time. Here the reason could be the accumulation of antibacterial agens which was gained by increasing the pre-treatment time from 5 up to 50 seconds. For sanitizing products chlorinated water is also used which includes several disadvantages. Other agents are hydrogen peroxide or lactic acid 27 28. The inactivation mode of lactic acid can be attributed to an acidification process causing depression of the inner pH of microbial cells by ionization of the undissociated acid molecules or disruption of the substrate transport by alteration of the cell-membrane permeability. Additionally food borne bacteria cannot grow at pH-values lower than about 4.0. During the treatment of bacterial suspension in this study an acidification on the PPW PPP and PPB was observed and may lead to a similar inactivation mode comparable to the observed lactic acid mode. Due to the plasma set-up and dry air below 32 relative humidity as working gas chemical reactions and species mainly based on RNS reactive nitrogen species are expected. Nitrogen and oxygen in air react to nitrogen monoxide NO which slide 11: International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-2 Issue-5 May- 2016 Page | 222 further leads to the generation of nitrogen dioxide NO 2 with oxygen O 2 . NO and NO 2 are two stable radicals with known antimicrobial effectivity. Nitrogen monoxide may also react with ozone O 3 to nitrogen dioxide and oxygen. Together both radicals NO NO 2 can form dinitrogen trioxide N 2 O 3 which may react with ozone to nitrogen trioxide radical NO 3 via dinitrogen hexoxide N 2 O 6 . Another product might be peroxynitrite ONOO - throughout the reaction of NO with superoxide radical O 2 - 29 30. All these reactions are possible in dry air after plasma ignition. Taking into account that this processed air was combined with 10 ml distilled water or other liquids other chemical reactions may happen. NO O 2 and water H 2 O react to nitrite NO 2 - and hydrogen H + . If instead of nitrogen monoxide NO 2 reacts with the other two molecules H + and nitrate NO 3 - are the products. N 2 O 3 is generated in gas and also gas/ water phase and may react with H 2 O to nitrite and hydrogen again. Two radicals NO 3 and NO 2 form dinitrogen pentoxide N 2 O 5 under the influence of water 29 30. The latter can react with water to nitrate and hydrogen. The occurrence of OH radicals was not detected. Reasons may be the absence of oxygen radicals O due to no energy intake. A further possibility may be water clusters such as H 2 ONO or H 2 OOH. The experiments showed a strong acidification which might be a result of nitrous acid HNO 2 and nitric acid HNO 3 the final end product of all reactions. Usually HNO 2 decays to hydrogen H + and nitrite NO 2 - but a pH-value beneath 2.75 could lead to a spontaneous forming of OH and NO radicals. Most of the mentioned ions radicals and molecules are highly toxic for microorganisms and the chemical cocktail as well as the pH shift may result in the gained inactivation. Further investigations on reactive species densities will provide a better insight into the chemical and biochemical processes underlying the antimicrobial effects observed and assumed in the presented work. Apart from that the exploration of the mechanisms of inactivation of the target microorganisms might reveal relevant details about the plasma inactivation process´s. Due to their different formation and composition of cell walls and membranes commonly gram-negative bacteria are less resistant than gram-positives which are followed by fungus conidiospores and endospores for the treatment by physical plasmas 31. This influence could also be observed in our results. However no significant difference in the inactivation kinetics for gram-negative E. coli as well as the gram-positive L. innocua is observed. Maybe E. coli has specific defense mechanisms against RNS and/or acidification. Additionally a higher impact of reactive oxygen species ROS like ozone or hydrogen peroxide in air and in water to affect bacteria walls due to lipid oxidation 32 33 may play a role compared to RNS which are needed to generate the PPW. Due to the fact that nitric acid could be generated in the plasma processed liquids especially in the PPW it was investigated separately with low pH-values. The results showed that only a very strong acidified HNO 3 solution led to comparable inactivation rates. An antimicrobial effect of PPW exclusively based on HNO 3 formation and increased acidification can be excluded. However the acidification strongly supports the inactivation process which was proved by using the buffering solutions PBS and nutrient broth. As described before the formation of RNS especially NO occurs in the presented plasma set-up. The achieved microbicide effects indicate the antimicrobial efficiency of generated RNS. V. CONCLUSIONS The new and innovative method for the generation of antimicrobial active water presented within this work showed a possible inactivation of 6 different microorganisms with microwave plasma processed water PPW based on distilled water with microwave plasma processed PBS PPP and with microwave plasma processed broth PPB. A significant dependency of inactivation efficiency due to used microorganism their resistance to plasma-chemical components acidification and the treatment times was detected. Buffering solutions and environments can affect the antibacterial efficacy of PPW. With regard to the final pH-value in the sanitizing solution this effect is not excluding this plasma process for decontamination processes. However the promising results and the advantages of plasma processed water low-temperature simple and cheap generation comparability to tap water rinsing ozonized water chlorinated water electrochemically activated water ECA offer a wide range of possible applications. The chemical interaction especially the function of water solved RNS and ROS with respect to microbial inactivation mechanisms should be further investigated. slide 12: International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-2 Issue-5 May- 2016 Page | 223 VI. CONCLUSION Gratefully we thank Dr. Marcel Erhard from the RIPAC-Labor GMBH for providing the direct isolate of Pseudomonas fluorescens. Finally we thank the Federal Ministry for Education and Research of Germany project funding reference number: 13N12428 for financial support. REFERENCES 1 WHO: Diet nutrition and the prevention of chronic diseases. 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