A study of the effect of gliding arc non-thermal plasma on almonds decontamination

Some foodstuffs such as dried fruits or nuts are rich sources of water-soluble vitamins and unsaturated fats while they provide energy and calorie in the nutrition pyramid for human body.¹ Therefore, hygiene and food safety improvement, especially for dried fruits, plays an essential role in the health of society. Various conditions such as ecological environment, farm and garden, seed and soil, the moisture and temperature, processing stage and packaging may result in the bacterial and fungal contamination of nuts.^2–4 Food poisoning due to common bacteria like Escherichia coli (E. coli),⁵ Salmonella, Shigella and pathogenic Fungi e.g. Aspergillus,⁶ contributes to death, even in advanced industrial countries in spite of modern diagnostic, laboratory and therapy modalities.² Scientists in nourishing or health field, food industry and microbiologists, are searching for new chemical, thermal, irradiation or physical methods for both detection and eradication of microbial contaminations.^7–12 Among different old disinfection methods, thermal methods can decrease pathogenic agents phenomenally, but unfortunately may alter color, smell, taste, freshness and quality of food.² In addition, chemical decontamination methods are not feasible for application in human food decontamination. In recent studies, non-thermal plasma has been identified as a new emerging method in food sterilization leading to cell destruction or programmed apoptosis phenomena by cascade decay and excessive production of free radicals.^13–17 

Plasma decontamination of various microorganism such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Bacillus atrophies, Cryptosporidium parvum, Geobacillus stearothermophilus, Salmonella enteritidis, Saccharomyces cerevisiae, Candida albicans, Aspergillus niger, bacterial endospores (B. subtilis and B. pumilus), and bacteriophage Phi X 174 has been studied on artificial surfaces such as glass, paper, nitrocellulose, and polypropylene.^6,18–23 Data from plasma treatment of fresh food, required in order to enhance the understanding of pathogen behavior, are scarce.

This method practically could destroy E. coli which is one of the most important common contaminants of Almonds.^24–26 The results of the study conducted for bacterial decontamination with an atmospheric-pressure plasma system indicated that Erwinia spp. and E. coli could be stopped in 6-8 minutes of treatment.²⁷ In another study, the 9-LOG reduction for 2.5 minutes and 10-LOG reduction for 5minute treatment for Erwinia on the potato were reported.²⁸ Moreover, using plasma flame, Salmonella spp. and Campylobacter contaminant of chicken skin was eradicated.²⁹ Furthermore, the effects of atmospheric-pressure plasma as a pasteurization method were examined for the inactivation of Salmonella enteritidis in almond.³⁰ After about 40 seconds of the process, a reduction in the bacterial population to 1.5 logarithmic cycles was observed. On the other side, the disinfection of Salmonella enteritidis and Salmonella typhimurium on the egg shells was investigated using plasma. The emission spectrum of plasma demonstrated the presence of positive ion species of nitrogen as well as OH and NO radicals. It was shown that the number of Salmonella enteritis was reduced to 2.5 and 4.5 logarithmic cycles, depending on humidity. Also, no negative effect was observed in the quality of eggs as a result of plasma process.³¹ Furthermore, atmospheric-pressure plasma was used to deactivate the inoculated Listeria monocytogenes on chicken and ham³² and the inoculated E. coli as a representative of pathogenic bacteria on carrots, tomatoes, and lettuce,³³ respectively. By increasing power and time of plasma process, microbial population was logarithmically decreased. Using an electron microscope, it was observed that the plasma process could break the cell membrane and change its shape.³³ 

In most of reported studies, argon or helium was used for plasma generation which increased the treatment cost. Therefore, using air as working gas could play a key role to industrialize plasma in food industry. Also, atmospheric pressure plasma has lower cost in comparison to low pressure plasma. Therefore, the most promising reactor for food decontamination is gliding arc plasma which works with air and produces more energetic electrons at atmospheric pressure. The main characteristics of this plasma system are their high electron temperature, high electron density, and yet the low gas temperature (less than 350K). The main goal of the present work is to study the treatment time and power effect of gliding arc plasma on almonds decontamination.

Almonds were purchased from Javid store (Gisha-Tehran-Iran). For each experiment, 3 almonds were subjected to UV radiation for 15 min bilaterally. Then 100 μL of E. coli (ATCC-29252) solution with turbidity equal to 0.5 Mac Farland standard was spotted on the surface of each Almonds and was dried for 20 min in laminar air flow cabinet. Finally, loaded samples were subjected treatment by gliding arc plasma.

Non thermal Plasma was generated by a gliding arc system.^13,34 The system consists of two steel electrodes in the form of knife edge with a thickness of 2 mm, length of 80 mm, width of 20 mm and gap distance of 6 mm. Also, a base made of Plexiglas was selected for keeping the electrodes. The plasma forming gas at 4 lit/min flow rate passes through them. A power supply with the frequency of 20 kHz and variable voltage (varying between 8 and 14 kV) was applied. By applying a high potential difference among the electrodes, the passing gases are ionized and plasma is formed. The main characteristics of these plasma systems are their high electron temperature (their high-energy), high electron density, and yet low gas temperature (less than 350 K). As shown in Fig. 1, the generated plasma is diverted upward due to high gas pressure and can process material surface. The sample distance to the end of electrodes is about 1.6 cm.³⁵ 

Optical emission spectroscopy (OES) is used for reactive plasma diagnostic. In this study, OES has been employed to monitor plasma to ensure that plasma has reached a steady state soon after plasma ignition and to characterize the excited species in the plasma phase. During the treatment of almond samples, the optical spectra were detected in the wavelength range from 200 to 1100 nm simultaneously by an optical spectrometer (Ava Spec-3648).

First, samples were taken in individual vial with sterile normal saline solution (due attention to sample weight), and then the mixture was shaken for 3 minutes and 100 microliter of solution was placed in a petri plate followed by incubation at 37°C for 20 hours. Plate count agar (PCA) for aerobic bacteria and selective agar media including Eosin-Methylene-Blue Aagar (EMBA) for E coli, Nutrient Agar (NA) for general bacteria and Sallmonella Shigella Agar (SSA) were used for detection and enumeration. Final results were reported as a LOG CFU/gr (forming units).

To determine morphology (roughness) and characterize the physical changes done on the surface by air plasma treatment, Scanning Electron Microscopy (SEM) is the best choice. Color characteristics of unexposed and plasma-exposed Almonds were determined by using a Hunter lab colorimeter; and values were expressed in terms of the L (lightness), a (redness) and b (yellowness). Also the color change (ΔE) was calculated.

All experiments were repeated in 3 times and whole statistical analyses were carried out using the SPSS software package (version IBM SPSS statistic 20) and the statistical significant (P<0.05) of the data was analyzed by a one-way Anova test.

The destruction of genetic material by UV indeed appears to play an important role in the case of certain plasmas. This can be explained by the absorption of VUV in the spores’ outer layers which protects the DNA core, while lower-wavelength (near 254 nm) photons are less absorbed in the outer layers and therefore much more sporadically effective. Therefore, depending on the choice of plasma parameters, particularly the gas composition (the presence of N₂ increase the UV radiation) and power, UV can play either a minimal or a decisive role in plasma sterilization. The synergistic effect of UV radiation with etching (e.g. oxygen atoms presence) appears to provide the most potent environment for killing microorganisms.^36–38 

Fig. 2 shows the light emission spectra of gliding arc plasma. The excited nitride oxide is present at the wavelengths 226, 236, and 246 nm but the intensity of these wavelengths are low, showing the minimal role of UV in plasma sterilization. The most predominant lines are related to $N2+$ ions at 354.7 nm and N₂ molecules at 336.1 nm, respectively.^39–41 Peaks at the wavelength of 777 nm and 844 nm imply the generation of oxygen radicals (singlet atomic oxygen, O). Also, the OH radial was detected in 306 nm. The addition of N₂ to O₂ plasma also boosts the yield of O and raises the intensity of UV emission, hence the sterilization effectiveness. Thus, in the present work, air was used as plasma forming gas.

During the plasma treatment the bombardment of microorganism surface by energetic electrons, ions and photons destroys the microorganism structure and increases the penetration of O and OH radicals, which could disrupt cellular metabolism. It is clear that increasing the treatment time increases the decontamination. The important point is the speed of decontamination. In the present work, we found decreasing the surviving spore density by 3 logs in 1 min at 482 w in both NA and EMB media. Also, after 3 min, 6 logs decrease of surviving spore density was obtained. Finally, Fig. 3 shows that after 5 min in NA media and 4 min in EMB media the decontamination of E.coli was done completely.

Rising P leads to an increase in electron density, n_e, hence to an increase in concentration of active species in plasma. However, excessive heating of the gas and the materials to be sterilized sets an obvious upper limit to the value of P. In addition, plasma turbulence increases according to the experimental observation by increasing the power and reduces the plasma contact with almond, which affects decontamination.³⁵ Also, increasing the power led to increasing the current density and increase the length of produced plasma between the electrodes. This increase in the length of the plasma line along with the gas flow causes turbulence behavior. Therefore, in the second step we changed the gliding arc power from 482 W to 670 W for 1 min plasma treatment and 482 to 590 W for 3 min. In NA media (Fig. 4.a) the decrease of surviving spore density reached to about 5 logs for 506 W which is a great value for decontamination. No significant change was observed for 590 W in comparison with 506 W. It could be attributed to an increase in plasma turbulence and consequently a decrease in exposure time of plasma on almond. In EMB media (Fig. 4.b) the decrease of surviving spore density was 6 logs for 506 W but again due to plasma turbulence it was 4 logs at 590 W. The best result was obtained in power 506 W with 6 logs decrease of surviving spore density in 1 min. Also, the effect of gliding arc power was investigated in 3 min (Fig. 5a and b). In both NA and EMB media at 506 W about 6 logs decrease of E.coli density was obtained. Also, for 590 W it was near to 5.5 logs. Therefore, for 482 W at 4 min plasma treatment, lower Log value was observed in compare to 506 W at 1 and 3 min. Then, the SEM and Salmonella and Shigella decontamination were done at 482 W and 4 min plasma treatment.

Optical emission spectroscopy in Fig. 6 shows the production of active radicals as a function of power. In fact, fiber optics was fixed during the power increase and proved the plasma turbulence in other power (590 W and 670 W).

In the third part, considering the optimum results, we chose 482 W and 4 min for study the salmonella and shigella decontamination. Table I shows the decrease of salmonella and shigella density from 8.17 logs to 3.59 and 4.09 logs, respectively, in NA media. The results for SSA media have been 3.03 and 2.97 logs, respectively. These results prove the gliding arc performance for fast decontamination.

The structure of almond after gliding arc processing was studied using electron microscopy. The structure is shown in Fig. 7. In general terms, the structure did not show important changes after processing. Fig. 7.a is related to before plasma treatment and Fig. 7.b shows the almond surface after 4 min gliding arc treatment. This result could be important for lifetime and color change of almond.

The color of Almonds did not change significantly due to gliding arc air plasma exposure for up to 4 minute. The average L (brightness), a (redness) and b (yellowness) was shown in Table II. Regarding to new finding, the range of changes in Hunter color number (ΔE) was from 0.7 to 2.44. Therefore, air plasma can be readily used for surface decontamination of almonds without noticeable color change. In fact, no obvious damage to the surface color of the plasma-exposed samples was observed. Finally, the collection of various content and information about food industrial decontamination method from multiple research groups, H. Mchugh, et.al (IR heating),⁴² powers and Kang, et.al (steam pasteurization),^43,44 Shinnichi kawamoto, etal (dry heat, hot water),⁴⁵ proved that almond disinfection using air plasma gliding arc can be offered as one of the effective emerging method transcending other routine food sterilization industrial techniques.

Gliding arc plasma reactor was used to decontaminate E.coli on almond. It was shown that increasing the treatment time increases the decontamination. In the present work, we found decreasing the surviving spore density by 3 logs in 1 min at 482 W in both NA and EMB media. Also, after 5 min in NA media and 4 min in EMB media the decontamination of E.coli was done completely. Then, in both media the decrease of surviving spore density was investigated for 1 and 3 min plasma treatments. The optimum result, 4 min was chosen for study of the salmonella and shigella decontaminations. Decrease of salmonella and shigella density from 8.17 logs to 3.59 and 4.09 logs, respectively, in NA media was observed. The results for SSA media are 3.03 and 2.97 logs, respectively. SEM and Hunter lab analysis did not show significant change in almond surface structure and color after 4 min plasma treatment.