Corn processing by pulsed electric fields with respect to microbial inactivation and improvement of seed vigour

doi:10.21203/rs.3.rs-3340989/v1

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Corn processing by pulsed electric fields with respect to microbial inactivation and improvement of seed vigour

https://doi.org/10.21203/rs.3.rs-3340989/v1

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Pulsed electric field (PEF) treatment of corn grains as an alternative to improve seed vigour and inactivation of endogenous microflora by energies ranging from 1.20 to 28.8 J were applied to determine effectiveness of applied energies on germination rate (GR), normal seedling rate (NSR), electrical conductivity (EC), ability to germinate under salt (100- and 200 mM salt) and cold (at 10 C for 7 days and at 25C for 5 days) stresses in addition to inactivation of total aerobic mesophilic bacteria (TAMB), total mold and yeast (TMY), and inactivation rate (%) of Aspergillus parasiticus. Increased energy provided 11.10% increase in GR, 21.22% increase in NSR, 95.50% increase in germination at 10 °C for 7 days, 32.53% increase in germination at 24 °C for 5 days, 68.35% increase under 100 mM- and 76% increase under 200 mM NaCI salt stresses. EC of the corn grains significantly affected by the measurement time rather than PEF treatment. Inactivation on the mean initial TAMB and TMY were approximately 9.25 and 7.93 log, respectively, with 63.33 ± 0.22% reduction in A. parasiticus culture. Optimization studies revealed 300 Hz, 28.80 J, and 19.78 sec as the most optimal processing parameters. PEF treated corn seedlings had stronger and taller body formation with stronger roots. PEF treatment carries a high potential to improve corn vigour with inactivation of surface microflora.

pulsed electric fields

corn

corn vigour

stress tolerance

microbial inactivation

Corn known as maize (Zea mays L.) is one of the valuable livestock used as human food, feed, and used in different industries as raw material. Corn is classified as thermophilic crop which is highly sensitive to cold (Haldimann 1998; Mao et al. 2017). The optimum growth temperature ranges from 21 to 27°C with suboptimal temperatures of 10–20°C. Growth temperatures below the optimum resulted in reduction in the biomass production and growth retardation. Irreversible damage and plant death occur at the temperatures below 10°C (Greaves 1996; Leipner and Stamp 2009). Soil salinity is one of the main factors to limit productivity of agricultural production, and almost 20% reduction in production is observed due to salinity alone in worldwide (Zhu 2001). Being one of the most salt sensitive cereals (Hanin et al. 2016; Machado and Serralheiro 2017) growth inhibition and retardation in development as well as reduction in protein synthesis, respiration, and photosynthesis (Meloni et al. 2003; Ashraf and Foolad 2007; Shtereva et al. 2015) in addition to oxidative stress leading to increase in reactive oxygen species (ROS) of hydroxyl radicals (OH), superoxide (O₂⁻) and hydrogen peroxide (H₂O₂) are observed in maize under salt stress (Alscher et al. 1997; Mittler 2002; Neill et al. 2002). In order to overcome crop loss and increase in production, development of resistant crops and the use of growth stimulators are practiced.

Pulsed electric fields (PEF) with the application of high voltage electric fields at certain frequency can be applied at various magnitude considered as lethal providing microbial inactivation when it is applied higher than 30 kV/cm (Toepfl et al. 2007) and nonlethal providing extraction, increase in drying (Lebovka et al. 2002; Ade-Omowaye et al. 2003; Chalermchat and Dejmek 2005), and improvement of seed vigour (Evrendilek and Tanasov 2017; Evrendilek et al. 2019; Atmaca et al. 2021) when it is applied lower than 30 kV/cm. Previous studies revealed that PEF treatment of wheat (Evrendilek et al. 2021), cucumber (Atmaca et al. 2021), red cabbage (Evrendilek et al. 2019), parsley, tomato, dill, garden rocket, and onion seeds (Evrendilek and Tanasov 2017) provided a significant inactivation on surface microflora in addition to increase in germination rate and resistance to cold and salt stresses in cucumber (Atmaca et al. 2021) and wheat (Evrendilek et al. 2021) but corn.

Aspergillus spp contamination of corn is one of the major safety concern with high economic risks due to production of aflatoxin (Robens and Cardwell 2003; Wu 2006). Thus, studies focused on inactivation of aflatoxin producing fungi were practiced with the reduction of Aspergillus niger by pulsed UV-light system in corn meal (Jun et al. 2003), inactivation of aflatoxin producing Aspergillus spp by atmospheric pressure fluidized bed plasma (Dasan et al. 2018), and Aspergillus parasiticus by pilot scale radio frequency (RF) in corn (Zheng et al. 2017). Effect of electro-ozonization on pre-sowing of corn seed were applied with the determination of its effect on germination ability, growing energy, and germination energy (Normov et al. 2019). Even though previous studies focused on inactivation of Aspergillus spp. and promotion of germination ability of corn; information regarding inactivation of aflatoxin producing Aspergillus paraciticus as well as endogenous microflora on corn grains in addition to increase in germination rate, normal seedling rate, and resistance to cold salt stress on corn grains treated by PEF are not reported in the literature. Thus, the objectives of this study were to treat corn grains by PEF to inactivate total mesophilic aerobic bacteria (TMAB), total mold and yeast (TMY), and A. paraciticus and provide an increase in the germination rate, normal seedling rate, and resistance to cold and salt stress.

Corn grain

Corn grain samples (Zea mays everta) were kindly obtained by a local producer (Bolu, Turkiye). The samples were kept in airtight container at room temperature until use.

PEF treatment system

A pilot-scale PEF system coupled with parallel plate electrode with 300x200 mm dimensions providing monopolar rectangular pulses was used. Distance between two electrodes was set to 0.6–0.7 cm to allow the corn grains pass through between two plates with no occlusion. PEF system enables to supply 20 kV electric field strength with 100–300 Hz oscillator frequency (Bulut et al. 2020). Corn grains were treated by the frequencies and energies in the range of 100–300 Hz and 1.20–28.90 J, respectively (Table 1).

Measurement of germination ability

Grains were planted on 10 cm moist soil, and germination was checked on a daily basis for three consecutive days. The mean germination rate (GR) was calculated (Evrendilek et al. 2021).

Normal seedling rate (%, NSR) was calculated based on the classification of the seedlings as normal and not normal after 5 days of the planting(Evrendilek et al. 2021).

Electrical conductivity

Grain samples were planted, and the water having the electrical conductivity (EC) of less than 5 µS/cm was added to each pot. EC of the grains were measured after 4, 8, and 24 h of the PEF treatment by the Sension 5 model held conductivity meter (HACH, CO, USA) (Evrendilek et al. 2021).

Adaptation to stress factors

Germination ability of the grains at both 10 and 25 ºC to determine adaptation to cold stress was enumerated after they were mixed with moistened soil and placed between two layers of rolled filter paper. Filter papers were placed inside the plastic bags and germination was controlled on a daily basis (Evrendilek et al. 2021; Atmaca et al. 2021).

All grains were planted, and 100 mL of water at first day and 50 mL on the following days at two EC levels (10.8 and 19.8 mS/cm) provided by 100- and 200-mM salt concentrations were given to grains placed in temperature-controlled cabinet to determine the germination ability under the salt stress (Evrendilek et al. 2021; Atmaca et al. 2021). Growth under salt and cold stresses was observed for 10 days.

Inactivation of surface microflora

Inactivation of the total aerobic mesophilic bacteria (TAMB) and total mold and yeast (TMY) were performed by surface plating method (Evrendilek et al. 2021). Results were reported as log cfu/g.

Aspergillus parasiticus culture and inoculation of the samples

Aflatoxin producing Aspergillus parasiticus culture isolated from red pepper was provided by Cukurova University (Adana, Turkiye). Spore formation was controlled by haemocytometer after 50 mL of distilled water and Tween 20 (Sigma, Sigma Aldrich, Steinheim, Germany) were added plates incubated at 22–24°C for 3–5 days.

Corn grains inoculated with A. parasiticus culture were incubated at 22–24°C for 3–5 days. Serial dilutions from both the control and PEF-treated samples were plated onto YGC-A, and the plates were incubated at 22–25°C for 24 h. Inactivation of A. parasiticus is calculated as % inhibition (Bulut et al. 2020; Mbah and Akueshi 2009).

Data analyses

To determine the mean significant differences among the PEF treatments and the control samples for the seed quality tests and microbial inactivation, Tukey’s multiple comparison tests following one-way analysis of variance (ANOVA) were performed at a 95% confidence level (p ≤ 0.05). Data analyses were performed using Minitab Version 17 (Minitab, Inc., State College, PA).

The best-fit multiple (non-)linear regression (MNLR) models in order to predict changes in the response variables as a function of the predictors in addition to their interactions were constructed as follows:

Y = β₀ + β₁χ₁ + β₂χ₂ + …+ β_nχ_n + ε (1)

where Y refers to the response variable; β₀ is the intercept; and β_{1 to n} are the slope coefficients. The joint optimization of all the responses was performed using the three operational predictors of treatment time (Trt), energy (E), and frequency (F) based on the composite desirability function (D) with the most optimal solution for the joint optimization.

PEF treatments provided a significant increase in the mean initial GR (60.11 ± 1.62%) of the corn samples on both 2^nd and 3^rd days of the germination studies (p < 0.05). GR for all treatments on 3^rd day were also significantly higher than that of the 2^nd day (p < 0.05). The highest GR of 80.00% by 9.59 and 28.8 J treatments at the 2^nd day and 100% by 6.00 and 10.80 J treatments at the 3^rd day of germination were observed after the treatments, respectively (Table 1). NSR of the corn grains was also significantly increased by all PEF treatments. NSR of the control samples (78.78 ± 1.73%) increased to 100.00 ± 0.00 by 18.0 J (Table 2). In addition, PEF-treated seedlings had stronger body and they were taller than that of the control samples (Fig. 1). Moreover, PEF-treated samples also had stronger and longer root formation than that of control samples (Fig. 2).

Control corn grain samples did not have ability to germinate at 10 °C for 7 days; whereas they had the germination rate of 62.22 ± 1.92% at 24 °C for 5 days. The highest germination rate of 2.22 ± 1.92% was observed by 15.90 and 28.80 J treatments at 10 °C for 7 days. Unlike germination at 10 °C for 7 days, all the PEF treatments provided a significant increase in the germination of corn grains at 24 °C for 5 days, and the highest germination ability of 92.22 ± 1.92% was observed by 28.80 J treatment (Table 3).

Except for the PEF treatments of 8.46 and 28.80 J; no significant difference was observed between the control (8.92 ± 1.61 µS/cmg) and the PEF-treated samples at 4^th h after the treatment (Table 4). Electric conductivity of the control samples at both 8^th (10.10 ± 1.72 µS/cmg) and 24^th (14.21 ± 0.63 µS/cmg) h after the PEF treatment, on the other hand, did not significantly changed by the PEF treatments. Although EC of the corn grains were not significantly changed by the applied PEF treatments; they were increased by the measurement time (Table 4).

Germination ability of the corn grains under 100 mM salt stress was greatly improved by PEF. Germination ability of the control samples were increased from 8.33 ± 0.60to 25.00 ± 1.20 from 3^rd to 10^th day; whereas the highest germination rates of the PEF treated samples improved from 16.67 ± 1.00% at 3^rd day by 8.46, 18.00, 22.56 and 28.80 J; 25.00% at 4^th day by 1.20, 18.00, 22.56, and 28.80 J; 41.67 ± 0.80% at 5^th day by 8.46 J; 58.33 ± 1.30% at 6^th day by 1.20 J; 66.67% at 7^th day by 1.20 and 9.54 J; 75.00 ± 0.00% at 8^th day by 9.54 J and 77.08 ± 1.00% at 9^th day by 9.54 J to 79.17 ± 1.46% by 9.54 J at 10^th day, respectively (p < 0.05) (Table 5).

Germination ability of the control corn grains increased from 0.00 ± 0.00 to 18.00 ± 0.78% from 3^rd to 10^th day under 200 mM salt stress; whereas the highest germination rates of the PEF treated samples increased from 16.67 ± 0.3% at 3^rd day by 18.00 J; 25.00% at 4^th day by 2.82, 8.46, 18.00, and 25.44 J; 33.33% at 5^th day by 2.82, 8.46, and 25.44 J; 41.67% at 6^th day 2.82, 8.46, 10.80, and 25.44 J; 58.33% at 7^th day by 1.20 and 8.46 J; 66.67% at both 8^th day by 1.20, 9.54, 25.44, and 28.80 J and 9^th day by 1.20, 9.54, 18.00, 25.44, and 28.80 J; and 75.00 ± 0.54 at 10^th day by 9.54 J, respectively. Germination ratio significantly increased by the time in both control and PEF-treated samples, and PEF-treated samples had significantly higher germination than that of the control samples under both 100- and 200-mM salt stress (p < 0.05) (Table 6). Grains treated by PEF and exposed to 100- and 200-mM salt stress had taller seedling than the control samples with stronger body and more leaves (Fig. 3 and 4).

Microbial inactivation increased with an increase in applied energy. While the mean initial TAMB count of the corn grains, 9.25 ± 0.59 log cfu/g, was reduced to undetectable level by the PEF treatments of 22.56 and 28.80 J applications (Fig. 5); the mean initial TMY count, 9.33 ± 0.103 log cfu/g, was lowered to 1.40 ± 0.18 log cfu/g by 28.80 J application (Fig. 6). The highest energy application 28.80 J caused the maximum inhibition of 63.33 ± 0.22% in A. parasiticus culture (Fig. 7).

The highest predictive power of the responses based on calculated as 100% for germination under 200 mM salt stress (3^rd day), 99.82% for % A. parasiticus inhibition, 99.75% for TMY inactivation, 99.50% for germination under 200 mM salt stress (4^th day), 99.32% for germination under 200 mM salt stress (8^th day), 99.21% germination under 200 mM salt stress (6^th day), 99.08 for germination under 100 mM salt stress (10^th day), 99.00% for germination under 200 mM salt stress (9^th day), 98.94% for germination under 100 mM salt stress (9^th day), 98.63% for germination under 100 mM salt stress (7^th day), 98.94% for germination under 100 mM salt stress (9^th day), 98.76% for germination under 200 mM salt stress (7^th day), 98.43% for TAMB inactivation, 98.26% germination under 100 mM salt stress (8^th day), 98.13% for germination under 200 mM salt stress (10^th day), 98.04% for germination under 200 mM salt stress (5^th day), respectively (Table 7).

Regression models for linear, square, 2- and 3-way interactions revealed that E and Trt*E interaction were significant for germination at 2nd day; whereas Trt, F, Trt*Trt, F*F, E*Trt, and E*F were significant for germination at 3rd day; F, Trt*Trt,F*F, E*E*E, Trt*Trt*Trt, F*F*F, and E*E*Trt were significant for normal seedling; E*E, Trt*F,E*E*E,Trt*Trt*Trt, E*E*Trt, and E*E*F were significant for cold test at 10 °C-7 day; E, Trt, F, E*E, Trt*Trt*, F*F, E*Trt, E*F, Trt*F, E*E*E, Trt*Trt*Trt, F*F*F, E*E*Trt, and E*E*E were significant for EC at 24 h; E, Trt, F, E*E, Trt*Trt*, F*F, E*Trt, E*F, Trt*F, E*E*E, F*F*F, and E*E*F were significant for TMAB inactivation; Trt, and Trt*Trt were significant for A. parasiticus inhibition; E, Trt,F, E*E, Trt*Trt, F*F, E*Trt, E*F, Trt*F, E*E*E, Trt*Trt*Trt, F*F*F, and E*E*F were significant for germination under 100 mM salt stress (3^rd day), E, Trt, E*Trt, E*F, Trt*F, Trt*Trt*Trt, and E*E*F were significant for germination under 100 mM salt stress (4^th day); E, E*E, E*Trt, E*F, Trt*F, Trt*Trt*Trt, F*F*F, and E*E*F were significant for germination under 100 mM salt stress (5^th day), Trt, F, Trt*Trt, F*F, Trt*Trt*Trt, F*F*F, and E*E*Trt were significant for germination under 100 mM salt stress (6^th day); E, Trt, F, E*E, Trt*Trt, F*F, E*Trt, E*F, Trt*F, E*E*E, F*F*F, E*E*Trt, and E*E*F were significant for germination under 100 mM salt stress (7^th day); E, Trt, F, E*E, Trt*Trt, F*F, E*Trt, E*F, Trt*F, E*E*E, Trt*Trt*Trt, F*F*F, E*E*Trt, and E*E*F were significant for germination under 100 mM salt stress (8^th day); E, Trt, F, E*E, Trt*Trt, F*F, E*Trt, E*F, Trt*F, E*E*E, F*F*F, E*E*Trt, and E*E*F were significant for germination under 100 mM salt stress (9^th day); E, Trt, F, E*E, Trt*Trt, F*F, E*Trt, E*F, Trt*F, E*E*E, F*F*F, E*E*Trt, and E*E*F were significant for germination under 100 mM salt stress (10^th day); E, Trt, F, E*E, Trt*Trt, F*F, E*Trt, E*F, Trt*F, E*E*E, Trt*Trt*Trt, F*F*F, E*E*Trt, and E*E*F were significant for germination under 200 mM salt stress (3^rd day); E, Trt, F, E*E, Trt*Trt, F*F, E*Trt, E*F, Trt*F, E*E*E, Trt*Trt*Trt, F*F*F, E*E*Trt, and E*E*F were significant for germination under 200 mM salt stress (4^th day); E, Trt, F, E*E, Trt*Trt, F*F, E*Trt, E*F, Trt*F, E*E*E, Trt*Trt*Trt, F*F*F, and E*E*F were significant for germination under 200 mM salt stress (5^th day); E, Trt, F, E*E, Trt*Trt, F*F, E*Trt, E*F, Trt*F, E*E*E, Trt*Trt*Trt, F*F*F, E*E*Trt, and E*E*F were significant for germination under 200 mM salt stress (6^th day); E, Trt, F, E*E, Trt*Trt, E*Trt, E*F, Trt*F, E*E*E, Trt*Trt*Trt, E*E*Trt, and E*E*F were significant for germination under 200 mM salt stress (7^th day); E, Trt, E*E, Trt*Trt, E*Trt, E*F, Trt*F, E*E*E, Trt*Trt*Trt, E*E*Trt, and E*E*F were significant for germination under 200 mM salt stress (8^th day); E, Trt, F, E*E, Trt*Trt, F*F, Trt*F, Trt*Trt*Trt, E*E*Trt, and E*E*F were significant for germination under 200 mM salt stress (9^th day); and E, Trt, F, E*E, F*F, E*Trt, E*F, Trt*F, E*E*E, Trt*Trt*Trt, F*F*F, E*E*Trt, and E*E*F were significant for germination under 200 mM salt stress (10^th day). Optimization studies revealed that 28.80 J energy, 19.78 sec treatment time and 300 Hz were the most optimal processing parameters with 0. 88 desirability to process corn grains by PEF.

Physiological responses of biological membrane to low intensity PEF considered as non-lethal causing stress is not fully understood yet but previous studies reported that PEF had effects on metabolism with reactive oxygen species (ROS) generation (Gabriel and Teissié 1994; Sabri et al. 1996) in addition to promotion of the production of secondary metabolites resulting in yield increase of a cytostatic compounds (Ye et al. 2004) as well as production of phytosterols and antioxidants from fruits and oil seeds (Guderjan et al. 2007; Eing et al. 2009). Modifications on growth, proliferation, and differentiation induced by electric fields (Berg 1993) depending on the physiological state of the treated cells and electrical parameters (Galindo 2017) were also observed. PEF treatment showed no adverse effect on radicle emergence and gross metabolic activity of germinating barley with no significant changes in proteins, but a decrease in alpha amylase concentration (Criddle et al. 1991).

Growth stimulating effects of high intensity electric fields in nanoseconds (nsPEF) have been observed on seven days old Arabidopsis thaliana seedlings (Eing et al. 2009). nsPEF treatments with the voltages exceeding 10000 V/cm and 30 pulses showed either no effect or an impairment of radicle elongation on germinating barley seeds (Criddle et al. 1991). Compare to untreated counterparts, PEF treatment of tomato seeds with 4-12 kV/cm AC electric fields had increased percent germination rate about 1.1–2.8 times (Moon and Chung 2000). Electrostatic field application on potato seeds prior to sowing for 12 min with 4 kV/cm had positive effects on germination (Cramariuc et al. 2005). Both higher germination and normal seedling rates with taller and stronger seedling and root formation on cucumber (Atmaca et al. 2021), wheat (Evrendilek et al. 2021), winter barley and better germination on red cabbage seeds (Evrendilek et al. 2019) with varying electric field strength and energy were also reported.

PEF impact is usually described with disruption on the cell membrane causing imbalance in membrane transport system with increased permeability which may yield increase in nutrient and water uptake from the soil. Increased membrane permeability measured by changes in EC. Usually increase in EC of the medium which surrounds the membrane indicates the electrolyte leakage from seeds into water, and thus, membrane damage (Marin et al. 2018). Significant increase in EC of corn grains by measurement time rather than PEF treatments indicated that cell damage is time dependent. Similar results were also reported for cucumber (Atmaca et al. 2021) and wheat (Evrendilek et al. 2021) grains that their EC were significantly increased by time rather than PEF treatment.

Salinity is one of the major factors to have lower crop yields. A hyperosmotic stress and ion imbalance due to high salt concentrations (Granella et al. 2018) require to control homeostasis, detoxification, and growth to develop salt tolerance. However, salinity tolerance is a complex mechanism which still remains unsolved. Increase in salt tolerance to 100- and 200-mM NaCl stresses provided by PEF may be the results of the activation of one of the stress tolerance mechanisms. In fact, increase in salt tolerance was observed in PEF treated cucumber seeds (Atmaca et al. 2021) and wheat grains (Evrendilek et al. 2021).

Inactivation of seedborne pathogens such as Drechslera graminea, Xanthomonas campestris pv. campestris, Fusarium graminearum, and Alternaria brassica on red cabbage seeds with increased treatment time and frequency (Evrendilek and Tanasov 2017), and significant inactivation on TAMB and TMY on cabbage, lettuce, garden rocket, wheat (Evrendilek and Tanasov 2017; Evrendilek et al. 2021), and cucumber (Atmaca et al. 2021) was obtained with increased energy. A. parasiticus inoculated in sesame seeds had significant decrease by increased PEF energy (Bulut et al. 2020).

Optimum processing parameters determined by the multi-objective optimization were differed among the seeds treated by PEF depending on the treatment parameters. While 161.8 Hz, 6.1 J, and 19.5 s as the optimal settings with 0.52 D were the optimum parameters for wheat (Evrendilek et al. 2021) wheat; 19.78 s and 17.28 J were the optimal settings for cucumber seeds based on best-fit Gaussian process (Atmaca et al. 2021) which show similarities to corn.

PEF treatment of corn grains with varying energy level demonstrated growth stimulative effect with increased resistance to cold and salt stresses. PEF treated seedlings had stronger body formation with longer and stronger roots. It is possible that strong root formation provide seedling to resist salt and cold stresses. Considering the economical dimension of the product loss due to lowered germination ability of the corn grains, PEF provides a great opportunity for corn with increased germination rate as well as resistance to stress conditions.

Microbial contamination of grains results in crop loss due to reduced germination ability, damage caused on grains, and food safety issues such as secondary metabolites, aflatoxin, produced by Aspergillus spp. Thus, it is important to inactivate endogenous and contaminant flora on grains with sustainable and environmentally friendly technologies such as PEF. PEF treatment applied at 28.80 J was very successful to inactivate surface microflora and aflatoxin producing A. paracisitcus. This study reports that PEF treatment of corn grains is good alternative to chemical applications, growth stimulators, and development of resistant crops, but further studies are required to dissolve the mechanisms of PEF on enzyme system, resistance development and increased germination ability of grains.

Acknowledgements

The financial support is provided by TUBITAK (Project no: 217O068).

Data Availability

Data will be available upon request.

Competing interests

The authors declare no competing interests.

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Table 1 Changes in the germination rate of the corn grains before and after PEF treatments

Energy	Days
(J)	2	3
0.00	60.11 ± 1.62^dB	88.90 ± 1.92^dA
1.20	72.22 ± 3.39^bB	97.78 ± 1.92^abA
2.82	68.89 ± 2.09^bB	96.67 ± 3.33^abA
3.18	70.00 ± 3.33^bB	93.33 ± 0.60^bcA
3.60	67.78 ± 1.27^cB	99.17 ± 0.40^bA
6.00	70.00 ± 2.02^bB	100.00 ± 0.28^aA
8.46	70.00 ± 3.33^bB	96.67 ± 3.33^abA
9.54	80.00 ± 0.08^aA	95.00 ± 0.26^bcA
10.80	65.56 ± 1.71^cB	100.00 ± 0.00^aA
14.10	76.67 ± 6.67^abB	96.67 ± 3.33^abA
15.90	73.33 ± 3.33^bB	95.56 ± 1.92^bA
18.00	72.22 ± 3.39^bB	96.67 ± 3.33^abA
22.56	65.33 ± 3.33^cB	97.78 ± 1.92^bA
25.44	75.56 ± 5.09^abB	96.67 ± 0.00^abA
28.80	80.00 ± 3.67^aB	97.78 ± 1.92^bA

*Data in the same column with lowercase superscript letter and data in the same row with uppercase superscript letter are significantly different (p ≤ 0.05)

Table 2 Normal seedling rate of corn grains before and after PEF treatment

Energy (J)	Normal seedling rate (%)
0.00	78.78 ± 1.73^f
1.20	86.40 ± 3.17^ef
2.82	91.87 ± 4.16^bcde
3.18	98.89 ± 1.92^ab
3.60	94.17 ± 1.67^abcde
6.00	93.33 ± 3.33^abcde
8.46	96.63 ± 3.33^abcd
9.54	93.78 ± 1.22^abcde
10.80	92.22 ± 1.92^abcde
14.10	90.88 ± 3.65^cde
15.90	97.66 ± 2.03^abc
18.00	100.00 ± 0.00^a
22.56	89.77 ± 3.45^cde
25.44	89.66 ± 0.00^de
28.80	96.59 ± 0.07^abcd

*Data in the same column with lowercase superscript letter are significantly different (p ≤ 0.05).

Table 3 Germination of corn grains before and after PEF treatment under cold stress

Energy (J)	Germination (%) at 10 °C for 7 days	Germination (%) at 24 °C for 5 days
0.00	0.00 ± 0.00^cB	62.22 ± 1.92^fA
1.20	0.00 ± 0.00^cB	73.33 ± 0.00^eA
2.82	0.00 ± 0.00^cB	85.56 ± 1.92^abcdA
3.18	0.00 ± 0.00^cB	86.67 ± 0.00^abcA
3.60	0.00 ± 0.00^cB	83.33 ± 1.67^bcdA
6.00	0.00 ± 0.00^cB	78.89 ± 3.89^deA
8.46	0.00 ± 0.00^cB	84.44 ± 1.92^bcdA
9.54	1.67 ± 0.00^aB	86.67 ± 0.00^abcdA
10.80	0.00 ± 0.00^cB	86.67 ± 0.00^abcA
14.10	0.00 ± 0.00^cB	80.00 ± 0.00^cdeA
15.90	2.22 ± 1.92^aB	85.56 ± 1.92^abcdA
18.00	0.00 ± 0.00^cB	90.00 ± 3.33^abA
22.56	0.00 ± 0.00^cB	84.44 ± 1.92^bcdA
25.44	0.00 ± 0.00^cB	84.44 ± 3.85^bcdA
28.80	2.22 ± 1.92^aB	92.22 ± 1.92^Aa

*Data in the same column with lowercase superscript letter and data in the same row with uppercase superscript letter are significantly different (p ≤ 0.05)

Table 4 Changes in the electrical conductivity of the corn grains by pulsed electric fields and time

	Electrical conductivity (µS/cmg)
Energy (J)	4^th hour	8^th hour	24^th hour
0.00	8.92 ± 1.61^abcB	10.10 ± 1.72^aB	14.21 ± 0.63^bcdA
1.20	9.69 ± 1.24^abB	10.83 ± 1.31^aB	15.67 ± 0.54^abcdA
2.82	6.79 ± 0.96^bcdeB	8.32 ± 0.72^aB	12.89 ± 0.82^cdA
3.18	6.82 ± 0.51^bcdeC	9.38 ± 0.49^aB	15.86 ± 0.72^abcdA
3.60	8.56 ± 1.21^abcdB	11.63 ± 0.58^aB	16.21 ± 0.53^abcA
6.00	10.95 ± 0.83^aB	12.05 ± 2.22^aB	17.07 ± 0.59^abA
8.46	5.51 ± 0.85^deC	10.72 ± 0.94^aB	15.19 ± 3.28^abcdA
9.54	6.61 ± 0.00^abcdeC	9.74 ± 0.00^aB	14.10 ± 0.00^abcdA
10.80	6.40 ± 0.95^cdeB	8.83 ± 0.44^aA	12.09 ± 1.25^cdA
14.10	7.51 ± 1.34^bcdeC	10.27 ± 0.97^aB	17.79 ± 1.90^abA
15.90	6.13 ± 1.03^cdeB	9.22 ± 2.44^aA	12.17 ± 1.40^dA
18.00	6.93 ± 1.43^bcdeB	8.86 ± 1.41^aB	16.50 ± 0.62^abcA
22.56	6.88 ± 0.54^bcdeC	8.52 ± 0.44^aB	14.42 ± 1.02^abcdA
25.44	4.86 ± 0.62^eC	7.91 ± 1.44^aB	13.99 ± 0.56^bcdA
28.80	6.30 ± 0.65^cdeC	9.30 ± 0.15^aB	16.89 ± 0.92^abA

*Data in the same column with lowercase superscript letter and data in the same row with uppercase superscript letter are significantly different (p ≤ 0.05)

Table 5 Germination ability of control and PEF treated corn grain under 100 mM NaCI salt stress

Energy	Germination (%) under 100mM NaCI salt stress
(J)	3. day	4. day	5. day	6. day	7. day	8. day	9. day	10. day
0.00	8.33 ± 0.60^bB	8.33 ± 0.00^eB	8.33 ± 0.00^fB	8.33 ± 0.00^iB	8.33 ± 0.00^hB	25.00 ± 0.00^hA	25.00 ± 0.00^iA	25.00 ± 1.20^fA
1.20	8.33 ± 0.60^bE	25.00 ± 0.00^bD	33.33 ± 1.00^cC	58.33 ± 1.30^aB	66.67 ± 1.00^aA	66.67 ± 1.00^bA	66.67 ± 1.20^bA	66.67 ± 0.00^bA
2.82	8.33 ± 0.60^bF	16.67 ± 1.00^dE	25.00 ± 1.00^dD	33.33 ± 1.00^fC	58.33 ± 1.20^bB	66.67 ± 1.00^bA	66.67 ± 1.00^bA	66.67 ± 0.00^bA
3.18	0.00 ± 0.00^cE	16.67 ± 1.00^dD	25.00 ± 1.00^dC	41.67 ± 0.90^eB	41.67 ± 1.10^dB	41.67 ± 1.20^fB	41.67 ± 1.40^fB	50.00 ± 1.00^dA
3.60	0.00 ± 0.00^cG	20.83 ± 2.08^cF	33.33 ± 4.00^bcE	45.83 ± 2.08^dD	50.00 ± 0.60^cC	54.17 ± 1.36^dB	62.50 ± 2.08^cA	66.67 ± 2.17^bA
6.00	0.00 ± 0.00^cE	8.33 ± 0.80^eD	16.67 ± 1.00^eC	16.67 ± 0.36^hC	16.67 ± 0.40^gC	33.33 ± 0.60^gB	33.33 ± 1.00^gB	50.00 ± 0.60^dA
8.46	16.67 ± 1.00^aF	33.33 ± 2.00^aE	41.67 ± 0.80^aD	50.00 ± 1.00^cC	58.33 ± 1.00^bB	58.33 ± 1.20^cB	66.67 ± 1.20^bA	66.67 ± 0.60^bA
9.54	0.00 ± 0.00^cG	20.83 ± 0.00^cF	35.42 ± 0.60^bE	56.25 ± 0.00^bD	66.67 ± 0.60^aC	75.00 ± 0.00^aB	77.08 ± 1.00^aA	79.17 ± 1.46^aA
10.80	0.00 ± 0.00^cE	16.67 ± 1.00^dD	25.00 ± 1.20^dC	25.00 ± 1.00^gC	33.33 ± 1.00^eB	50.00 ± 0.60^eA	50.00 ± 1.00^eA	50.00 ± 0.00^dA
14.10	0.00 ± 0.00^cF	8.33 ± 0.60^eE	16.67 ± 0.80^eD	16.67 ± 1.00^hD	25.00 ± 1.00^fC	41.67 ± 0.80^fB	50.00 ± 1.00^eA	50.00 ± 0.00^dA
15.90	0.00 ± 0.00^cG	16.67 ± 1.00^dF	25.00 ± 1.00^dE	25.00 ± 1.20^gE	25.00 ± 1.00^fD	33.33 ± 1.20^gC	50.00 ± 1.40^eB	58.33 ± 0.60^cA
18.00	16.67 ± 1.00^aF	25.00 ± 1.60^bE	33.00 ± 1.00^cD	41.67 ± 0.80^eC	50.00 ± 1.60^cB	58.33 ± 0.80^cA	58.33 ± 1.40^dA	58.33 ± 1.00^cA
22.56	16.67 ± 1.00^aD	25.00 ± 1.40^bC	25.00 ± 1.00^dC	33.33 ± 1.00^fB	33.33 ± 0.00^eB	33.33 ± 1.00^gB	50.00 ± 1.00^eA	50.00 ± 1.00^dA
25.44	0.00 ± 0.00^cD	16.67 ± 1.00^dC	25.00 ± 1.20^dB	25.00 ± 1.00^gB	33.33 ± 0.00^eA	33.33 ± 1.26^gA	33.33 ± 1.32^gA	33.33 ± 0.80^eA
28.80	16.67 ± 1.00^aE	25.00 ± 1.00^bD	33.33 ± 1.40^cC	41.67 ± 1.80^eB	50.00 ± 1.60^cA	50.00 ± 1.00^eA	50.00 ± 0.00^eA	50.00 ± 0.00^dA

*Data in the same column with lowercase superscript letter and data in the same row with uppercase superscript letter are significantly different (p ≤ 0.05)

Table 6 Germination ability of control and PEF treated corn grain under 200 mM NaCI salt stress

Energy	Germination (%) under 200mM NaCI salt stress
(J)	3. day	4. day	5. day	6. day	7.day	8.day	9.day	10.day
0.00	0.00 ± 0.00^cE	0.00 ± 0.0.00^eD	3.33 ± 0.32^fC	3.33 ± 0.08^hC	16.67 ± 0.72^gB	16.60 ± 0.96^hB	16.67 ± 0.08^fB	18.00 ± 0.78^gA
1.20	8.33 ± 0.00^bF	16.67 ± 0.42^bE	25.00 ± 0.32^bD	33.33 ± 0.10^cC	58.33 ± 1.38^aB	66.67 ± 0.33^aA	66.67 ± 1.05^aA	66.67 ± 0.54^bA
2.82	0.00 ± 0.00^cE	25.00 ± 0.32^aC	33.33 ± 0.46^aB	41.67 ± 0.32^aA	16.67 ± 0.09^gD	16.67 ± 0.44^hD	16.67 ± 0.05^fD	33.33 ± 0.82^fB
3.18	0.00 ± 0.00^cF	8.33 ± 0.00^dE	16.67 ± 0.08^dD	25.00 ± 0.09^dC	25.00 ± 1.44^fC	25.00 ± 0.00^gC	33.33 ± 0.04^eB	58.33 ± 0.90^cA
3.60	0.00 ± 0.00^cH	12.50 ± 2.08^cG	20.83 ± 2.08^cF	37.50 ± 0.54^bE	45.83 ± 2.08^cD	50.00 ± 0.84^cC	58.33 ± 1.26^bB	66.67 ± 2.20^bA
6.00	0.00 ± 0.00^cD	16.67 ± 0.80^bC	16.67 ± 0.06^dC	16.60 ± 1.02^fC	33.33 ± 1.02^eB	33.33 ± 0.00^fB	33.33 ± 0.38^eB	41.67 ± 1.30^eA
8.46	0.00 ± 0.00^cE	25.00 ± 1.00^aD	33.33 ± 0.30^aC	41.67 ± 0.81^aB	58.33 ± 1.04^aA	58.33 ± 1.02^bA	58.33 ± 0.44^bA	58.33 ± 0.76^cA
9.54	0.00 ± 0.00^cF	8.33 ± 0.00^dE	20.83 ± 0.08^cD	20.83 ± 0.02^eD	58.42 ± 1.44^aC	66.67 ± 1.11^aC	66.67 ± 0.02^aB	75.00 ± 2.01^aA
10.80	0.00 ± 0.00^cEf	16.67 ± 0.82^bD	25.00 ± 0.00^bC	41.67 ± 0.46^aB	41.67 ± 0.09^dB	50.00 ± 1.56^cA	50.00 ± 0.00^cA	50.00 ± 1.42^dA
14.10	0.00 ± 0.00^cC	8.33 ± 0.00^dB	8.33 ± 0.06^eB	8.33 ± 0.08^gB	41.67 ± 0.09^dA	41.67 ± 0.07^dA	41.67 ± 2.08^dA	41.67 ± 0.68^eA
15.90	0.00 ± 0.00^cF	16.67 ± 0.40^bE	25.00 ± 0.60^bD	25.00 ± 0.02^dD	25.00 ± 1.22^fD	38.33 ± 0.00^eC	58.33 ± 0.89^bB	66.67 ± 1.46^bA
18.00	16.67 ± 0.36^aF	25.00 ± 0.40^aE	25.00 ± 0.36^bE	33.00 ± 0.00^cD	50.00 ± 2.22^bC	58.33 ± 0.03^bB	66.67 ± 2.18^aA	66.67 ± 1.43^bA
22.56	0.00 ± 0.00^cE	16.67 ± 0.56^bD	25.00 ± 0.24^bC	25.00 ± 0.30^dC	25.00 ± 2.00^fC	33.33 ± 0.07^fB	33.33 ± 1.00^eB	41.67 ± 1.38^eA
25.44	0.00 ± 0.00^cF	25.00 ± 0.42^aE	33.33 ± 0.66^aD	41.67 ± 0.32^aC	50.00 ± 0.00^bB	66.67 ± 0.33^aA	66.67 ± 2.16^aA	66.67 ± 2.09^bA
28.80	0.00 ± 0.00^cF	8.33 ± 0.06^dE	16.67 ± 0.08^dD	25.00 ± 0.22^dC	41.67 ± 0.60^dB	66.67 ± 0.33^aA	66.67 ± 1.52^aA	66.67 ± 2.00^bA

*Data in the same column with lowercase superscript letter and data in the same row with uppercase superscript letter are significantly different (p ≤ 0.05)

Table 7 The best-fit multiple (non-)linear regression models for the corn grains treated by pulsed electric fields

Response variable	Equation		(%)	(%)	SE
Germination at 2^nd day (%)	=61.11-12031E+197Trt-3.82F-17.7EE-6.73TrtTrt+0.0279FF+30.8ETrt +0.509EF+57.0TrtF+0.025EEE+0.021TrtTrtTrt-0.000050FFF-0.052EETrt+0.0154EEF	56.46	36.15	2.04	6.45
Germination at 3^rd day (%)	=88.89+3462E-80.6Trt+1.838F+5.40EE+6.96TrtTrt-0.01356FF11.39ETrt -0.2132EF-16.21TrtF-0.0344EEE-0.0487TrtTrtTrt+0.000025FFF +0.0772EETrt-0.00331EEF	72.33	59.42	37.74	2.06
Normal seedling rate (%)	=78.77-2721E+148.8Trt-3.086F-3.87EE-13.33TrtTrt+0.02271FF+16.21ETrt +0.312EF+12.2TrtF+0.1242EEE+0.2046TrtTrtTrt-0.000041 FFF-0.2858EETrt-0.00233EEF	86.47	80.16	69.56	2.51
Cold test 10 °C-7 day (%)	=0.000+1702E-0.2Trt+0.067F+3.59EE-0.030TrtTrt-0.00050FF-2.23ETrt +0.0014EF-8.26TrtF+0.0509EEE+0.0518TrtTrtTrt+0.000001FFF -0.0979EETrt-0.00687EEF	65.19	48.95	21.68	0.71
Cold test 24°C-5 day (%)	=62.22+1103E+24.4Trt-0.420F+1.23EE-2.26TrtTrt+0.00408FF+1.13ETrt + 0.0491EF-5.50TrtF+0.0436FEE+0.0774TrtTrtTrt-0.000008FFF - 0.1008EETrt-0.00308EEF	94.44	91.85	87.50	2.09
E.C- 4 hours (µS/cmg)	=8.919+113E-6.4Trt+0.199F-0.54EE+0.46Trt-Trt-0.00152FF+0.11ETrt-0.0110EF-0.52TrtF-0.0040EEE-0.0136TrtTrtTrt+0.000003FFF +0.0103EETrt+0.00105EE*F	78.69	68.74	52.05	1.01
E.C- 8 hours (µS/cmg)	=10.099-81E+4.1Trt-0.113F-0.04EE-0.59TrtTrt+0.00028FF+0.31ETrt -0.0243E-F+0.41TrtF+0.0007EEE+0.0126TrtTrtTrt+0.000001FFF -0.0084EETrt+0.00054EE*F	57.43	37.56	4.22	1.23
E.C- 24 hours (µS/cmg)	=14.212-5324E+44.8Trt-0.889F-11.34EE-3.94TrtTrt+0.00671FF+12.38E Trt+0.1552EF+25.46TrtF-0.0598EEE-0.0856TrtTrtTrt-0.000013FFF +0.1303EETrt+ 0.01532EEF	75.22	63.66	44.25	1.21
TAMB inactivation (log cfu/g)	=9.179+1743E-25.37Trt+0.588F+3.166EE+2.194TrtTrt-0.00417FF-4.25ETrt-0.0512EF- 8.30TrtF+0.01702EEE+0.00397TrtTrtTrt +0.000007FFF-0.0253EETrt-0.004451EEF	98.43	97.70	96.47	0.43
TMY inactivation (log cfu/g)	=9.1880+46E+0.17Trt+0.0037F+0.206EE+0.062TrtTrt+0.000090 FF -0.112ETrt+0.00369EF-0.234TrtF+0.00065EEE-0.00051TrtTrtTrt-0.000000FFF-0.00213EETrt -0.000354EEF	99.75	99.63	99.43	0.14
A. parasiticus % inhibition	=-0.000-549E+2.3Trt+0.367F-0.70EE+0.02TrtTrt-0.00197FF+1.76ETrt+ 0.0593EF+2.57TrtF+0.0194EEE-0.0249TrtTrtTrt+0.000003FFF -0.0206EETrt-0.00137EEF	99.82	99.73	99.59	0.97
Germination under 100 mM salt stress (3^rd day)	=8.33333+7546.90E-104.929Trt+1.09945F+7.75127EE+5.30632TrtTrt -0.012164FF-18.5833ETrt-0.712218EF-35.2605TrtF-0.13168EEE +0.132060TrtTrtTrt+0.000029FFF+0.112649EETrt+0.010145EEF	100	100	100	0.00
Germination under 100 mM salt stress (4^th day)	=8.333+6273E-0.3Trt-0.384F+11.61EE-2.86TrtTrt-0.00095FF-11.03ETrt -0.3873EF-29.90TrtF+0.0377EEE+0.2915TrtTrtTrt +0.000008FFF -0.2032EETrt-0.00777EEF	98.04	97.13	95.60	1.20
Germination under 100 mM salt stress (5^th day)	=16.67+6064E-30.7Trt+0.181F+13.52EE+0.27TrtTrt-0.00550FF-15.88E Trt-0.4915EF-28.64TrtF-0.0071EEE+0.2382TrtTrtTrt+0.000018 FFF -0.1151EETrt-0.00764EEF	95.05	92.76	88.90	1.94
Germination under 100 mM salt stress (6^th day)	=16.67-1353E+159.2Trt-3.382F-0.39EE-8.08TrtTrt+0.02018FF+11.85E Trt-0.122EF+6.1TrtF+0.0589EEE+0.4192TrtTrt-0.000027FFF -0.3118EETrt+0.00592EE*F	97.02	95.62	93.29	2.74
Germination under 100 mM salt stress (7^th day)	=25.00+8455E-244.2Trt+4.627F+14.30EE+17.78TrtTrt-0.03716FF-37.57 ETrt-1.1380EF-38.61TrtF-0.3058EEE-0.0402TrtTrtTrt+0.000075 FFF+0.4381EETrt+0.01340EEF	98.63	97.99	96.92	2.15
Germination under 100 mM salt stress (8^th day)	=25.00+10462E-405.6Trt+8.350F+14.38EE+33.32TrtTrt-0.06188FF-50.83 ETrt-1.301EF-47.54TrtF-0.4225EEE -0.3515TrtTrtTrt+0.000115FFF +0.7678EETrt+0.01745EEF	98.26	97.44	96.08	2.21
Germination under 100 mM salt stress (9^th day)	=25.000+24034E-461.3Trt+0.598F+40.38EE+36.33TrtTrt-0.07296FF -72.59ETrt-1.5884EF-112.81TrtF-0.1481EEE-0.0057TrtTrtTrt +0.000139FFF+0.2005EETrt-0.02354EEF	98.94	98.45	97.62	1.70
Germination under 100 mM salt stress (10^th day)	=25.000+24015E-319.7Trt+6.893F+42.94EE+24.61TrtTrt-0.05279FF -60.62ETrt-1.1996EF113.76TrtF+0.0692EEE+0.2328TrtTrtTrt +0.000102FFF-0.2307EETrt-0.04074EEF	99.08	98.64	97.92	1.52
Germination under 200 mM salt stress (3^rd day)	=-0.000000-4304.99E+20.0580Trt-0.515374F-8.87300EE-2.21736TrtTrt +0.003241FF+6.80069ETrt-0.045235EF+20.8550TrtF-0.137016EEE -0.072508TrtTrtTrt-0.000004FFF+0.212002EETrt+0.019635EEE	100	100	100	0.00
Germination under 200 mM salt stress (4^th day)	=16.667+12613E-275.45Trt+4.963F+25.035EE+24.347TrtTrt-0.03828FF -48.19ETrt-0.9625EF-58.89TrtF-0.22327EEE-0.0947TrtTrtTrt +0.000072FFF+0.3587EETrt-0.01045EEF	99.50	98.87	99.26	0.53
Germination under 200 mM salt stress (5^th day)	=25.000+18489E-Trt+5.121F+38.06EE+24.22TrtTrt- 0.03985FF-57.17ETrt-0.9793EF-87.36TrtF-0.0650EEE+0.0962TrtTrtTrt +0.000076FFF+0.0491EETrt-0.03417EEF	98.04	97.13	95.60	1.20
Germination under 200 mM salt stress (6^th day)	=33.333+15787E-337.0Trt+6.163F+36.01EE+30.57TrtTrt-0.04839FF -64.01ETrt-1.1690EF-73.76TrtF-0.2525EEE-0.0851TrtTrtTrt +0.000093FFF+0.4159EETrt-0.02255EEF	99.21	98.84	98.23	1.20
Germination under 200 mM salt stress (7^th day)	=33.333-18526E+56.6Trt-1.919F-27.88EE-6.12TrtTrt+0.00717FF+17.64 ETrt-0.3249EF+90.15TrtF-0.5130EEE-0.3084TrtTrtTrt-0.000002 FFF +0.8791EETrt+0.06356EEF	98.76	98.18	97.20	1.70
Germination under 200 mM salt stress (8^th day)	=33.333-14706E+43.8Trt-1.234F-18.68EE-4.76TrtTrt+0.00199FF+10.36 ETrt-0.3317EF+71.68TrtF-0.4149EEE-0.2152TrtTrtTrt+0.000008 FFF+0.6996EETrt+0.04675EEF	99.32	99.00	98.47	1.61
Germination under 200 mM salt stress (9^th day)	=33.33-4420E+155.7Trt-3.135F+7.79EE-14.54TrtTrt+0.01536FF+3.78 ETrt-0.1068EF+21.04TrtF+0.0414EEE+0.3212TrtTrtTrt-0.000015FFF-0.2332EETrt-0.00938EEF	99.00	98.53	97.74	1.94
Germination under 200 mM salt stress (10^th day)	=41.67-7117E+354.2Trt-7.061F+3.34EE-31.35TrtTrt+0.04696FF+27.53 ETrt+0.5417EF+32.51TrtF+0.3018EEE+0.5759TrtTrtTrt-0.000076FFF-0.7371EETrt-0.02184EEF	98.13	97.25	95.79	2.15

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Corn processing by pulsed electric fields with respect to microbial inactivation and improvement of seed vigour

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