Effect of Ozone Application on the Fungal Count and Lipid Quality of Peanut Grains

Peanut is susceptible to fungal contamination at all stages of its production chain, which can lead to aflatoxin production, which can cause serious health problems for consumers. In this sense, post-harvest ozonation of grains has the potential to reduce contaminant microorganisms, but it may cause oxidative damage, degrading organic constituents. Thus, factors influencing the reduction of fungal contamination by gaseous ozone in peanuts (grains and grains in pods) and changes in lipid and oil quality of grain were investigated. The analyzed variables were total fungi count, electrical conductivity, peroxide index, and 2-thiobarbituric acid test. Ozone concentration (10, 30, and 50 ppm) and ozonation time (30, 45, and 60 minutes) significantly affected fungal count (p < 0.05). The maximum fungal reductions were 75.79% for grains and 82.66% for grains in pods at a concentration of 50 ppm and exposure of 60 minutes. The electrical conductivity of exudates was affected by ozone concentration. There was degradation of lipids at a cellular level, but no differences were observed in the peroxide index of treated grains.


Introduction
Peanut (Arachis hypogaea L.) is consumed worldwide because of its high nutritional value, as a source of lipids and proteins, but is susceptible to fungal contamination, which can produce aflatoxins (Martins et al., 2017;Power et al., 2017;Wang, Lien, & Ling, 2018).The fungal species Aspergillus flavus and Aspergillus parasiticus are the main producers of mycotoxins in peanuts.The species A. flavus produces the aflatoxins B 1 and B 2 and A. parasiticus is able to produce the aflatoxins G 1 and G 2 , presenting a highly toxic character for human and animal health (Sahab, Hassanien, El-Nemr, Abdel-Alim, & Abdel-Wahhab, 2013).
Many methods have been used to reduce or remove these fungal species, but the consolidation of a highly effective method is far from being defined (Granella, Christ, Werncke, Bechlin, & Coelho, 2018).The methods involve chemical (Saalia & Phillips, 2011), physical (Mao et al., 2016), and biological processes (Chen, Kong, Chi, Shan, & Guan, 2015), but are not practical and are based on laboratory analysis, not large-scale.
In the peanut processing industry, pods coming from the field are dried in order to facilitate the removal of grains.Pods or grains with a low water content can be stored until processed.Thus, our study sought to evaluate the effect of ozonation not only on grains but also on grains in the pod, which naturally have a physical barrier.
The studies reported the application of ozone in peanut grains, so our results can help the processing industry in the decision to apply the gas in grains or grains in the pod, in a way that facilitates the industrial process.
Thus, the main objective of this study was to evaluate the different ozonation conditions in peanuts (grains and grains in pods) under levels of fungi reduction, electrical conductivity, peroxide index, and 2-thiobarbituric acid test.

Samples
Samples of peanuts (grains and grains in pods) (approximately 8% wb water content) of the variety Runner Granoleico, obtained from the commercial production in northwestern Paraná (with averages of temperature and precipitation during planting and harvesting of 27 °C and 146 mm, respectively,), were stored at 7 °C until the beginning of the tests.The experiment was conducted at the Laboratory of Quality Control of Agricultural Products (LACON) of the Western Paraná State University (UNIOESTE), Campus of Cascavel, Paraná, Brazil.

Ozonation System
Ozone (O 3 ) gas was obtained by a Philozon SKID-20 industrial ozonator, with measurement of the ozone concentration generated and capacity of 20 g O 3 h -1 through a corona discharge effect with forced air-cooling of 2 L min -1 .The input used for ozone production was pure oxygen in a PSA (pressure swing adsorption) system.Samples with 200 g per replicate of pods and grains were packed in a support (50 L capacity) with flow and pressure control in the inlet and outlet and the generated O 3 was injected.Ozone gas was introduced into the system at different concentrations and times, according to experimental design.

Experimental Design
The experimental design was a central composite design (CCD) with two factors, i.e., ozone concentration (X1) and application time (X2).Each factor in the experiment was established and coded at three levels, i.e., lower (-1), medium (0), and higher (+1), with values of X1 of 10, 30, and 50 ppm and X2 of 30, 45, and 60 min.Seven tests were performed, including three replications at the central point (Table 1).The total fungal count, electrical conductivity, peroxide index, and 2-thiobarbituric acid test were selected as dependent variables of the process.The results were analyzed using the software Statistica 10 (StatSoft Inc., Tulsa, OK, USA).The significance test and analysis of variance (ANOVA) were used to evaluate the quality of fit of the model from Equation (1): where, Ŷ is the estimated response, b 0 is the intercept term, b 1 and b 2 are the coefficients of linear terms, b12 is the coefficient of the interaction term, and X 1 and X 2 are the factors.
The coefficient of determination (R 2 ) was determined for the model and the model accuracy was established.
Three control samples were used (not included in the CCD).

Total Fungal Count and Identification
Ozonized peanut samples were analyzed for the evaluation of the effects of ozonation on fungal decontamination, as in Beuchat and Cousin (2001).Samples of 25 g were transferred to an Erlenmeyer flask containing 225 mL of peptone saline solution (0.1%).The samples were homogenized for 60 seconds, corresponding to the dilution 10 -1 .From this dilution, dilutions 10 -2 and 10 -3 were prepared using test tubes containing 9 mL of peptone saline solution (0.1%).Aliquots of 0.1 mL were plated on an acidified (10% tartaric acid) potato dextrose agar surface and then incubated for five days at 25 °C.After this period, the colonies were counted and the results expressed as CFU g -1 of grains.
For identification of fungi the direct plating method on filter paper was used.The beans were individually arranged on layers of filter paper dampened inside containers with transparent lids.Then, they were placed in incubation chamber for 24 hours at 20±2 ºC in a regime of 12 hours of light and 12 hours of darkness.Subsequently, the plates containing the beans were stored in a freezer (-20 ºC) for 24 hours.At the end of the freezing period, the plates were again returned to the incubation chamber, under the same conditions as before, for a further 5 days.After the entire incubation period, the samples were examined under an optical microscope for the identification of fungal structures (MAPA, 2009).

Electrical Conductivity
Three replications of 50 grains were used per treatment.Samples were pre-weighed on an analytical balance, placed in plastic containers (200 mL) with 75 mL of deionized water, and maintained at 25 °C for 24 hours.The electrical conductivity of the solution was determined using a conductivity meter (Vieira, Tekrony, Egli, & Rucker, 2001).The results were expressed as µS cm -1 g -1 .

Peroxide Index
Ozonized peanut grains were submitted to cold pressing for oil extraction (10 to 50 kN).Oil samples were analyzed according to the standards AOCS ( 2009), Cd 8-53.The peroxide index (PI) was calculated by Equation ( 2): where, N is the normality of the sodium thiosulphate solution (Na 2 S 2 O 3 ), f is the correction factor of the Na 2 S 2 O 3 solution, Va is the volume of standard 0.1 N Na 2 S 2 O 3 spent on sample titration (mL), Vs is the volume of standard 0.1 N Na 2 S 2 O 3 spent on the titration without sample (mL), and m is the sample mass (g).

2-thiobarbituric Acid Test
Ozonized samples of peanut grains and oil were used to perform the 2-thiobarbituric acid test (TBA).Samples of 0.25 g were homogenized in 2 mL of 0.1% trichloroacetic acid (TCA).The homogenized was centrifuged for 10 min at 10,000 rpm (4 °C).The supernatant was collected (250 µL) and mixed with 3 mL of 0.5% TBA solution and 20% TCA solution, then incubated at 95 °C for 35 minutes for color development.The reaction was stopped by cooling and lipid peroxidation was determined at 535 nm and 600 nm in a spectrophotometer.The results were expressed in mg of malonaldehyde (MA) kg of fresh mass (FM) (Silva, Borges, & Ferreira, 1999) using the extinction coefficient of 1.56 × 10 -5 cm -1 (Michaowicz, Posmyk, & Duda, 2009).

Water Content of Grains
The water content was determined by a forced air circulation oven at 105±1 °C for 24 hours from three samples of 25 g of seeds for each replication (MAPA, 2009).The values were expressed as the percentage of wet basis (%, wb).

Effect of Ozonation on Total Fungal Count
Fungi of the genus Aspergillus (A. flavus and A. parasiticus), Rhizopus, Cladosporium, and Penicillium were identified in the peanut samples.
For calculating the fungal reduction, the total counts of control samples were used as a reference, corresponding to 9.5 × 10 -1 CFU g -1 for peanut grains and 7.9 × 10 -2 CFU g -1 for peanut grains in the pod.
The effects of each selected independent variable, in addition to its interactions with the levels of % of fungal reduction, evaluated after grain ozonation were studied using the central composite design (CCD).
The Pareto diagram shown in Figure 1 presents the terms considered significant by the t-test for the % of fungal

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Figure 3.

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The content of TBA was reduced as a function of the ozone application time, which can be explained by a change in the fatty acid profile of membranes.Linoleic (18:2) and linolenic acids (18:3) are the main fatty acids of the plant membrane (Taiz & Zeiger, 2017) and their peroxidation results in the formation of the 4-hydroxy-2-nonenal (HNE) and malonaldehyde (MA), respectively (Møller, Jensen, & Hansson, 2007).Peanut oil has a high oleic acid (18:1) content (Sarvamangala, Gowda, & Varshney, 2011), which forms fewer degradation products reactive with TBA (Nawar, 1996).Initial membrane peroxidation may reduce the formation of MA at the end of the ozonation process and explain the reduction of TBA content since MA is the main product detected by this test.Scussel et al. (2011) observed that MA values decreased and remained constant during the storage of ozonized nuts due to ozone oxidation and attributed these results to the amount of oleic (monounsaturated) and linoleic (polyunsaturated) acids.
In addition, a higher TBARS content was observed in the analysis of grains in relation to the pure oil.This is also due to the character of the test, which can quantify other aldehydes from sugars, as acetaldehyde and Maillard reaction compounds (Nawar, 1996), which are not formed in the extracted oil.
Therefore, the oxidative reactions originate from the double bonds present in fatty acid molecules, which make up the lipid fraction of food.In peanut grains, unsaturated fatty acids represent the major part of the lipid fraction, with a high possibility of occurring oxidative reactions in these grains due to the structure of their molecules (Sarvamangala et al., 2011).

Conclusions
The results of this study suggested that the longer the exposure time to O 3 and the higher its concentration, the fungicidal effect is increased.These conditions corresponded to the ozone concentration of 50 ppm and exposure time of 60 minutes.However, this increase may cause leakage of exudates, directly related to the deterioration of the cell membrane.Therefore, this effect should be considered by processing industry in case of using higher concentrations.In addition, lipid degradation occurred at the cellular level, but differences in the peroxide index were not observed in ozonized grains.

Table 1 .
Matrix of the central composite design with the actual and coded values