Evaluation of Oxidation Process by Ozonation and Glucose Oxidase Enzyme on the Degradation of Benzoquinone in Wheat Flour

2.1. Insect Cultures

Tribolium castaneum was obtained from a stock culture maintained at the stored grain and product pests Department, Plant Protection Research Institute, Agriculture Research Center. It was kept at temperature (28 ±2°C, and 65 ±5% R.H) on wheat flour for two months.

2.2. Chemicals and Solvents

GOX from Aspergillus niger and (MBQ and EBQ) were obtained from Sigma- Aldrich, Lyon, France. All solvents in this study were HPLC-grade. The water was double distilled with a Millipore water purification system (Bedford, MA).

2.3. Infestation of Wheat Flour by T. Castaneum

One kilogram of wheat flour was used and divided into glass jars having the capacity of 100gm. T.castaneum infected these jars with unsexed pairs of insect adults. The jars were left on the bench in the laboratory for three months under laboratory temperature conditions (with an average of 28±2ºC and 65±5%R.H). The previous design was replicated to obtain sufficient quantities of flour to complete the experiment. After three months, the jars containing the flour were sieved thoroughly by 40 wire mesh size to separate the insects. Then the (MBQ, EBQ) were determined in wheat flour before and after treatment by ozone gas and GOX enzyme for each of them.

2.4. Ozonation of Contaminated Samples

The wheat flour was treated with ozone gas using an ozone generator (Model OZO 6 VTTL OZO Max Ltd, Shefford, Quebec Canada; (http://www. ozomax.com). Flour samples (about 250 g) were placed in a cylindrical acrylic container having an inlet valve at one end and an outlet valve at the opposite end. All samples were exposed to ozone gas at (10, 20, and 40 ppm) for 15, 30 and 45 min. Finally, ozone-treated samples were placed in plastic bags, then extraction and determination of MBQ, EBQ were performed by HPLC according to Tomoskozi-Farkas and Daood method [17].

2.5. Adding of GOX Enzyme to Flour

GOX was added to flour at concentrations (10, 15, and 20 ppm), then mixed until the dough was formed, and left for 15, 30, and 45 min before determination of the MBQ and EBQ in samples. The control samples were without the GOX enzyme. The percentages of degradation were calculated from three replicates for each treatment, and the equation used is as follows:

The percentage of degradation BQs =

Where: C is concentrations of MBQ and EBQ in the samples without ozonation or adding GOX.

T is concentrations of MBQ and EBQ in samples after ozonation or adding GOX.

2.6. Statistical Analyses

The general Linear Model procedure of the SPSS ver. 18 (IBM Corp, NY) was used to analyse statistically. The significance of the differences among treatment groups was determined by Waller–Duncan k-ratio. All statements of significance were dependent on the probability of P-value ≤0.05, which was considered to be statistically significant.

3.1. Impact of Addition GOX Enzyme on BQs

Data presented in Fig. (2) show the percentages of degradation of MBQ after adding GOX at 10 ppm were 9.4, 13.25, and 22.8% after mixing for 15, 30, and 45 min, respectively. The results indicated that the percentages of degradation of MBQ increase to 13.7, 20.23, and 39.6% after mixing for 15, 30, and 45 min, respectively, when the GOX dose increased to 20 ppm. Concerning EBQ, the results in Fig. (3) indicated that the addition of GOX at 20 ppm degraded the EBQ to 13.6, 18.9, and 35.9% after 15, 30, and 45 min from mixing time, respectively. According to previous studies, GOX at these concentrations does not affect the rheological and technological properties of flour and dough; but improves them [18-20]. Hydrogen peroxide is produced from oxidized glucose by glucose oxidase, as Equation 1. H₂O₂ promotes different reactions in the dough, like disulfide bonding between the water-soluble protein fraction and dityrosine bonding in gluten protein. Accordingly, Hydrogen peroxide reacting with alternating C=C, C=O, and C–C bonds in BQs molecule produces or forms some compounds such as aldehydes. On the other hand, the presence of many minerals and their ions in the chemical composition of flour enhances the work of hydrogen peroxide to decrease the BQs. Furthermore, MBQ is sensitive toward both strong mineral acids and alkali, which cause condensation and decomposition of the compound [21-23]. Finally, the BQs molecule responds to H₂O₂ through the double bond between C3-C4 in the benzene ring, changing into 2, 3-epoxy-p-benzoquinone. Under conditions of oxidation, 2, 3-epoxy-p-benzoquinone transformed into aldehydes, ketones, acids, and CO₂ [24, 25]. On the other hand, the ANOVA analyses of the results Table 1 show that the reduction or degradation of MBQ and EBQ depends significantly on the concentration of the GOX enzyme and the time of mixing (Eq. 1).

(1)

3.2. Ozonolysis Efficiency for BQs in Flour

Data presented in Figs. (4 and 5) show the percentages of degradation of MBQ and EBQ after ozonation at 10, 20 and 40 ppm. The results indicated that the ozonation of wheat flour for 45 min at 10, 20 and 40 ppm degraded the MBQ to 49.8, 63.04, and 84.1%, respectively (Fig. 4). On the other hand, EBQ degraded to 40.8, 57.4, and 78.8% at the same dose and exposure times (Fig. 5). The analysis of ANOVA (Table 2) shows that the doses of ozone gas and exposure time of ozonation have a significant effect on the content of BQs. These results reflect that the ozone gas has shown high efficiency in the degradation of the MBQ and EBQ; this may be due to the high oxidizing power of ozone, which opens the ring and double bonds. The degradation of BQs increases with the excess of the exposure time of ozonation, which may be due to depleted ozone gas with some components of the chemical composition of wheat flour such as protein and carbohydrate, and others. Ozone gas can interact with many compounds and chemical bonds like in the furan ring in aflatoxin, while ozone attacks the double bond at C8–C9. As a result of this reaction, aflatoxin molozonide was formed, which converted to aflatoxin ozonide. This last compound was unstable and changed to aldehydes, ketones, acids, and CO₂ [26]. According to this, ozone gas may react with double bonds in BQs, also combine with the C3 in MBQ and EBQ after the peroxo bond breaking [27]. According to many previous studies, treatment of flour by ozone gas at this dose and exposure time has positive effects on rheological and technology properties to obtain improved bakery products [28-30]. The carbonyl group such as aldehydes, ketones, and carboxylic acids were intermediates products for degradation of BQs by ozonation or OH [31].

3.3. Characterization of MBQ after Ozonation in vitro

According to obtained results, the best treatment was chosen, which was ozonation of MBQ, as representative for BQs at 40 ppm for 45 min, especially since the difference in structure between methyl and ethyl group only. So, in this study, UV-Visible Spectrophotometer and Fourier Transform Infrared Spectroscopy (FTIR) were used to describe the changes that occurred in the structure for BQs. The UV–vis spectroscopy for MBQ untreated showed the absorption peak at 250 nm. The sample revealed many peaks as secondary products after ozonation, as shown in Fig. (6), which may be due to some side reactions, which led to a loss of the basic structure of BQs [32]. FTIR spectrum analysis for MBQ after oxidation by ozone gas indicated the formation of the products of oxidation and the break of the double bond between C=O in C1 and C4, which showed a high absorbance at 1620 cm^-1 in the untreated sample. The results of FTIR analysis to the treated sample indicated that many groups appeared like aldehyde at 1750cm^-1 and carboxylic at 3550cm^-1, etc. (Fig. 7).