Effect of different ozone treatments on the degradation of deoxynivalenol and flour quality in Fusarium-contaminated wheat

ABSTRACT

Deoxynivalenol (DON) in wheat (Emai596) was degraded by five ways of ozone treatments, including ozone-gas treated wheat flour, wheat kernels before and after tempering, and tempering and washing with ozonated-water. For lab-scale milling, the effect on flour quality was investigated. The DON was quantified via high-performance liquid chromatography after extraction with immunoaffinity columns. The measure indicated all treatments could decrease the DON content of flour below 1.00 mg/kg. Ozone-gas treated wheat kernels after tempering had the highest DON degradation rate 33.33%. Ozonation produced no significant difference in gluten qualities, dynamic viscoelasticity, pasting and mixing properties, when wheat kernels were treated with ozone-gas or ozonated-water. Although ozone-gas treated flour had the highest whiteness (85.56), but it had negative effect on the rest of quality indicators. The results suggested the treatment of Ozone-gas treated tempered wheat kernels could not only degrade DON most efficiently but also ensure quality of flour.

Resumen

Para el presente estudio se degradó el desoxinivalenol (DON) presente en el trigo (Emai596), empleando cinco tratamientos diferentes con ozono, entre ellos: harina de trigo tratada con gas ozono, granos de trigo antes y después del templado, el templado y el lavado con agua ozonizada. Se investigó el efecto en la calidad de la harina, para realizar la molienda a escala de laboratorio. El DON se cuantificó mediante cromatografía líquida de alto rendimiento, después de la extracción con columnas de inmunoafinidad. Las mediciones indicaron que todos los tratamientos aplicados pueden disminuir el contenido de DON de la harina por debajo de 1.00 mg/kg. Los granos de trigo tratados con gas ozono después del templado registraron la mayor tasa de degradación de DON, 33.33%. Cuando los granos de trigo fueron tratados previamente con gas ozono o agua ozonizada, la ozonización no produjo diferencias significativas en las cualidades del gluten, la viscoelasticidad dinámica y las propiedades de pegado y mezclado. Aunque la harina tratada con gas ozono mostró la mayor blancura (85.56), el efecto del tratamiento fue negativo en el resto de los indicadores de calidad. Los resultados indican que el tratamiento de los granos de trigo templados con gas ozono no sólo puede degradar el DON de manera más eficiente, sino que también garantiza la calidad de la harina.

KEYWORDS:

• Ozone treatment
• deoxynivalenol
• wheat flour quality
• detoxification
• wheat tempering
• wheat washing
• ozonated-water

PALABRAS CLAVE:

• Tratamiento con ozono
• desoxinivalenol
• calidad de la harina de trigo
• desintoxicación
• templado de trigo
• lavado de trigo
• agua ozonizada

1. Introduction

Deoxynivalenol (DON), also known as vomitoxin, is a type B trichothecene, and the occurrence of DON is associated primarily with Fusarium graminearum and F. roseum. Fusarium is the main pathogen that causes Fusarium head blight (FEB) in wheat (Shin et al., 2012). As a worldwide disease, it not only contributes to the loss of grain production, but also inflicts an adverse impact on human and animal health (Savi et al., 2014). The occurrence of FEB in wheat entails warm and humid climatic conditions, so it occurs many times in the main wheat-producing areas of the Yangtze River basin in China, and brings heavy losses to the majority of growers. FEB, wheat stripe rust, and powdery mildew have become three major wheat diseases in the world (Kouadio et al., 2005; Pestka & Smolinski, 2005).

DON could degrade to be metabolites by acetylation, oxidation, or glycosylation, and the metabolites still retains the toxicological effects (Khaneghah et al., 2018). Therefore, accurate detection methods for the content of DON and its metabolites are particularly important. The analytical steps for DON in grain generally include toxin extraction from samples using an extraction solvent, followed by a clean-up step intended to eliminate interference, and a final detection of the target toxin by suitable analytical methods (Ran et al., 2013). Since DON and its metabolites are polar compounds, they are usually extracted by polar solvents or mixtures of several polar solvents (such as methanol, ethanol, acetonitrile, and water). In the clean-up step, solid-phase extraction (SPE), immunoaffinity columns (IAC), and multifunctional columns (MFC) are being widely applied (Pascale, 2009). Available analytical methods of DON and its derivatives in grains are mainly based on principles of immunoassay, chromatography, and mass spectrometry. The polar nature of DON molecule and its solubility in organic solvents and water make HPLC a most common technique in cereal DON detection (M. M. Li et al., 2014; Santos Alexandre et al., 2018). Kostelanska et al. (2010) employing DONdedicated immunoaffinity columns (IAC) for the purification of beer samples, the results show that the IAC can separate DON, DON-3-Glc and 3-AcDON at the same time, and the recovery rate is high. Our current study selected IAC-HPLC to detect the content of DON and its metabolites.

At present, three methods are predominant in DON detoxification: physical (Dhillon et al., 2009), chemical (Abramson et al., 2005), and biological (Karlovsky, 2011) detoxification. Despite of the low cost, physical detoxification does not really shine, and has certain impact on the nutrition of grain. So, it is not universally applicable in the food industry. Biological detoxification has high cost and unstable effect, thereby limiting the wide application of this method. Ozone treatment is a widely studied chemical detoxification method, and ozone can easily react with some compounds including aflatoxin, ochratoxin A, and melamine (Piemontese et al., 2018). Compared with other bacteriostatic chemicals, ozone is generally considered safe in food because it is easy to decompose into oxygen with no residues. It can get around the shortcomings of common chemical methods in food safety (Panagiota & Graham, 2002; Thalmann et al., 2018). In recent years, extensive studies have turned the spotlight on the use of ozone in sterilization (Dhillon et al., 2009), disinsection (Piemontese et al., 2018), pesticide residue (S. Wang et al., 2019), and toxin degradation in grain processing (Wang, Shao et al., 2016). Ozone gas is very effective in deactivating wheat-related fungi, and the ozone dose required to cause significant inactivation of fungi is much lower than the threshold for destroying the germination ability of wheat (Wu et al., 2006). The oxidation ability of ozone gas has great potential in the removal of various mycotoxins, and the degradation rate of DON increases significantly with the increase of ozone concentration and exposure time (Wang, Luo et al., 2016). Ozone treatment does not reduce the quality of wheat, but has the advantage of improving flour quality (M. M. M. M. Li et al., 2014). Through the evaluation of toxicity in mice, it is found that ozone gas can effectively reduce the harm of DON, but has a slight toxic effect on animals in this process (Wang, Wang et al., 2016). Notwithstanding the wide study of ozone gas in the degradation of grain toxins, there are few reports on the use of ozonated water, and emphasis is primarily placed on employing ozonated water to reduce the content of microorganisms in grains, fruits, and vegetables (Dhillon et al., 2009).

Ozone is a strong oxidant, on the one hand, and it can oxidize and degrade DON in wheat. On the other hand, it may change the physical and chemical properties of wheat flour and dough. There are many researchers who had studied the effect of ozone on wheat during storage, processing, and toxin degradation, but the research was generally limited to a single ozone treatment method. In this study, ozone gas and ozonated water were selected to treat Fusarium-contaminated wheat (a total of five ways) in the processes of tempering, washing, and milling, The objective of the paper was to determine the ozone treatment which was the most effective on DON degradation and the best maintenance of wheat flour quality.

2. Materials and methods

2.1. Raw materials

Wheat (Emai 596) samples naturally contaminated with DON (11.28% of original moisture content and 1.289 mg/kg of DON) were provided by Hubei Grain and Oil Inspection Station. DON (CAS: 51481–10-8) standard was purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyethylene glycol and methanol were supplied by Fisher (Waltham, MA, USA). Ultrapure water was obtained from a Millipore-Q SP Re- agent Water system (Millipore, Bedford, MA, USA). All other chemicals (analytical grade) such as silver nitrate (AgNO3), potassium iodide, and sulfuric acid were provided by Sinopharm Chemical Reagent Co. Ltd (Shanghai, China).

2.2. Ozone Gas and Ozonated Water Preparation

Ozone gas was generated from an adjustable ozone generator (HW-ET-21 G, Guangzhou Huanwei Environmental Protection Technology Co., Ltd., Guangdong, China). This device has its own oxygen generator can be used by power supply without adding more air source. The concentration of ozone gas produced was 20–80 mg/L. The ozone in the experiment was determined by the ozone concentration detection sensor (EST-10-O3, Shenzhen Wanyi Technology Co., Ltd., Guangdong, China).

Ozonated water was generated by passing 60 mg/L ozone gas through 500 mL of distilled water by means of the Venturi tube which was combined with air stone, followed by 1 h of aeration under the condition of ice bath conditions to generate 20 mg/L ozonated water. The ozonated water in the experiment was determined by the ozonated water concentration detector (KNF-2024, Shenzhen Wanyi Technology Co., Ltd., Guangdong, China). The plastic wrap was then used to seal the mouth of the bottle and it was kept in an ice bath for later use.

2.3. Experimental design of five ozone treatments

A total of five ways were designed to treat wheat with ozone (ozone gas or ozonated water): treatment of wheat (sample A) with ozone gas before tempering; treatment of wheat (sample B) with ozone gas after tempering; direct treatment of milled wheat flour (sample C) with ozone gas; cleaning of wheat (sample D) with ozonated water; tempering of wheat (sample E) with ozonated water. Details of the five ozone treatments steps and procedures were shown in Table 1.

Table 1. Details of the five ozone treatments steps and procedures.

Tabla 1. Detalles de los cinco pasos y procedimientos aplicados para los tratamientos de ozono

Sample	Ozone gas/ozonated water	Wheat kernels/wheat flour	Before tempering/after tempering	Other parameters
control	\	\	\	500 g of wheat sample without impurities was tempered until the moisture content was 16.0%, and was made into wheat flour by using the experimental mill LabMill (CHOPIN Technologies). All the flour was mixed evenly and put into a zip-lock bag and stored in a refrigerator at 4°C.
Sample A	Ozone gas (60 mg/L)	Wheat kernels	Before tempering	500 g of wheat sample was treated with ozone gas for 2 h, shaken every other 15 min, and then tempered to the moisture content of 16.0%. The wheat flour was made by using the experimental mill LabMill (CHOPIN Technologies), and all the flour was mixed evenly and then put into a zip-lock bag and stored in a refrigerator at 4°C.
Sample B	Ozone gas (60 mg/L)	Wheat kernels	After tempering	First, 500 g of wheat sample was taken to temper the wheat until the moisture content was 16.0%, and then the tempered wheat sample was treated with ozone gas for 2 h and shaken every 15 min. The wheat flour was made by using the experimental mill LabMill (CHOPIN Technologies), and all the flour was mixed evenly and then put into a zip-lock bag and stored in a refrigerator at 4°C.
Sample C	Ozone gas (60 mg/L)	Wheat flour	\	The milled wheat flour sample (500 g) was directly treated with ozone gas for 2 h and shaken every 15 min. The treated flour was mixed evenly and then put into a zip-lock bag and stored in a refrigerator at 4°C.
Sample D	Ozonated water (20 mg/L)	Wheat kernels	\	500 g of wheat sample was mixed with 500 mL of ozonated water in a 2.5 L container to clean 30 min. The surface of the wheat sample was dried with 30°C hot air. The wheat was tempered to the moisture content of 16.0% and made into flour by the experimental mill LabMill (CHOPIN Technologies), and all the flour was mixed evenly and then put into a zip-lock bag and stored in a refrigerator at 4°C.
Sample E	Ozonated water (20 mg/L)	Wheat kernels	Tempering 24 h under the condition of avoiding light at 25°C	500 g of wheat sample was tempered with ozonated water for 24 h to the moisture content of 16.0% and made into flour by the experimental mill LabMill (CHOPIN Technologies), and all the flour was mixed evenly and then put into a zip-lock bag and stored in a refrigerator at 4°C.

2.4. Extraction, purification and determination of DON

The DON determination was based on Chinese National Standard “GB 5009.111–2016 Determination of Deoxynivalenol and Its Acetylated Derivatives in Food” (China national standard (GB), 2016), the major methods included immunoaffinity chromatography and high-performance liquid chromatography (HPLC).

Extraction: 25 ± 0.01 g of sample and 5 g of polyethylene glycol were added to 125 mL of water to ensure that the sample/water ratio was 1:5, and the mixture was homogenized with homogenizer (XW-18D, Hangzhou Qiwei Instrument Co., Ltd., Zhejiang, China) for 1 min, filtered to obtain the supernatant, and then filtered once again with fiberglass filter paper to get the loading buffer.

Purification: 2.5 mL of extracting solution was accurately pipetted and DON immunoaffinity chromatography column (Beijing Meizheng Biological Technology Co., Ltd., Beijing, China) was used for solid-phase extraction. After drying, 20 mL of ultra-pure water was added and the water was completely dried before 1 mL of methanol was added to elute DON. Finally, the eluted methanol was dried with nitrogen evaporators (N-EVAP, Organomation Associates, Inc., USA). After drying, it was re-dissolved in mobile phase (methanol/water = 1:4) and passed through 0.45 μm filter membrane to HPLC vial (Waters e2695 with PDA detector, Milford, MA, USA).

HPLC conditions: Waters Symmetry C18 (Milford, MA, USA) chromatographic column (5 μm, 250 mm×4.6 mm); mobile phase (methanol/water = 1:4); flow rate (0.8 mL/min); column temperature (35°C); injection volume (20 μL); detection wavelength (218 nm). The total run time was 20 min.

In HPLC analysis, the peak area of DON at a retention time of 11.2 minutes was calculated, and the DON content in the samples were quantified from the calibration curve (DON standard solutions were 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 μg/mL; Correlation coefficients R² = 0.9993).

The degradation rate of DON was calculated as follows:

(1) $Dr=\frac{C_k-C_s}{C_k}$(1)

Dr is the degradation rate of DON, C_k is the DON content in wheat kernels, C_s is the DON content in measured samples.

2.5. Chemical and physical analysis

Protein, starch, fat, and moisture content were determined according to AACC methods 46–30, 76–13, 30–25, and 44–15A (American Association of Cereal Chemists (AACC), 2000), respectively. The AACC method 56–60 (2000) was used for sedimentation test. Falling number (FN) was determined according to the AACC method 56–81B (2000) with the apparatus model 1900 (Perten, Sweden). The wet gluten content (WGC) and gluten index (GI) were tested using AACC method 38–12A (2000).

2.6. Determination of pasting properties

The pasting properties of wheat flour were determined using the Rapid Visco Analyser (RVA-Super 4, Newport Scientific Pvt. Ltd., Australia), according to AACC method 76–21 (2000). Wheat flour was mixed with two solvents (25 mL), respectively (corrected to compensate for a 14% moisture basis). Two solvents were used: distilled water, 1 mM silver nitrate (AgNO₃) solution.

2.7. Measurement of mixing behaviors

The mixing behaviors of flour dough were investigated using Mixolab (Chopin, Tripette &Renaud, Paris, France) according to AACC 54–60.01 (2000). The Mixolab device was used to determine the following: water absorption (g/kg), stability time (min), development time (min), protein weakening (C₂, Nm), peak torque of starch gelatinization (C₃, Nm), cooking stability (C₄, Nm) and starch gelling (C₅, Nm), starch gelatinization during heating phases (C₃-C₂, Nm), amylase activity (C₃-C₄, Nm), starch gelatinization during cooling phases (C₅-C₄, Nm).

2.8. Analysis of colors

Colors of the wheat flour were measured using a Hunter Lab colorimeter (Ultra Scan VIS, Hunterlab, USA). The color differences (∆E) between ozone-treated samples (S) and the control samples (C) is calculated as follows (László et al., 2008):

(2) $\mathrm{\Delta }E=\sqrt{{\left(L_s^{\ast }-L_c^{\ast }\right)}^2+{\left(a_s^{\ast }-a_c^{\ast }\right)}^2+{\left(b_s^{\ast }-b_c^{\ast }\right)}^2}$(2)

The whiteness indexes (WI) were calculated as follows (Saberi et al., 2016):

(3) $WI=100-\sqrt{{\left(100-L^{\ast }\right)}^2+a^{\ast 2}+b^{\ast 2}}$(3)

where L* indicates lightness value of color (from 100 to 0), a* indicates a red (+a*) to a green (-a*), and b* indicates a yellow (+b*) to a blue (-b*), respectively. The colorimeter was used to evaluate L*, a*, and b*.

2.9. Dynamic viscoelasticity

The test was determined using a DHR-2 Rheometer (TA Instruments, New Castle, USA). Prepare the dough samples using Farinograph mixer (Model 8101, Brabender OHG, Duisburg, Germany). Dough samples were rested for 30 min before the rheological measurement. A 50 mm diameter of standard steel parallel plate probe (1 mm-gap distance) was selected. The conditions of frequency sweep tests were carried out at 30°C (temperature) and 0.1–10 Hz (frequency), following an initial equilibration of samples for 5 min at 30°C, the strain sweep test at constant frequency (1 Hz).

2.10. Statistics analysis

Data from the three repeated experiments were analyzed to determine whether the variances were statistically homogeneous, and the results were expressed as mean± SD. Statistical comparison was made by one-way ANOVA followed by a Duncan’s multiple range test using SPSS software. Differences were considered to be significant when the p-values were under 0.05.

3. Results and discussion

3.1. DON detoxification

The DON concentration was detected in wheat flour, which was milled after using ozone to treat wheat kernels. The data in Figure 1 indicated all the ozone treatment methods could reduce the DON content to be less than 1.00 mg/kg (Chinese national standards and the US FDA stipulate that the safety limit for DON in food is 1.00 mg/kg). The DON degradation rate of control was the lowest (10.85%), sample B had the highest rate (33.33%). The results showed that the five ozone treatment methods degraded the DON in contaminated wheat effectively. The only difference of samples A and B was that ozone gas was used to treat sample A before tempering, while sample B was treated after tempering. However, sample A had higher DON content than sample B. This result was in agreement with the report of M. M. M. M. Li et al. (2014) who found that ozone could be quickly decomposed into atomic oxygen with strong oxidation and hydroxyl ions in water, which were effective in oxidative degradation of DON. Hence, for the sample with higher moisture content undergoing the same condition of ozone treatment, the rate of degradation of DON was higher after ozone treatment.

Figure 1. Effects of five ways of ozone treatments on the degradation of DON in wheat flour. The DON content: control, 1.15 (mg/kg); sample A, 0.97 (mg/kg); sample B, 0.86 (mg/kg); sample C, 0.92 (mg/kg); sample D, 0.98 (mg/kg); sample E, 0.96 (mg/kg); the wheat, 1.29 (mg/kg).

Figura 1. Efectos de los diferentes tratamientos de ozono en la degradación del DON en la harina de trigo. Contenido de DON: control, 1.15 (mg/kg); muestra A, 0.97 (mg/kg); muestra B, 0.86 (mg/kg); muestra C, 0.92 (mg/kg); muestra D, 0.98 (mg/kg); muestra E, 0.96 (mg/kg); el trigo, 1.29 (mg/kg)

3.2. Chemical composition

The effect of different ozone treatment methods on chemical composition of wheat flour (Table 2) showed that starch, protein, or fat content were not significantly different between the treated and untreated samples (p > .05), which was consistent with the result reported by Dubois et al. (2006) and Wang et al. (2016).

Table 2. Chemical composition, gluten quality, α-amylase activity, and colors of wheat flour as affected by five ways of ozone treatments.

Tabla 2. Composición química, calidad del gluten, actividad de la α-amilasa y colores de la harina de trigo afectados por los cinco tratamientos de ozono

	control	Sample A	Sample B	Sample C	Sample D	Sample E
Composition
Starch (%)	75.09 ± 0.04^a	75.06 ± 0.01^a	75.05 ± 0.05^a	75.04 ± 0.02^a	75.07 ± 0.10^a	75.04 ± 0.02^a
Protein (%)	12.23 ± 0.20^ab	12.08 ± 0.04^b	12.41 ± 0.04^a	12.04 ± 0.04^b	12.27 ± 0.18^ab	12.20 ± 0.16^ab
Fat (%)	1.31 ± 0.07^a	1.35 ± 0.02^a	1.32 ± 0.02^a	1.38 ± 0.03^a	1.36 ± 0.01^a	1.33 ± 0.04^a
Gluten quality
WGC (%)	30.90 ± 1.27^ab	30.00 ± 0.42^a	30.50 ± 0.28^a	10.35 ± 0.23 ^c	31.10 ± 0.14^b	29.55 ± 0.07^a
GI	0.95 ± 0.02^a	0.93 ± 0.03^a	0.92 ± 0.01^a	0.23 ± 0.06^b	0.92 ± 0.00^a	0.93 ± 0.00^a
SV (mL)	24.82 ± 0.21^a	23.51 ± 0.16^b	23.36 ± 0.22^b	15.03 ± 0.30 ^c	24.53 ± 0.12^a	23.49 ± 0.07^b
α-amylase activity
FN(s)	389.5 ± 3.5^b	372.0 ± 7.1^bc	348.0 ± 8.5^c	472.0 ± 13.9^a	365.5 ± 3.5^bc	358.0 ± 1.4^bc
colors
L*	83.78 ± 0.15^a	84.66 ± 0.16^b	84.74 ± 0.07^b	87.14 ± 0.25 ^c	84.52 ± 0.26^b	84.23 ± 0.64^b
a*	5.08 ± 0.06 ^c	4.76 ± 0.10^b	4.81 ± 0.01^b	3.08 ± 0.07^a	4.85 ± 0.08^ab	4.87 ± 0.08^ab
b*	7.44 ± 0.07^b	7.67 ± 0.07^ab	7.85 ± 0.08^ab	5.81 ± 0.03 ^c	7.58 ± 0.09^b	7.69 ± 0.10^ab
∆E	-	0.97	1.08	4.24	0.80	0.56
WI	81.44	82.20	82.18	85.56	82.10	81.79

Note: Values are presented as mean ± SD (n = 3). Values with different letters are significantly different (p < 0.05). WGC, Wet Gluten Content; GI, Gluten Index; SV, Sedimentation Volume; FN, Falling Number; L*, lightness value of color; a*, red-green value of color; b*, yellow-blue value of color; ∆E, color differences; WI, whiteness indexes; sample A, treatment of wheat kernels with ozone gas before tempering; sample B, treatment of wheat kernels with ozone gas after tempering; sample C, direct treatment of milled wheat flour with ozone gas; sample D, cleaning of wheat with ozonated water; sample E, tempering of wheat with ozonated water.

Nota: Los valores se presentan como medias ± DE (n = 3). Los valores con letras distintas son significativamente diferentes (p < 0.05). WGC, contenido de gluten húmedo; GI, índice de gluten; SV, volumen de sedimentación; FN, número de caída; L*, valor de luminosidad del color; a*, valor de color rojo-verde; b*, valor de color amarillo-azul; ∆E, diferencias de color; WI, índices de blancura; muestra A, tratamiento de los granos de trigo con gas de ozono antes del templado; muestra B, tratamiento de los granos de trigo con gas ozono después del templado; muestra C, tratamiento directo de la harina de trigo molida con gas ozono; muestra D, limpieza del trigo con agua ozonizada; muestra E, templado del trigo con agua ozonizada.

3.3. Gluten qualities

The Wet Gluten Content (WGC) and Gluten Index (GI) of all samples can be seen in Table 2. There was no significant difference in WGC and GI between the ozone-treated samples and control, when the wheat kernels were treated by ozone gas or ozone water. However, the sample C (wheat flour treated with 60 mg/L ozone gas for 2 h) was ozonated with ozone gas directly, WGC and GI of sample C fell by 66.5% and 75.8%, respectively. When the ozone gas concentration was 60 mg/L after wheat flour was treated for 2 h (sample C), it had the stronger oxidation, which affected the formation of gluten in wheat dough. Similar results were also reported by Mei et al. (2016) who found a decrease in wet gluten when ozone treatment time was further increased, which destroyed the gluten structure and the water-holding capacity decreased rapidly. There was a similar report in Lee et al. (2017).

The quality of wheat gluten is not only related to WGC and GI, but to the Sedimentation Volume (SV), the higher the SV of wheat flour, the better the quality of gluten protein and flour (Ibanoglu, 2002). It was found from Table 2 that the SV of ozone-treated wheat (sample D) with short contact time was not significantly different from that of the control, while the SV of the other ozonated samples decreased to different degrees. The sample C had the lowest SV, decreasing by 39.4%, while the SV of samples A, B, and E decreased slightly (5.3%, 6.0%, and 5.4%, respectively) due to protection of wheat seed coat.

In determination of SV, the lactic acid solution has the function of expanding the wheat gluten protein, which can increase the viscosity of the flour gel, thereby changing the sedimentation speed of flour. According to Man. Li et al. (2012), the potential for polymerization of low molecular weight proteins was found after ozone gas was used to treat wheat four. Additionally, ozonation caused a significant reduction in the SDS solubility of the wheat prolamins (Gozé et al., 2017). The results in Table 2 were analyzed, and it was found that ozone treatment enhanced the polymerization of low molecular weight proteins and reduced the solubility of prolamins in wheat flour. These changes made the SV of ozonated samples decrease to different degrees, even though ozone did not cause the change of WGC and GI in wheat, except for sample C.

3.4. Dynamic viscoelasticity

This testing simultaneously measures the storage modulus (G’) and loss modulus (G”) represent dynamic elasticity and dynamic viscous of dough, and loss tangent (tan δ). The results of Dynamic Viscoelasticity (DV) were shown in Figure 2. All samples showed an increase in G’ and G” with increasing frequency from 0.1 to 10 Hz, and the tan δ < 1.0 indicated a solid-like behavior. Compared with control, the ozone-treated wheat kernels did not change the G’, G” and tan δ significantly. Nevertheless, the G’ and G” of sample C were much higher than the other samples, the tan δ was lowest. Dynamic rheological parameters are able to indicate the quality of wheat glutens. Glutens from good quality wheat are rheologically characterized as more elastic (G’) and less viscous (G”) (Song & Zheng, 2007). Eventhough the sample C had the most elastic, but also had the highest viscous (G”) and the lowest tan δ. Generally, the dough made from good quality flour has tan δ values lower than dough made from poor quality flour (song & Zheng, 2007). The tan δ is the ratio of G” to G’, it can be used to describe the proportion of polymer in the dough, the value was smaller, the degree of polymerization was higher (Tunick, 2011). It was found that the quality of sample C was inferior to other samples. Presumably, the stronger oxidation (sample C) caused gluten proteins in wheat flour to accumulate more easily, which led to poorer quality of wheat flour.

Figure 2. Dynamic responses of five ways of ozone treatments of wheat flour, showing changes in G’(a), G” (b), and tan δ(c). G’, storage modulus; G”, loss modulus; tan δ, ratio of G” to G’.

Figura 2. Respuestas dinámicas de los diferentes tratamientos de ozono aplicados a la harina de trigo, mostrando los cambios en G’(a), G” (b), y tan δ(c). G’, módulo de almacenamiento; G”, módulo de pérdida; tan δ, relación de G” a G’

3.5. amylase activity

Falling number (FN) is an important index to measure the α-amylase activity of wheat flour. The results in Table 2 indicated that the ozone treatment caused different degrees of changes to FN. The FN of sample C increased significantly to 472 s, an increase of 21.2% compared with control. The other ozonated samples had lower FN than control, but it was not significant. In the report by Ibanoglu (2002), using ozonated water for washing did not alter FN of either soft or hard wheat grains. But Mei et al. (2016) showed the FN rose almost to 400 s after 2 h of ozone treatment. Piechowiak et al. (2018) also reported that the activity of α-amylase declined with increasing ozone concentration and prolonging process.

Generally, higher FN means lower α-amylase activity in wheat flour, the results of FN indicated the stronger ozone treatment (sample C) had an inhibitory effect on the α-amylase activity. In this experiment, the results showed the protection of wheat seed coat, the α-amylase activity did not change in the other four ozonated samples. A study found that the ozone could deactivate α-amylase through fast reaction with aromatic and sulfur-containing amino acids residues of enzyme (Martínez-Gallegos et al., 2014). Ozone can have an inhibitory effect on many enzymes like polyphenol oxidase, lipoxygenase, lipase, α-amylase (Dubois et al., 2006).

3.6. Colors of wheat flour

The color of wheat flour is an important factor in consumer acceptance of flour and its products (Li et al., 2013). L*(lightness) is a quantity that measures the percentage of total solar spectral reflectance in relation to a pure white surface (Sandhu et al., 2011). The carotenoid in flour is one of the main factors affecting whiteness (Humphries et al., 2004), and ozone in gaseous or aqueous form can react with double bonds in carotenoid pigments, including β-carotene, xanthophyll, and flavones (Wang, Shao et al., 2016).

Colors of samples were analyzed in Table 2, all ozone-treated samples showed a significant increase in L* values (p < .05). The sample C had the highest L* value, an increase of 4.0% compared with control. There was a significant difference in a* and b* values between sample C and control, a decrease of 39.3% and 21.9% compared with control, respectively. There was a similar report by Sandhu et al. (2011) whose color analysis indicated that L* value increased and b* value decreased with exposure time of flour treated with ozone gas. The ∆E and WI in Table 2 showed that all ozone treatments made the wheat flour becoming whiter. Sample C had the highest ∆E (4.24) and WI (85.56), and the other four ozone treatments caused the lower ∆E and WI. It indicated that after the wheat flour (sample C) was exposed to ozone gas for 2 h, the white significantly increased, it tended to turn blue and green, and the tone became cold. The rest of the samples had a slight increase in white hue (WI value) compared with control under different ozone treatments, but a* and b* values did not change significantly.

3.7. Pasting properties

During the wheat flour cooking process, the viscosity during the gelatinization of starch affects the quality of wheat products such as bread, noodles (Chen et al., 2012). Table 3 shows the pasting properties of wheat flour in two solvents (distilled water and silver nitrate (AgNO₃) solution). In distilled water, the peak viscosity, trough viscosity, final viscosity, breakdown, setback, pasting temperature, and peak time of sample C were significantly different (p < .05) from the other samples. The sample C exhibited the highest peak viscosity and breakdown; the other pasting indices of sample C were the lowest. The rest of four ozonated samples (A, B, D, E) and control showed a notable difference in their pasting properties. According to the test results, the oxidation mode of sample C had obviously changed the pasting properties of wheat flour.

Table 3. Effect of five ways of ozone treatments on pasting properties of wheat flour.

Tabla 3. Efecto de cinco formas de tratamiento con ozono en las propiedades de pegado de la harina de trigo

Parameters	control	Sample A	Sample B	Sample C	Sample D	Sample E
Distilled water
Peak viscosity (cP)	2044 ± 45^a	2044 ± 101^a	1958 ± 5^a	2160 ± 37^b	2005 ± 61^a	2062 ± 29^a
Trough viscosity (cP)	1283 ± 4^b	1277 ± 66^b	1290 ± 11^b	920 ± 21^a	1318 ± 34^b	1300 ± 17^b
Breakdown (cP)	762 ± 42^b	767 ± 35^b	668 ± 16^a	1240 ± 16 ^c	687 ± 27^a	762 ± 45^b
Final viscosity (cp)	2322 ± 64^b	2348 ± 101^b	2221 ± 20^b	1296 ± 24^a	2289 ± 73^b	2421 ± 3^bc
Setback (cP)	1040 ± 61^bc	1071 ± 35 ^c	931 ± 31^b	377 ± 4^a	972 ± 40^b	1122 ± 14 ^c
Peak time (min)	6.17 ± 0.04 ^c	6.10 ± 0.03^bc	6.19 ± 0.02 ^c	5.67 ± 0.00^a	6.20 ± 0.08 ^c	6.07 ± 0.00^b
Pasting temperature (°C)	90.00 ± 0.45 ^c	89.25 ± 0.63^bc	89.90 ± 0.30 ^c	68.53 ± 0.03^a	90.73 ± 0.28 ^c	88.8 ± 0.08^b
Silver nitrate (AgNO3) solution
Peak viscosity (cP)	2684 ± 23^b	2857 ± 8^c	2819 ± 47 ^c	2193 ± 4^a	2821 ± 18^c	2806 ± 73 ^c
Trough viscosity (cP)	1891 ± 4^b	2040 ± 18 ^c	2035 ± 44 ^c	941 ± 1^a	2047 ± 30 ^c	2049 ± 46 ^c
Breakdown (cP)	793 ± 20^b	816 ± 10 ^c	784 ± 3a^b	1252 ± 5^d	774 ± 12^ab	757 ± 28^a
Final viscosity (cp)	3144 ± 46^b	3389 ± 9^c	3320 ± 118 ^c	1354 ± 34^a	3437 ± 76 ^c	3364 ± 79 ^c
Setback (cP)	1253 ± 50^b	1348 ± 26 ^cd	1385 ± 74^d	413 ± 33^a	1390 ± 46^d	1277 ± 33^bc
Peak time (min)	6.37 ± 0.04^b	6.40 ± 0.00^b	6.40 ± 0.00^b	5.64 ± 0.04^a	6.43 ± 0.03^b	6.40 ± 0.07^b
Pasting temperature (°C)	89.15 ± 0.45^b	90.03 ± 0.38 ^c	89.60 ± 0.10^bc	68.15 ± 0.35^a	89.80 ± 0.20^bc	89.65 ± 0.00^bc

Note: Values are presented as mean ± SD (n = 3). Values with different letters are significantly different (p < 0.05). sample A, treatment of wheat kernels with ozone gas before tempering; sample B, treatment of wheat kernels with ozone gas after tempering; sample C, direct treatment of milled wheat flour with ozone gas; sample D, cleaning of wheat with ozonated water; sample E, tempering of wheat with ozonated water.

Nota: Los valores se presentan como media ± DE (n = 3). Los valores con letras distintas son significativamente diferentes (p < 0.05). Muestra A, tratamiento de los granos de trigo con gas de ozono antes del templado; muestra B, tratamiento de los granos de trigo con gas de ozono después del templado; muestra C, tratamiento directo de la harina de trigo molida con gas de ozono; muestra D, limpieza del trigo con agua ozonizada; muestra E, templado del trigo con agua ozonizada.

The α-amylase activity has a significant effect on the pasting properties of wheat flour. Mariotti et al. (2005) used AgNO₃ solution to inhibit α-amylase activity in normal wheat flour. In the subsequent RVA test, the distilled water was replaced by 0.085 g/L AgNO₃ solution. Apart from sample C, the four ozonated samples (A, B, D, E) were considered to have higher viscosity (peak viscosity, trough viscosity, final viscosity) than control when α-amylase activity was inhibited, but pasting temperature or peak time was not significantly different from that of control. Gozé et al. (2017) reported that ozone-treated samples exhibited higher carboxyl content than control, and a slight increase in carboxyl groups was noticed by increasing ozonation. It could change swelling power of wheat starch to increase the viscosity. Sample C had test parameters similar to the pasting properties in distilled water. It was observed that α-amylase activity was almost completely inhibited when ozone gas was used to directly treat wheat flour, which could be further confirmed by the detection results of FN (increased by 21.2%).

3.8. Mixing behaviors

According to the test results of all samples in Table 4, the dough stability time and development time, C₃, C₄, C₃-C₂, C₃-C₄, C₅-C₄ of sample C were significantly different from the other four samples and control. In the Mixolab parameters, the water absorption, stability time, and development time are important indices of flour quality and processing characteristics, and these indexes of Mixolab were related to farinograph (Blandino et al., 2015). The development time refers to the strength of gluten, the stability time reflects the dough elasticity (Teng et al., 2014). Compared with control, the stability time and development time of sample C decreased by 73% and 80%, respectively. The observed reduction indicated that the gluten strength and the elasticity of dough were affected significantly by the direct ozone treatment of wheat flour. According to the results of WGC and GI, it was found that the gluten quality declined after the ozone treatment of sample C, which led to a significant decrease in the dough formation time and stabilization time.

Table 4. Effect of five ways of ozone treatments on mixing behaviors of wheat flour.

Tabla 4. Efecto de cinco tratamientos diferentes con ozono en el comportamiento de la mezcla de harina de trigo

Parameters	control	Sample A	Sample B	Sample C	Sample D	Sample E
Water absorption (%)	65.5	65.8	66.0	66.8	65.4	65.2
Stability time (min)	10.28	9.90	9.83	2.75	9.85	10.25
Development time (min)	6.47	7.65	6.97	1.30	7.12	7.57
C2 (Nm)	0.63	0.61	0.57	0.51	0.56	0.62
C3 (Nm)	1.80	1.82	1.84	2.12	1.79	1.87
C4 (Nm)	1.29	1.27	1.34	0.78	1.30	1.31
C5 (Nm)	2.54	2.53	2.54	2.56	2.48	2.64
C3-C2 (Nm)	1.17	1.21	1.27	1.61	1.23	1.25
C3-C4 (Nm)	0.51	0.55	0.5	1.34	0.49	0.56
C5-C4 (Nm)	1.25	1.26	1.2	1.78	1.18	1.33

Note: Values are presented as mean ± SD (n = 3). Values with different letters are significantly different (p < 0.05). C₂, protein weakening; C₃, peak torque of starch gelatinization; C₄, cooking stability; C₅, starch gelling; C₃-C₂, starch gelatinization during heating; C₃-C₄, amylase activity; C₅-C₄, starch gelatinization during cooling; sample A, treatment of wheat kernels with ozone gas before tempering; sample B, treatment of wheat kernels with ozone gas after tempering; sample C, direct treatment of milled wheat flour with ozone gas; sample D, cleaning of wheat with ozonated water; sample E, tempering of wheat with ozonated water.

Nota: Los valores se presentan como media ± DE (n = 3). Los valores con letras distintas son significativamente diferentes (p < 0.05). C₂, debilitamiento de la proteína; C₃, par máximo de gelatinización del almidón; C₄, estabilidad en la cocción; C₅, gelificación del almidón; C₃-C₂, gelatinización del almidón durante el calentamiento; C₃-C₄, actividad de la amilasa; C₅-C₄, gelatinización del almidón durante el enfriamiento; muestra A, tratamiento de los granos de trigo con gas de ozono antes del templado; muestra B, tratamiento de los granos de trigo con gas ozono después del templado; muestra C, tratamiento directo de la harina de trigo molida con gas ozono; muestra D, limpieza del trigo con agua ozonizada; muestra E, templado del trigo con agua ozonizada.

Mixolab determination of the pasting properties was expressed by three points: C₃–C₂, C₃–C₄ and C₅–C₄ related to C₃, C₄, C₅, respectively. The higher value of C₃–C₂ corresponded to the higher viscosity during the gelatinization of flour starch. The lower C₃-C₄ value showed the stronger stability of starch gel and amylase activity. The higher value of C₅-C₄ indicated that starch was more prone to retrogradation. In this paper, both C₃ and C₄ values of sample C were different among other samples, and it indicated that starch gelatinization or amylase activity was significantly altered. The C₃–C₂, C₃–C₄, and C₅–C₄ of sample C were the highest, corresponding to the RVA parameters (peak viscosity, breakdown viscosity, setback viscosity, respectively) in distilled water and linked to FN. These changes indicated that sample C had the highest viscosity, lowest amylase activity, and bad stability of flour gel, easier to go stale.

4. Conclusions

The research of this paper showed that all the five ozone treatments could effectively degrade the DON content of contaminated wheat to be within the limit of safety (<1.00 mg/kg), and ozone had the best effect on the degradation of DON after tempering wheat (degradation rate 33.33%). By comparing the quality indexes of wheat flour, it was found that due to the protection of wheat epidermis, when ozone gas or ozonated water treated wheat kernels, it did not cause significant changes in the gluten qualities, dynamic viscoelasticity, pasting and mixing properties except lightly beneficial changes in colors and development time of dough. When the wheat flour treated with 60 mg/L ozone gas for 2 h (sample C), it had the stronger oxidation, which reduced WGC, GI and SV by 66.5%, 75.8%, and 39.4%, respectively. Furthermore, the sample C had the bad stability of flour gel, lowest stability time and development time of dough. The stronger oxidation reaction led to poorer quality of wheat flour. According to the degradation effect of DON and the quality of flour, the ozone gas was selected to treat the tempered wheat (sample B), which could not only degrade DON to the maximum extent, but also ensure that the quality of wheat was basically unchanged. This study provides a reference for the application of ozone in the degradation of wheat toxins.

Declaration of interest statement

The authors declare that they do not have any conflict of interest.

Additional information

Funding

This work was supported by the National Key Research and Development Program of China [2018YFD0401002) and National Natural Science Foundation of China [31801577].