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Changes in common wheat grain milling behavior and tissue mechanical properties following ozone treatment
ARTICLE IN PRESS
Journal of Cereal Science 47 (2008) 245–251 www.elsevier.com/locate/jcs
Changes in common wheat grain milling behavior and tissue mechanical properties following ozone treatment C. Desvignesa, M. Chauranda, M. Duboisb, A. Sadoudia, J. Abecassisa, V. Lullien-Pellerina, a
UMR 1208 Inge´nierie des Agropolyme`res et Technologies Emergentes, CIRAD-INRA-Montpellier SupAgro-Universite´ Montpellier II, 2 place Pierre Viala, F-34000 Montpellier, France b Goe¨mar, ZAC La Madeleine, F-35400 Saint Malo Cedex, France Received 26 December 2006; received in revised form 21 March 2007; accepted 4 April 2007
Abstract Ozone treatment (10 g/kg) of common wheat grains with a new patented process, Oxygreens, used before milling was found to significantly reduce (by 10–20%) the required energy at breaking stage whatever the grain hardness and without changes in the flour yield. Detailed study of each of the milling steps undertaken on a hard type cultivar showed that both the breaking and the reduction energy were decreased. Reduction of the coarse bran yield was also observed concomitantly with an increase in the yield of white shorts. Biochemical characterization of the milling fractions pointed out changes in technological flour properties as starch damage reduction, aleurone content enrichment and increase of insoluble glutenin polymers. Measurement of wheat grain tissue mechanical properties showed that ozone treatment leads to reduction of the aleurone layer extensibility and affects the local endosperm resistance to rupture. These data as well as the direct effect of ozone oxidation on biochemical compounds could explain the observed changes in milling energy, bran and shorts yield and flour composition. r 2007 Elsevier Ltd. All rights reserved. Keywords: Aleurone; Common wheat; Mechanical properties; Milling; Ozone
1. Introduction Ozone (O3) is a strong oxidant recognized since 1997 as a GRAS (generally recognized as safe) substance and used in a number of applications in the food industry for destruction or detoxification of chemicals or microorganisms (Graham, 1997; Kim et al., 1999; Liangji, 1999). These applications include the surface decontamination, storage and preservation of perishable foods as well as water or manufacturing equipment and packaging sterilization (Khadre et al., 2001). Abbreviations: Dmax, maximum displacement associated to rupture, E0 , tensile modulus; eela, elastic strain; emax, maximum strain to rupture, fela, lineic strength to elastic deformation; fmax, lineic strength to rupture or maximum accepted force to rupture; K0 , grinding index (required energy to produce 1 kg of flour); SE-HPLC, size-exclusion HPLC; NIRS, near infrared spectroscopy; NTP, normal temperature pressure; V, cone print volume in penetrometry assay. Corresponding author. Tel.: +33 4 99 61 31 05; fax: +33 4 99 61 30 76. E-mail address: [email protected] (V. Lullien-Pellerin). 0733-5210/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2007.04.004
Treatments of wheat grains with ozone either generated from air or pure oxygen and applied as a gas (Wu et al., 2006) in fumigation or dissolved in water in washing (Ibanoglu, 2002) or tempering steps (Ibanoglu, 2001) were already shown to be effective on bacteria or fungi inactivation. Furthermore, wheat grain or flour biochemical composition as well as flour yield or technological properties appeared to be unaffected in these conditions (Ibanoglu, 2001, 2002; Mendez et al., 2003). Recently, a new process (Oxygreens) based on ozone treatment of wheat grains after pre-moistening in a closed batch reactor was patented (Coste et al., 2005; Yvin et al., 2001). It could be easily introduced between wheat grain cleaning and milling in place of the classical tempering step and was considered as safe and approved by the French Food Safety Authority for some specific flour uses (Gaou et al., 2005). However, tempering constitutes an important step in milling as it helps the separation between the starchy endosperm and the other tissues increasing their differences in mechanical properties (Abecassis, 1993).
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Thus, physical and biochemical conditions at this step appear critical for tissue mechanical behavior and efficient tissue separation. Indeed, mechanical properties of peripheral wheat grain tissues were already found to be influenced by oxidative conditions (Peyron et al., 2001, 2002) and consequently could affect the milling behavior (Peyron et al., 2002). As an oxidative treatment, Oxygreens effect on wheat grain milling behavior has to be analyzed specifically in terms of potential tissue mechanical property changes and consequences on biochemical composition of the obtained fractions. This paper describes this study and analyzes the results in relation to the observed changes of wheat grain tissue mechanical properties.
stopped when ozone consumption reached 5 or 10 g/kg of wet grain mass. Total reaction time was between 100 and 300 min depending on the ozone consumption dose except for the soft wheat which showed shorter treatment duration (80–180 min). After ozone treatment and sorting with an air classifier (NSP classifier, Tripette et Renaud, France), grains were collected and maintained at +4 1C in a dry room for further analysis or used in milling after a 24 h resting time. A control sample for each wheat batch was also treated in the reactor but without ozone addition either 15 min (Table 1) or the same duration as the corresponding ozone treatment (Table 2). 2.3. Micromilling
2. Materials and methods 2.1. Wheat samples Common wheat (Triticum aestivum L.) grains from three distinct cultivars with distinct kernel hardness (measured by NIRS analysis, approved method 39-70A, AACC, 2000), Camp Remy and Caphorn as medium hard and Crousty as soft, were used in this study. All samples were harvested in France in 2003 or 2005 and purchased or kindly supplied by Arvalis-Institut du Ve´ge´tal (Paris) or Danone Vitapoˆle (Palaiseau). Grains were cleaned to remove impurities and stored at room temperature before use. 2.2. Wheat grain treatment with ozone using Oxygreens process The Oxygreens treatment was performed on 10 kg of grains as already described in Dubois et al. (2006). Water added, in the reactor, was equal to 4% of the initial wet grain mass. After water addition, ozone was introduced into the reactor, with pure O2 as carrier gas, at concentration of 89 g of O3/m3 NTP of O2. The consumption of O3 was measured by two BMT963 analyzers (Messtechnik, Berlin, Germany), the first one at the inlet of the reactor, the second one at the outlet of the reactor. Treatment was
Each wheat grain sample (200 g) that was already tempered by water addition in the Oxygreens reactor in order to reach 15–18% (w/w) moisture content taking into account its initial water content, as milled according to the process already described in Greffeuille et al. (2006a). Milling diagram included two breaking stages, one sizing and one reduction stage and allowed obtaining four flour fractions, coarse and fine bran fractions as well as two short fractions. The micromill was equipped with torque transducers allowing the one-line measurement of mechanical energy consumption at each stage. Energy needed to produce 1 kg of flour was calculated and is represented by the K0 index (kJ/kg of flour, Pujol et al., 2000). 2.4. Biochemical analysis 2.4.1. Moisture, protein and ash contents Moisture, protein and ash content of the grains or milling fractions were determined according to approved methods 44-19, 46-12 with Nx5.7 and 08-12 (AACC, 2000), respectively. 2.4.2. Phytic acid, total starch and damaged starch contents Phytic acid was determined at 500 nm from acidic extract of milling fractions using a colorimetric method modified by Vaintraub and Lapteva (1988). A standard curve was
Table 1 Comparison of the milling behavior at breaking stage from distinct wheat samples after only tempering with water (0 g/kg) in the reactor or treated with ozone (5 or 10 g/kg)
Ozone consumption (g/kg) Break flour yield(1) (% w/w) Total energy(2) (kJ/kg) K0 index(3) (kJ/kg flour)
Soft type
Hard type
Crousty*
Caphorn*
0 26.8a 14.8a 55.4a
5 27.0a 12.5b 46.2b
10 27.1a 13.2c 48.2c
0 20.6a 27.1a 131a
Camp Remy* 5 20.7a 27.8a 134a
10 20.3a 25.2b 124b
0 29.9a 24.8a 82.9a
5 29.7a 22.9b 77.0b
Camp Remy** 10 29.5a 22.3c 75.6c
0 23.8a 20.4a 85.8a
5 23.8a 19.0b 79.8b
10 23.9a 17.1c 71.7c
Distinct letters for an identical parameter and the same wheat sample indicate significant differences mean values with Po0.05 taking into account coefficients of variation of the considered parameter: (1)o2%, (2), (3)o5%. Ozone consumption, break flour yield and corresponding required energy were expressed relative to the wet grain mass except K0 index, which corresponds to the energy required relative to the wet flour mass obtained after two breaking steps. Wheat grains belong to distinct hardness class and were cultivated in 2003 (*) or in 2005 (**).
ARTICLE IN PRESS C. Desvignes et al. / Journal of Cereal Science 47 (2008) 245–251 Table 2 Effect of ozone treatment on the milling behavior of wheat grains from the Camp Remy cultivar Hard wheat Camp Remy (2005) Milling stages
Energy(1) (kJ/kg of grains) K0 index(2) (kJ/kg of flour)
Control +ozone Control +ozone
Break
Sizing
Reduction
20.4a 17.1b 78.1a 71.7b
39.1a 33.0b 74.5a 65.4b
22.2a 17.2b 30.4a 24.9b
18.2a 19.2a
28.4a 28.7a
Flours Flour yield(3) (% w/w)
Control +ozone
26.0a 23.9a Brans Coarse
Fraction yield (% w/w)
(4)
Control +ozone
a
7.6 5.6b
Shorts Fine a
9.3 9.7a
Brown a
4.3 4.9a
White 6.2a 8.0b
Distinct letters for an identical parameter and the same milling fraction indicate significant differences between mean values with Po0.05 taking into account coefficients of variation of the considered parameter: (3), (4) o2%, (1), (2)o5%. Milling energy and K0 index obtained from untreated (control) or ozone treated grains (10 g consumed ozone/kg of grains) at each milling stage and yield of each fraction obtained at break, sizing and reduction steps. Values were expressed relative to wet grain or flour mass.
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crossing crease on one side of the grain delimiting two flat endosperm surfaces to perform the tests. Before mechanical assay, samples were equilibrated in a temperate chamber (25 1C) under 75% relative humidity for 48 h in order to assure a 15% moisture content as controlled by measurement. Indentation tests were performed using a tractioncompression static-machine (Zwick 2.5, Zwick/Roell, Roissy, France) equipped with a conical tip (angle of point 301). Each sample was placed directly under the tip and subjected to four distinct assays at sufficient spacing between cone penetrations in order to avoid interferences between indentations due to local damage. The assay included a first step 0.2 N pre-charge of the sample at an imposed speed (0.5 mm/s) followed by an imposed displacement (0.8 mm) at a 0.02 mm/s speed until maximum force (fmax) was reached which defined the sample rupture. The data capture rate was 20 points/s. The following parameters were obtained from the strength (N)/displacement (mm) curves: maximum accepted force (fmax, N), maximum displacement until rupture (Dmax, mm). Sample volume subjected to the cone penetration ( ¼ cone print) was calculated using the equation V ¼ ðP=3Þa2 D3max with a ¼ tan (cone radius). V/fmax ratio was calculated and expresses the sample mechanical behavior for an equal strength of 1 N.
2.4.3. Characterization of proteins from flours by sizeexclusion chromatography Proteins extracted from flours with sodium phosphate buffer (pH 6.9) containing 1% sodium dodecyl sulfate (SDS) were analyzed by size exclusion high performance liquid chromatography (SE-HPLC) according to Morel et al. (2000). The chromatogram was divided into five fractions (F1–F5) that were attributed to soluble high molecular size (HMW) glutenins (F1) and low molecular size (LMW) glutenins (F2), o-gliadins (F3) or a-, b-, ggliadins (F4) and water and salt soluble proteins (F5), respectively. Insoluble proteins (Fi) were also quantified by SE-HPLC after solubilization of the insoluble protein pellet and sonication.
2.5.2. Preparation of wheat outer layers and mechanical assays Peripheral tissues and the aleurone layer were isolated by hand according to radial grain orientation, equilibrated in water content and cut as already described in Antoine et al. (2003). Uniaxial tension tests were performed using dynamic mechanical thermal analysis (DMTA Mk III, Rheometrics Inc., Piscataway, USA) at a rate of 0.05 mm/s until sample breakage. Before tension assays, the stability of the dynamic modulus was used as an indicator of the sample equilibration in temperature (30 1C) and water content (17%) after a dynamic test at imposed strain (strain ¼ 0.01%, total time ¼ 12 min, frequency ¼ 1.59 Hz). Mechanical properties of the samples were determined from obtained curves expressing strength according to strain taking into account the tissue thickness which is measured with a micrometer (Braive Instruments, Checy, France). The measured parameters were lineic strength to rupture (fmax) and lineic strength to elastic deformation (fela), maximum strain (emax) and elastic strain (eela), tensile modulus (E0 ), and total rupture energy (Wtot). Lineic strength corresponds to the strength exerted per unit of tissue width necessary to cause reversible deformation or rupture. Assays in which rupture did not occur in the middle of the strip were discarded.
2.5. Mechanical properties measurement
2.6. Statistical analysis
2.5.1. Preparation of wheat grains and mechanical assays Wheat grains were abraded longitudinally with sand-paper on the two sides of the grain. The obtained sections (3 mm thickness) preserve the peripheral tissues and present a
Variance analysis was performed using Minitab.13 software (Minitab Inc, PA, USA). To discriminate among the means, Fisher’s least significant difference (LSD) procedure was used.
obtained with corn phytate (P-8810, Sigma) solutions of known concentrations. Damaged and total starch content of flour or bran were estimated using Megazyme kits (Megazyme International Ireland Ltd., Ireland) according to approved AACC methods 76-31 and 76-13, respectively (AACC, 2000).
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3. Results and discussion 3.1. Milling behavior after ozone treatment of wheat grains Comparisons of the milling behavior obtained after the first two breaking stages with four distinct wheat grain samples either untreated or treated with ozone were summarized in Table 1. Results showed that flour yield was not affected by the ozone treatment whatever the amount of ozone consumption by grains, or the type of wheat sample. These results are in accordance with previous data obtained by Ibanoglu (2001), even if distinct concentrations of ozone and milling conditions were applied. Furthermore, a significant decrease (10–20%) in the energy required for grain breaking was observed for all the samples with the highest amount (10 g/kg) of applied ozone. This effect was also noticed at the lower ozone dose and appears to depend on the wheat cultivar but not on wheat hardness. Consequently, the K0 index which expresses the energy required to obtain 1 kg of flour was reduced for ozone treated grains. It is interesting to note that following UV irradiation of wheat grains (T. durum in this case) which is also considered as an oxidative treatment, a same order decrease in the breaking energy was observed at milling; however, in that case UV effect was partly attributed to drying effect that did not occur in Oxygreens conditions (Peyron et al., 2002). However, ozone treatment in the Oxygreens reactor led to slight level of grain mass loss even if it was generally less than 1–2%. Furthermore, time to reach the desired ozone amount in the reactor is not negligible and could contribute to this mass loss due to contact between grains or the reactor grain mixing device (see Materials and Methods). Therefore, an untreated control staying the same time in the reactor as the ozone treated sample was tested in order to better analyze the ozone effect. In these conditions, ozone (10 g/kg) effect on each step of the milling process was studied with one of the wheat grain samples (Camp Remy, 2005) and summarized in Table 2. Comparison between the results obtained from untreated or ozone-treated wheat grains already confirms the significant energy saving for a comparable flour yield at breaking stage. Analysis of the other milling steps (sizing and reduction) also pointed out the decrease in the energy required for an identical level of flour production. A significant reduction in the coarse bran yield (around 30%) was also observed following ozone wheat grain treatment which seems balanced by the increase in white shorts yield. Therefore, ozone could be suggested to enhance bran friability or the ability to separate bran from the starchy endosperm.
3.2. Biochemical characterization of the milling fractions In order to further analyze the milling products, characterization of the main biochemical compounds was
undertaken in the fractions obtained from either untreated or ozone treated wheat grains (Table 3). Ash and phytic acid content was found to slightly increase in break flours concomitantly with a significant decrease of the ash content in coarse bran and a decrease of the phytic acid content in white shorts. As these biochemical compounds could be considered as efficient markers of wheat grain outer layers (Greffeuille et al., 2005), this flour enrichment could either be linked to a greater reduction in size of the outer layers at milling or a distinct tissue separation between the starchy endosperm and peripheral tissues. Total starch measurement in brans and shorts obtained from untreated or ozone-treated wheat grains showed that all of the fractions except fine brans contain a higher starch concentration following ozone treatment which appears in accordance with the above hypothesis. Moreover, starch damage was found significantly reduced (around 10%) in sizing and reduction flours that could be linked to the decrease in required milling energy in these steps. Protein content of the different flours was, however, not affected by the ozone treatment and found around 10–12%. But, detailed study of the protein size distribution by SE-HPLC after extraction with sodium phosphate buffer (pH 6.9) containing 1% SDS according to Morel et al. (2000) reveals an increase in the non-soluble fraction (Fi) in milling flours obtained from the ozone treated grains. These changes were found to correlate with a corresponding significant decrease in the soluble glutenin fractions (F1 and F2) whereas the other protein fractions were unchanged. Therefore, ozone treatment in these conditions was shown to lead to an increase in glutenin insolubility. This event probably occurs through disulfide bonds due to oxidation of the cysteine residues or other potential covalent links through oxidation of the aromatic amino acids already demonstrated to occur following ozone exposition (Cataldo, 2003). As quality and value of flours are related to the amount of glutenin insoluble fraction (Don et al., 2006; Mac Ritchie, 1999), ozone treatment could be a way to modulate the flour properties. Changes in the flour properties (i.e. dough strengthening) obtained from grains treated with ozone was indeed already observed (Coste et al., 2005). Furthermore, reduction of the level of starch damage in flours from the ozone treated grains may influence the potential starch enzymatic degradation and water absorption during dough making (Dexter et al., 1994; Mariotti et al., 2006). 3.3. Outer layers and starchy endosperm mechanical properties In order to better understand the decrease in energy required at each milling step and the changes in biochemical composition of the obtained fractions, mechanical properties of the outer layers as well as of the endosperm were explored and results summarized in Tables 4 and 5, respectively.
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Table 3 Biochemical characterization of the milling fractions obtained from Camp Remy untreated (control) or ozone treated grains (10 g/kg) Hard wheat Camp Remy (2005) Brans
Shorts
Flours
Coarse
Fine
Brown
White
Break 1
Break 2
Sizing
Reduction
Ash(1) (%/d.m.) Phytic acid(2) (mg/g d.m.) Total starch(3) (%/d.m.) Starch damage(4) (%/d.m.)
Control +ozone Control +ozone Control +ozone Control +ozone
5.10a 4.68b 43.0a 42.5a 29.2a 30.0b nd nd
5.36a 5.41a 42.9a 44.7a 22.5a 21.7a nd nd
4.90a 5.08a 38.1a 38.5a 21.4a 23.1b nd nd
2.42a 2.17a 16.3a 13.4b 56.0a 57.7b nd nd
0.57a 0.62b 1.8a 2.7b nd nd 3.7a 3.8a
0.52a 0.54a 1.6a 2.0b nd nd 4.1a 4.0a
0.55a 0.58a 1.9a 2.1a nd nd 5.9a 5.2b
0.51a 0.52a 1.6a 1.2a nd nd 4.3a 3.8b
Total proteins(5) (%/d.m.) Insoluble proteins(6) (%/total proteins) Soluble glutenins(7) (%/total proteins) Other soluble proteins(8) (%/total proteins)
Control +ozone Control +ozone Control +ozone Control +ozone
nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd
11.2a 11.0a 17.2a 23.6b 24.8a 19.1b 58.0a 57.2a
12.1a 12.0a 20.8a 26.0b 24.0a 19.4b 55.2a 54.6a
11.1a 11.1a 16.2a 21.3b 25.6a 21.0b 58.2a 57.7a
10.8a 10.7a 20.7a 26.9b 23.8a 19.5b 55.5a 53.6a
Distinct letters for an identical parameter and the same milling fraction indicate significant differences between mean values with Po0.05 taking into account coefficients of variation of the considered parameter: (1), (2), (5), (6), (7), (8)o2%, (3), (4)o5%. Insoluble proteins are determined in Fi. Soluble glutenins are HMW and LMW glutenins; other proteins: a, b, g, o gliadins, albumins and globulins. nd, not determined. Ash and phytic acid content of each of the fractions, total starch content of brans and shorts, percent of starch damage and total protein content into flours and changes in distribution between glutenins and the other protein classes extracted from flours and analyzed by SE-HPLC. Values are means with n ¼ 2 or 4 (starch measurements).
Table 4 Mechanical properties of the outer layers isolated according to radial orientation from untreated (control) or ozone treated (10 g/kg) wheat grains (Camp Remy cultivar) and measured using traction tests
Cam Remy Outer layer without pericarp Aleurone layer
Control +ozone Control +ozone
fela (N/mm)
fmax (N/mm)
eelas (%)
emax (%)
E0 (N/mm)
Wtot/102 (N/mm)
0.970.1a 1.070.2a 0.370.1a 0.670.1b
1.770.1a 1.870.4a 0.770.1a 0.970.1a
3.170.9a 3.471.0a 2.170.4a 1.970.1a
18.972.6a 17.270.9a 19.074.0a 10.772.5b
34.9711.9a 30.8713.1a 19.775.7a 33.079.3b
24.372.3a 22.075.5a 10.372.3a 10.771.4a
Distinct letters for an identical parameter and the same type of tissue indicate distinct groups determined by ANOVA multiple range test with Po0.01. Mechanical characteristics are lineic strength and strain to elastic deformation (felast, eelast), lineic strength and strain to rupture (fmax, emax), tensile modulus (E0 ), and total energy to rupture (Wtot). Values are means and standard deviations with n ¼ 8.
Table 5 Mechanical properties of grain slices obtained by abrasion from untreated (control) or ozone (10 g/kg) treated Camp Remy grains and tested by penetrometry assays
Control +ozone
fmax accepted (N)
Dmax (mm)
Vcone print (103 mm3)
[Vcone print/fmax] (104 mm3/N)
12.6471.72a 8.2370.78b
0.3070.07a 0.1770.04b
2.0370.02a 0.3770.00b
1.6070.34a 0.4470.03b
Distinct letters for an identical parameter indicate distinct groups determined by ANOVA multiple range test with Po0.05. Values are means and standard deviations with n ¼ 15.
To avoid possible heterogeneity between individual wheat grains as the Oxygreens treatment could lead to slight grain mass loss, mechanical properties of the handisolated outer layers from untreated and ozone treated
grains after removal of the outer pericarp or the aleurone layers were analyzed in traction tests (Table 4). All analyzed tissues exhibited the typical elasto-plastic rheological behavior characterized by a biphasic strain/
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lineic strength curve as previously reported for corresponding tissues from cultivated common wheat cultivars (Antoine et al., 2003; Greffeuille et al., 2006b). Significant differences were pointed out between mechanical properties of the aleurone layer isolated from ozone-treated grains compared to untreated grains but could not be detected in the outer layers from which only the outer pericarp was removed. An apparent reduction of the aleurone layer extensibility and an increase in tissue rigidity were observed as shown by the decrease of maximum strain to rupture and the increase of tensile modulus, respectively. These changes in mechanical properties appear in accordance with the significant increase of the phytic acid content in break flours pointed out in Table 3. Indeed, measurement of a lower strain to rupture for outer layers isolated from distinct wheat cultivars was already found correlated with a higher amount of phytic acid in break flours obtained after milling (Greffeuille et al., 2006b). Thus, ozone treatment of wheat grains appears to particularly affect the mechanical behavior of the aleurone layer. However, as the aleurone layer rupture could also be influenced by the overall breakage pattern of the endosperm and as the required milling energy was reduced at each step, endosperm mechanical measurements were also undertaken on wheat grain slices by penetrometry assays (Table 5). Ozone-treated grains were found to allow an applied maximum strength (fmax) until rupture around 8 N whereas non-treated samples were found to break at around 12 N. Furthermore, maximum displacement (Dmax) associated with rupture was also found to be reduced in ozone-treated grains by about twofold which results in a significant reduction of the sample volume (V) subjected to the cone penetration. Thus, data clearly pointed out the lesser resistance of the endosperm samples obtained from ozonetreated grains as calculated ratio (V/fmax) show that sample volume subjected to the cone penetration was reduced by more than threefold for an equal strength of 1 N. This changes in mechanical behavior could be explained by an increase of the endosperm friability. Reduction of the required energy observed for milling could thus be related to changes in the wheat grain tissue mechanical properties. Indeed, ozone treatment was found to affect clearly the aleurone layer extensibility and also mechanical resistance of the endosperm, probably of the sub-aleuronic part. Therefore, in addition to a potential role on bacteria and fungi inactivation, ozone treatment (10 g/kg) appears to allow gain in milling energy. Furthermore, the ozone treatment was found to lead to interesting biochemical changes in flours, i.e. starch damage reduction, aleurone content enrichment, increase of insoluble glutenin polymer, that could either be due to tissue mechanical properties’ changes or direct effect of ozone. More detailed study of the ozone effect on wheat grain tissue and targeted molecules susceptible to oxidation thus appears interesting to investigate in order to analyze their potential relationship with the observed changes in tissue mechanical behavior and consequences on milling fraction properties.
Acknowledgments We gratefully thank both INRA (CEPIA department) and the Languedoc-Roussillon region for the PhD financial support of C. Desvignes. The authors also acknowledge A.G. Despres (Goe¨mar, Saint Malo) and G. Maraval (INRA, Montpellier) for their help for ozone treatment and wheat grain milling respectively. We also thank C. Olive´ (SupAgro, Montpellier), S. Volle (Goe¨mar grant) and J. Bonicel (INRA, Montpellier) for their skilful technical assistance. References AACC, 2000. American Association of Cereal Chemists Approved Methods, 10th ed. The Association, St Paul, MN. Abecassis, J., 1993. Nouvelles possibilite´s d’appre´cier la valeur meunie`re et la valeur semoulie`re des ble´s. Industrie des Ce´re´ales 81, 25–37. Antoine, C., Peyron, S., Mabille, F., Lapierre, C., Bouchet, B., Abecassis, J., Rouau, X., 2003. Individual contribution of grain outer layers and their cell wall structure to the mechanical properties of wheat bran. Journal of Agricultural and Food Chemistry 51, 2026–2033. Cataldo, F., 2003. On the action of ozone on proteins. Polymer Degradation and Stability 82, 105–114. Coste, C., Bailli, A., Dubois, M., 2005. Method for ozone treatment of plant materials. French Patent FR 2845015 (A1). Dexter, J.E., Preston, K.R., Martin, D.G., Gender, E.J., 1994. The effects of protein content and starch damage on the physical dough properties and bread making quality of Canadian durum wheat. Journal of Cereal Science 20, 139–151. Don, C., Mann, G., Bekes, F., Hamer, R.J., 2006. HMW-GS affect the properties of glutenin particles in GMP and thus flour quality. Journal of Cereal Science 44, 127–136. Dubois, M., Coste, C., Despres, AG., Efstathiou, T., Nio, C., Dumont, E., Parent-Massin, D., 2006. Safety of Oxygreens, an ozone treatment on wheat grains. Part 2. Is there a substantial equivalence between Oxygreen-treated wheat grains and untreated wheat grains? Food Additives and Contaminants 23, 1–15. Gaou, I., Dubois, M., Pfohl-Leszkowicz, A., Coste, C., De Jouffrey, S., ParentMassin, D., 2005. Safety of Oxygreens, an ozone treatment on wheat grains. Part 1—a four week toxicity study in rats by dietary administration of treated wheat. Food Additives and Contaminants 22, 1113–1119. Graham, D.M., 1997. Use of ozone for food processing. Food Technology 51, 72–75. Greffeuille, V., Abecassis, J., Bar L’Helgouac’h, C., Lullien-Pellerin, V., 2005. Differences in the aleurone layer fate between hard and soft common wheats at grain milling. Cereal Chemistry 82, 138–143. Greffeuille, V., Abecassis, J., Rousset, M., Oury, F.X., Faye, A., Bar L’Helgouac’h, C., Lullien-Pellerin, V., 2006a. Grain characterization and milling behavior of near-isogenic lines differing by hardness. Theoretical Applied Genetics 114, 1–12. Greffeuille, V., Abecassis, J., Lapierre, C., Lullien-Pellerin, V., 2006b. Bran size distribution at milling and mechanical and biochemical characterization of common wheat grain outer layers: a relationship assessment. Cereal Chemistry 83, 641–646. Ibanoglu, S., 2001. Influence of tempering with ozonated water on the selected properties of wheat flour. Journal of Food Engineering 48, 345–350. Ibanoglu, S., 2002. Wheat washing with ozonated water: effects on selected flour properties. International Journal of Food Science and Technology 37, 579–584. Khadre, M.A., Yousef, A.E., Kim, J.G., 2001. Microbiological aspects of ozone applications in food: a review. Journal of Food Science 66, 1242–1252. Kim, J.G., Yousef, A.E., Dave, S., 1999. Application of ozone for enhancing the microbiological safety and quality of foods: a review. Journal of Food Protection 62, 1071–1087.
ARTICLE IN PRESS C. Desvignes et al. / Journal of Cereal Science 47 (2008) 245–251 Liangji, X., 1999. Use of ozone to improve the safety of fresh fruits and vegetables. Food Technology 53, 58–61. Mac Ritchie, F., 1999. Wheat proteins: characterization and role in flour functionality. Cereal Foods World 44, 188–193. Mariotti, M., Pagani, M.A., Lucisano, M., 2006. Starch damage. An interesting index for the definition of wheat flour quality. Tecnica Molitoria 57, 397–403. Mendez, F., Maier, D.E., Mason, L.J., Woloshuk, C.P., 2003. Penetration of ozone into columns of stored grains and effects on chemical composition and processing performance. Journal of Stored Products Research 39, 33–44. Morel, M.H., Dehlon, P., Autran, J.C., Leygue, J.P., Bar-L’Helgouac’h, C., 2000. Effects of temperature, sonication time and power settings on size distribution and extractability of total flour proteins as determined by size-exclusion high performance liquid chromatography. Cereal Chemistry 77, 685–691. Peyron, S., Abecassis, J., Autran, J.C., Rouau, X., 2001. Enzymatic oxidative treatments of wheat bran layers: effects on ferulic acid
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composition and mechanical properties. Journal of Agricultural and Food Chemistry 49, 4694–4699. Peyron, S., Abecassis, J., Autran, J.C., Rouau, X., 2002. Influence of UV exposure on phenolic acid content, mechanical properties of bran, and milling behavior of durum wheat (Triticum durum Desf.). Cereal Chemistry 79, 726–731. Pujol, R., Le´tang, C., Lempereur, I., Chaurand, M., Mabille, F., Abecassis, J., 2000. Description of a micromill with instrumentation for measuring characteristics of wheat grain. Cereal Chemistry 77, 421–427. Vaintraub, I.A., Lapteva, N.A., 1988. Colorimetric determination of phytate in unpurified extracts of seeds and the products of their processing. Analytical Biochemistry 175, 227–230. Wu, J., Doan, H., Cuenca, M.A., 2006. Investigation of gaseous ozone as an anti-fungal fumigant for stored wheat. Journal of Chemical Technology and Biotechnology 81, 1288–1293. Yvin, J.C., Bailli, A., Joubert, J.M., Bertaud, O., 2001. Method and installation for making flour from ozone-treated grains. French patent FR2802390 (A1).
Journal of Cereal Science 47 (2008) 245–251 www.elsevier.com/locate/jcs
Changes in common wheat grain milling behavior and tissue mechanical properties following ozone treatment C. Desvignesa, M. Chauranda, M. Duboisb, A. Sadoudia, J. Abecassisa, V. Lullien-Pellerina, a
UMR 1208 Inge´nierie des Agropolyme`res et Technologies Emergentes, CIRAD-INRA-Montpellier SupAgro-Universite´ Montpellier II, 2 place Pierre Viala, F-34000 Montpellier, France b Goe¨mar, ZAC La Madeleine, F-35400 Saint Malo Cedex, France Received 26 December 2006; received in revised form 21 March 2007; accepted 4 April 2007
Abstract Ozone treatment (10 g/kg) of common wheat grains with a new patented process, Oxygreens, used before milling was found to significantly reduce (by 10–20%) the required energy at breaking stage whatever the grain hardness and without changes in the flour yield. Detailed study of each of the milling steps undertaken on a hard type cultivar showed that both the breaking and the reduction energy were decreased. Reduction of the coarse bran yield was also observed concomitantly with an increase in the yield of white shorts. Biochemical characterization of the milling fractions pointed out changes in technological flour properties as starch damage reduction, aleurone content enrichment and increase of insoluble glutenin polymers. Measurement of wheat grain tissue mechanical properties showed that ozone treatment leads to reduction of the aleurone layer extensibility and affects the local endosperm resistance to rupture. These data as well as the direct effect of ozone oxidation on biochemical compounds could explain the observed changes in milling energy, bran and shorts yield and flour composition. r 2007 Elsevier Ltd. All rights reserved. Keywords: Aleurone; Common wheat; Mechanical properties; Milling; Ozone
1. Introduction Ozone (O3) is a strong oxidant recognized since 1997 as a GRAS (generally recognized as safe) substance and used in a number of applications in the food industry for destruction or detoxification of chemicals or microorganisms (Graham, 1997; Kim et al., 1999; Liangji, 1999). These applications include the surface decontamination, storage and preservation of perishable foods as well as water or manufacturing equipment and packaging sterilization (Khadre et al., 2001). Abbreviations: Dmax, maximum displacement associated to rupture, E0 , tensile modulus; eela, elastic strain; emax, maximum strain to rupture, fela, lineic strength to elastic deformation; fmax, lineic strength to rupture or maximum accepted force to rupture; K0 , grinding index (required energy to produce 1 kg of flour); SE-HPLC, size-exclusion HPLC; NIRS, near infrared spectroscopy; NTP, normal temperature pressure; V, cone print volume in penetrometry assay. Corresponding author. Tel.: +33 4 99 61 31 05; fax: +33 4 99 61 30 76. E-mail address: [email protected] (V. Lullien-Pellerin). 0733-5210/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2007.04.004
Treatments of wheat grains with ozone either generated from air or pure oxygen and applied as a gas (Wu et al., 2006) in fumigation or dissolved in water in washing (Ibanoglu, 2002) or tempering steps (Ibanoglu, 2001) were already shown to be effective on bacteria or fungi inactivation. Furthermore, wheat grain or flour biochemical composition as well as flour yield or technological properties appeared to be unaffected in these conditions (Ibanoglu, 2001, 2002; Mendez et al., 2003). Recently, a new process (Oxygreens) based on ozone treatment of wheat grains after pre-moistening in a closed batch reactor was patented (Coste et al., 2005; Yvin et al., 2001). It could be easily introduced between wheat grain cleaning and milling in place of the classical tempering step and was considered as safe and approved by the French Food Safety Authority for some specific flour uses (Gaou et al., 2005). However, tempering constitutes an important step in milling as it helps the separation between the starchy endosperm and the other tissues increasing their differences in mechanical properties (Abecassis, 1993).
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Thus, physical and biochemical conditions at this step appear critical for tissue mechanical behavior and efficient tissue separation. Indeed, mechanical properties of peripheral wheat grain tissues were already found to be influenced by oxidative conditions (Peyron et al., 2001, 2002) and consequently could affect the milling behavior (Peyron et al., 2002). As an oxidative treatment, Oxygreens effect on wheat grain milling behavior has to be analyzed specifically in terms of potential tissue mechanical property changes and consequences on biochemical composition of the obtained fractions. This paper describes this study and analyzes the results in relation to the observed changes of wheat grain tissue mechanical properties.
stopped when ozone consumption reached 5 or 10 g/kg of wet grain mass. Total reaction time was between 100 and 300 min depending on the ozone consumption dose except for the soft wheat which showed shorter treatment duration (80–180 min). After ozone treatment and sorting with an air classifier (NSP classifier, Tripette et Renaud, France), grains were collected and maintained at +4 1C in a dry room for further analysis or used in milling after a 24 h resting time. A control sample for each wheat batch was also treated in the reactor but without ozone addition either 15 min (Table 1) or the same duration as the corresponding ozone treatment (Table 2). 2.3. Micromilling
2. Materials and methods 2.1. Wheat samples Common wheat (Triticum aestivum L.) grains from three distinct cultivars with distinct kernel hardness (measured by NIRS analysis, approved method 39-70A, AACC, 2000), Camp Remy and Caphorn as medium hard and Crousty as soft, were used in this study. All samples were harvested in France in 2003 or 2005 and purchased or kindly supplied by Arvalis-Institut du Ve´ge´tal (Paris) or Danone Vitapoˆle (Palaiseau). Grains were cleaned to remove impurities and stored at room temperature before use. 2.2. Wheat grain treatment with ozone using Oxygreens process The Oxygreens treatment was performed on 10 kg of grains as already described in Dubois et al. (2006). Water added, in the reactor, was equal to 4% of the initial wet grain mass. After water addition, ozone was introduced into the reactor, with pure O2 as carrier gas, at concentration of 89 g of O3/m3 NTP of O2. The consumption of O3 was measured by two BMT963 analyzers (Messtechnik, Berlin, Germany), the first one at the inlet of the reactor, the second one at the outlet of the reactor. Treatment was
Each wheat grain sample (200 g) that was already tempered by water addition in the Oxygreens reactor in order to reach 15–18% (w/w) moisture content taking into account its initial water content, as milled according to the process already described in Greffeuille et al. (2006a). Milling diagram included two breaking stages, one sizing and one reduction stage and allowed obtaining four flour fractions, coarse and fine bran fractions as well as two short fractions. The micromill was equipped with torque transducers allowing the one-line measurement of mechanical energy consumption at each stage. Energy needed to produce 1 kg of flour was calculated and is represented by the K0 index (kJ/kg of flour, Pujol et al., 2000). 2.4. Biochemical analysis 2.4.1. Moisture, protein and ash contents Moisture, protein and ash content of the grains or milling fractions were determined according to approved methods 44-19, 46-12 with Nx5.7 and 08-12 (AACC, 2000), respectively. 2.4.2. Phytic acid, total starch and damaged starch contents Phytic acid was determined at 500 nm from acidic extract of milling fractions using a colorimetric method modified by Vaintraub and Lapteva (1988). A standard curve was
Table 1 Comparison of the milling behavior at breaking stage from distinct wheat samples after only tempering with water (0 g/kg) in the reactor or treated with ozone (5 or 10 g/kg)
Ozone consumption (g/kg) Break flour yield(1) (% w/w) Total energy(2) (kJ/kg) K0 index(3) (kJ/kg flour)
Soft type
Hard type
Crousty*
Caphorn*
0 26.8a 14.8a 55.4a
5 27.0a 12.5b 46.2b
10 27.1a 13.2c 48.2c
0 20.6a 27.1a 131a
Camp Remy* 5 20.7a 27.8a 134a
10 20.3a 25.2b 124b
0 29.9a 24.8a 82.9a
5 29.7a 22.9b 77.0b
Camp Remy** 10 29.5a 22.3c 75.6c
0 23.8a 20.4a 85.8a
5 23.8a 19.0b 79.8b
10 23.9a 17.1c 71.7c
Distinct letters for an identical parameter and the same wheat sample indicate significant differences mean values with Po0.05 taking into account coefficients of variation of the considered parameter: (1)o2%, (2), (3)o5%. Ozone consumption, break flour yield and corresponding required energy were expressed relative to the wet grain mass except K0 index, which corresponds to the energy required relative to the wet flour mass obtained after two breaking steps. Wheat grains belong to distinct hardness class and were cultivated in 2003 (*) or in 2005 (**).
ARTICLE IN PRESS C. Desvignes et al. / Journal of Cereal Science 47 (2008) 245–251 Table 2 Effect of ozone treatment on the milling behavior of wheat grains from the Camp Remy cultivar Hard wheat Camp Remy (2005) Milling stages
Energy(1) (kJ/kg of grains) K0 index(2) (kJ/kg of flour)
Control +ozone Control +ozone
Break
Sizing
Reduction
20.4a 17.1b 78.1a 71.7b
39.1a 33.0b 74.5a 65.4b
22.2a 17.2b 30.4a 24.9b
18.2a 19.2a
28.4a 28.7a
Flours Flour yield(3) (% w/w)
Control +ozone
26.0a 23.9a Brans Coarse
Fraction yield (% w/w)
(4)
Control +ozone
a
7.6 5.6b
Shorts Fine a
9.3 9.7a
Brown a
4.3 4.9a
White 6.2a 8.0b
Distinct letters for an identical parameter and the same milling fraction indicate significant differences between mean values with Po0.05 taking into account coefficients of variation of the considered parameter: (3), (4) o2%, (1), (2)o5%. Milling energy and K0 index obtained from untreated (control) or ozone treated grains (10 g consumed ozone/kg of grains) at each milling stage and yield of each fraction obtained at break, sizing and reduction steps. Values were expressed relative to wet grain or flour mass.
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crossing crease on one side of the grain delimiting two flat endosperm surfaces to perform the tests. Before mechanical assay, samples were equilibrated in a temperate chamber (25 1C) under 75% relative humidity for 48 h in order to assure a 15% moisture content as controlled by measurement. Indentation tests were performed using a tractioncompression static-machine (Zwick 2.5, Zwick/Roell, Roissy, France) equipped with a conical tip (angle of point 301). Each sample was placed directly under the tip and subjected to four distinct assays at sufficient spacing between cone penetrations in order to avoid interferences between indentations due to local damage. The assay included a first step 0.2 N pre-charge of the sample at an imposed speed (0.5 mm/s) followed by an imposed displacement (0.8 mm) at a 0.02 mm/s speed until maximum force (fmax) was reached which defined the sample rupture. The data capture rate was 20 points/s. The following parameters were obtained from the strength (N)/displacement (mm) curves: maximum accepted force (fmax, N), maximum displacement until rupture (Dmax, mm). Sample volume subjected to the cone penetration ( ¼ cone print) was calculated using the equation V ¼ ðP=3Þa2 D3max with a ¼ tan (cone radius). V/fmax ratio was calculated and expresses the sample mechanical behavior for an equal strength of 1 N.
2.4.3. Characterization of proteins from flours by sizeexclusion chromatography Proteins extracted from flours with sodium phosphate buffer (pH 6.9) containing 1% sodium dodecyl sulfate (SDS) were analyzed by size exclusion high performance liquid chromatography (SE-HPLC) according to Morel et al. (2000). The chromatogram was divided into five fractions (F1–F5) that were attributed to soluble high molecular size (HMW) glutenins (F1) and low molecular size (LMW) glutenins (F2), o-gliadins (F3) or a-, b-, ggliadins (F4) and water and salt soluble proteins (F5), respectively. Insoluble proteins (Fi) were also quantified by SE-HPLC after solubilization of the insoluble protein pellet and sonication.
2.5.2. Preparation of wheat outer layers and mechanical assays Peripheral tissues and the aleurone layer were isolated by hand according to radial grain orientation, equilibrated in water content and cut as already described in Antoine et al. (2003). Uniaxial tension tests were performed using dynamic mechanical thermal analysis (DMTA Mk III, Rheometrics Inc., Piscataway, USA) at a rate of 0.05 mm/s until sample breakage. Before tension assays, the stability of the dynamic modulus was used as an indicator of the sample equilibration in temperature (30 1C) and water content (17%) after a dynamic test at imposed strain (strain ¼ 0.01%, total time ¼ 12 min, frequency ¼ 1.59 Hz). Mechanical properties of the samples were determined from obtained curves expressing strength according to strain taking into account the tissue thickness which is measured with a micrometer (Braive Instruments, Checy, France). The measured parameters were lineic strength to rupture (fmax) and lineic strength to elastic deformation (fela), maximum strain (emax) and elastic strain (eela), tensile modulus (E0 ), and total rupture energy (Wtot). Lineic strength corresponds to the strength exerted per unit of tissue width necessary to cause reversible deformation or rupture. Assays in which rupture did not occur in the middle of the strip were discarded.
2.5. Mechanical properties measurement
2.6. Statistical analysis
2.5.1. Preparation of wheat grains and mechanical assays Wheat grains were abraded longitudinally with sand-paper on the two sides of the grain. The obtained sections (3 mm thickness) preserve the peripheral tissues and present a
Variance analysis was performed using Minitab.13 software (Minitab Inc, PA, USA). To discriminate among the means, Fisher’s least significant difference (LSD) procedure was used.
obtained with corn phytate (P-8810, Sigma) solutions of known concentrations. Damaged and total starch content of flour or bran were estimated using Megazyme kits (Megazyme International Ireland Ltd., Ireland) according to approved AACC methods 76-31 and 76-13, respectively (AACC, 2000).
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3. Results and discussion 3.1. Milling behavior after ozone treatment of wheat grains Comparisons of the milling behavior obtained after the first two breaking stages with four distinct wheat grain samples either untreated or treated with ozone were summarized in Table 1. Results showed that flour yield was not affected by the ozone treatment whatever the amount of ozone consumption by grains, or the type of wheat sample. These results are in accordance with previous data obtained by Ibanoglu (2001), even if distinct concentrations of ozone and milling conditions were applied. Furthermore, a significant decrease (10–20%) in the energy required for grain breaking was observed for all the samples with the highest amount (10 g/kg) of applied ozone. This effect was also noticed at the lower ozone dose and appears to depend on the wheat cultivar but not on wheat hardness. Consequently, the K0 index which expresses the energy required to obtain 1 kg of flour was reduced for ozone treated grains. It is interesting to note that following UV irradiation of wheat grains (T. durum in this case) which is also considered as an oxidative treatment, a same order decrease in the breaking energy was observed at milling; however, in that case UV effect was partly attributed to drying effect that did not occur in Oxygreens conditions (Peyron et al., 2002). However, ozone treatment in the Oxygreens reactor led to slight level of grain mass loss even if it was generally less than 1–2%. Furthermore, time to reach the desired ozone amount in the reactor is not negligible and could contribute to this mass loss due to contact between grains or the reactor grain mixing device (see Materials and Methods). Therefore, an untreated control staying the same time in the reactor as the ozone treated sample was tested in order to better analyze the ozone effect. In these conditions, ozone (10 g/kg) effect on each step of the milling process was studied with one of the wheat grain samples (Camp Remy, 2005) and summarized in Table 2. Comparison between the results obtained from untreated or ozone-treated wheat grains already confirms the significant energy saving for a comparable flour yield at breaking stage. Analysis of the other milling steps (sizing and reduction) also pointed out the decrease in the energy required for an identical level of flour production. A significant reduction in the coarse bran yield (around 30%) was also observed following ozone wheat grain treatment which seems balanced by the increase in white shorts yield. Therefore, ozone could be suggested to enhance bran friability or the ability to separate bran from the starchy endosperm.
3.2. Biochemical characterization of the milling fractions In order to further analyze the milling products, characterization of the main biochemical compounds was
undertaken in the fractions obtained from either untreated or ozone treated wheat grains (Table 3). Ash and phytic acid content was found to slightly increase in break flours concomitantly with a significant decrease of the ash content in coarse bran and a decrease of the phytic acid content in white shorts. As these biochemical compounds could be considered as efficient markers of wheat grain outer layers (Greffeuille et al., 2005), this flour enrichment could either be linked to a greater reduction in size of the outer layers at milling or a distinct tissue separation between the starchy endosperm and peripheral tissues. Total starch measurement in brans and shorts obtained from untreated or ozone-treated wheat grains showed that all of the fractions except fine brans contain a higher starch concentration following ozone treatment which appears in accordance with the above hypothesis. Moreover, starch damage was found significantly reduced (around 10%) in sizing and reduction flours that could be linked to the decrease in required milling energy in these steps. Protein content of the different flours was, however, not affected by the ozone treatment and found around 10–12%. But, detailed study of the protein size distribution by SE-HPLC after extraction with sodium phosphate buffer (pH 6.9) containing 1% SDS according to Morel et al. (2000) reveals an increase in the non-soluble fraction (Fi) in milling flours obtained from the ozone treated grains. These changes were found to correlate with a corresponding significant decrease in the soluble glutenin fractions (F1 and F2) whereas the other protein fractions were unchanged. Therefore, ozone treatment in these conditions was shown to lead to an increase in glutenin insolubility. This event probably occurs through disulfide bonds due to oxidation of the cysteine residues or other potential covalent links through oxidation of the aromatic amino acids already demonstrated to occur following ozone exposition (Cataldo, 2003). As quality and value of flours are related to the amount of glutenin insoluble fraction (Don et al., 2006; Mac Ritchie, 1999), ozone treatment could be a way to modulate the flour properties. Changes in the flour properties (i.e. dough strengthening) obtained from grains treated with ozone was indeed already observed (Coste et al., 2005). Furthermore, reduction of the level of starch damage in flours from the ozone treated grains may influence the potential starch enzymatic degradation and water absorption during dough making (Dexter et al., 1994; Mariotti et al., 2006). 3.3. Outer layers and starchy endosperm mechanical properties In order to better understand the decrease in energy required at each milling step and the changes in biochemical composition of the obtained fractions, mechanical properties of the outer layers as well as of the endosperm were explored and results summarized in Tables 4 and 5, respectively.
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Table 3 Biochemical characterization of the milling fractions obtained from Camp Remy untreated (control) or ozone treated grains (10 g/kg) Hard wheat Camp Remy (2005) Brans
Shorts
Flours
Coarse
Fine
Brown
White
Break 1
Break 2
Sizing
Reduction
Ash(1) (%/d.m.) Phytic acid(2) (mg/g d.m.) Total starch(3) (%/d.m.) Starch damage(4) (%/d.m.)
Control +ozone Control +ozone Control +ozone Control +ozone
5.10a 4.68b 43.0a 42.5a 29.2a 30.0b nd nd
5.36a 5.41a 42.9a 44.7a 22.5a 21.7a nd nd
4.90a 5.08a 38.1a 38.5a 21.4a 23.1b nd nd
2.42a 2.17a 16.3a 13.4b 56.0a 57.7b nd nd
0.57a 0.62b 1.8a 2.7b nd nd 3.7a 3.8a
0.52a 0.54a 1.6a 2.0b nd nd 4.1a 4.0a
0.55a 0.58a 1.9a 2.1a nd nd 5.9a 5.2b
0.51a 0.52a 1.6a 1.2a nd nd 4.3a 3.8b
Total proteins(5) (%/d.m.) Insoluble proteins(6) (%/total proteins) Soluble glutenins(7) (%/total proteins) Other soluble proteins(8) (%/total proteins)
Control +ozone Control +ozone Control +ozone Control +ozone
nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd
11.2a 11.0a 17.2a 23.6b 24.8a 19.1b 58.0a 57.2a
12.1a 12.0a 20.8a 26.0b 24.0a 19.4b 55.2a 54.6a
11.1a 11.1a 16.2a 21.3b 25.6a 21.0b 58.2a 57.7a
10.8a 10.7a 20.7a 26.9b 23.8a 19.5b 55.5a 53.6a
Distinct letters for an identical parameter and the same milling fraction indicate significant differences between mean values with Po0.05 taking into account coefficients of variation of the considered parameter: (1), (2), (5), (6), (7), (8)o2%, (3), (4)o5%. Insoluble proteins are determined in Fi. Soluble glutenins are HMW and LMW glutenins; other proteins: a, b, g, o gliadins, albumins and globulins. nd, not determined. Ash and phytic acid content of each of the fractions, total starch content of brans and shorts, percent of starch damage and total protein content into flours and changes in distribution between glutenins and the other protein classes extracted from flours and analyzed by SE-HPLC. Values are means with n ¼ 2 or 4 (starch measurements).
Table 4 Mechanical properties of the outer layers isolated according to radial orientation from untreated (control) or ozone treated (10 g/kg) wheat grains (Camp Remy cultivar) and measured using traction tests
Cam Remy Outer layer without pericarp Aleurone layer
Control +ozone Control +ozone
fela (N/mm)
fmax (N/mm)
eelas (%)
emax (%)
E0 (N/mm)
Wtot/102 (N/mm)
0.970.1a 1.070.2a 0.370.1a 0.670.1b
1.770.1a 1.870.4a 0.770.1a 0.970.1a
3.170.9a 3.471.0a 2.170.4a 1.970.1a
18.972.6a 17.270.9a 19.074.0a 10.772.5b
34.9711.9a 30.8713.1a 19.775.7a 33.079.3b
24.372.3a 22.075.5a 10.372.3a 10.771.4a
Distinct letters for an identical parameter and the same type of tissue indicate distinct groups determined by ANOVA multiple range test with Po0.01. Mechanical characteristics are lineic strength and strain to elastic deformation (felast, eelast), lineic strength and strain to rupture (fmax, emax), tensile modulus (E0 ), and total energy to rupture (Wtot). Values are means and standard deviations with n ¼ 8.
Table 5 Mechanical properties of grain slices obtained by abrasion from untreated (control) or ozone (10 g/kg) treated Camp Remy grains and tested by penetrometry assays
Control +ozone
fmax accepted (N)
Dmax (mm)
Vcone print (103 mm3)
[Vcone print/fmax] (104 mm3/N)
12.6471.72a 8.2370.78b
0.3070.07a 0.1770.04b
2.0370.02a 0.3770.00b
1.6070.34a 0.4470.03b
Distinct letters for an identical parameter indicate distinct groups determined by ANOVA multiple range test with Po0.05. Values are means and standard deviations with n ¼ 15.
To avoid possible heterogeneity between individual wheat grains as the Oxygreens treatment could lead to slight grain mass loss, mechanical properties of the handisolated outer layers from untreated and ozone treated
grains after removal of the outer pericarp or the aleurone layers were analyzed in traction tests (Table 4). All analyzed tissues exhibited the typical elasto-plastic rheological behavior characterized by a biphasic strain/
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lineic strength curve as previously reported for corresponding tissues from cultivated common wheat cultivars (Antoine et al., 2003; Greffeuille et al., 2006b). Significant differences were pointed out between mechanical properties of the aleurone layer isolated from ozone-treated grains compared to untreated grains but could not be detected in the outer layers from which only the outer pericarp was removed. An apparent reduction of the aleurone layer extensibility and an increase in tissue rigidity were observed as shown by the decrease of maximum strain to rupture and the increase of tensile modulus, respectively. These changes in mechanical properties appear in accordance with the significant increase of the phytic acid content in break flours pointed out in Table 3. Indeed, measurement of a lower strain to rupture for outer layers isolated from distinct wheat cultivars was already found correlated with a higher amount of phytic acid in break flours obtained after milling (Greffeuille et al., 2006b). Thus, ozone treatment of wheat grains appears to particularly affect the mechanical behavior of the aleurone layer. However, as the aleurone layer rupture could also be influenced by the overall breakage pattern of the endosperm and as the required milling energy was reduced at each step, endosperm mechanical measurements were also undertaken on wheat grain slices by penetrometry assays (Table 5). Ozone-treated grains were found to allow an applied maximum strength (fmax) until rupture around 8 N whereas non-treated samples were found to break at around 12 N. Furthermore, maximum displacement (Dmax) associated with rupture was also found to be reduced in ozone-treated grains by about twofold which results in a significant reduction of the sample volume (V) subjected to the cone penetration. Thus, data clearly pointed out the lesser resistance of the endosperm samples obtained from ozonetreated grains as calculated ratio (V/fmax) show that sample volume subjected to the cone penetration was reduced by more than threefold for an equal strength of 1 N. This changes in mechanical behavior could be explained by an increase of the endosperm friability. Reduction of the required energy observed for milling could thus be related to changes in the wheat grain tissue mechanical properties. Indeed, ozone treatment was found to affect clearly the aleurone layer extensibility and also mechanical resistance of the endosperm, probably of the sub-aleuronic part. Therefore, in addition to a potential role on bacteria and fungi inactivation, ozone treatment (10 g/kg) appears to allow gain in milling energy. Furthermore, the ozone treatment was found to lead to interesting biochemical changes in flours, i.e. starch damage reduction, aleurone content enrichment, increase of insoluble glutenin polymer, that could either be due to tissue mechanical properties’ changes or direct effect of ozone. More detailed study of the ozone effect on wheat grain tissue and targeted molecules susceptible to oxidation thus appears interesting to investigate in order to analyze their potential relationship with the observed changes in tissue mechanical behavior and consequences on milling fraction properties.
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