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Effect of Oxygreen® wheat ozonation process on bread dough quality and protein solubility
Journal of Cereal Science 55 (2012) 392e396
Contents lists available at SciVerse ScienceDirect
Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Effect of OxygreenÒ wheat ozonation process on bread dough quality and protein solubility F. Violleau a, *, A.-G. Pernot b, O. Surel a a b
Université de Toulouse, INPT, Ecole d’Ingénieurs de Purpan, Laboratoire d’Agro-Physiologie, UPSP/DGER 115, 75 voie du TOEC, BP 57611, F-31076 Toulouse Cedex 03, France Green Technologies, Z.A.C. La Madeleine, Avenue Général Patton, 35418 Saint Malo, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 November 2010 Received in revised form 5 October 2011 Accepted 28 January 2012
OxygreenÒ ozonation process for wheat grain is efficient to modify its technological properties. Experiments have been realized to evaluate the influence of operational conditions (humidification rate, ozone pressure and ozone concentration in the inlet flow). Dough alveographic data and protein solubility have been pursued. Ozonated samples exhibited W between 231 104 and 289 104 J. An increase of W, and P/L was observed; nevertheless, ozonation treatment had to be moderated. In fact, high ozone concentration and pressure had a negative impact on dough strength. For treatment pursued with low ozone pressure and concentration, the Un-extractable Polymeric Protein/Extractable Protein ratio (UPP/EP) was higher (from 0.45 to 0.65) than that for the control (0.49). It was lower (from 0.41 to 0.57) after a harsh treatment. Our study shows that the OxygreenÒ wheat ozone treatment leads to flours with the force and the tenacity higher, and extensibility lower than the control. Protein oxidation by ozone is the main suggested phenomenon which could explain the modification of protein solubility and technological properties of flours. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Ozone Wheat Protein Oxygreen
1. Introduction Ozone has been used for years in water treatment. This powerful oxidant has made it possible to destroy micro-organisms and organic molecules. Recognized GRAS in 1997, this product has been used more and more often in the food industry (Guzel-Seydim et al., 2004). There have been numerous application areas of ozone in the industry but research has been rarely performed using ozone on cereals and cereal-based products (Tiwari et al., 2010). Ozone has been used as a fumigant to disinfest stored products (Sousa et al., 2008) as maize (Kells et al., 2001). The effects of long exposure to a high ozone concentration (50 ppm) on grain quality for end-users have been studied (Mendez et al., 2003). Treatment of grains with 50 ppm ozone for 30 days has no detrimental effect on popping volume of popcorn, fatty acid and amino acid composition of soybean, wheat and maize milling characteristics, baking characteristics of wheat, and stickiness of rice. Ozone in the gas phase was used for wheat flour treatment. This treatment led to bread with greater specific volume and whiter Abbreviations: EP, extractable protein; UPP, un-extractable polymeric protein. * Corresponding author. Tel.: þ33 5 61 15 29 78; fax: þ33 5 61 15 30 60. E-mail address: [email protected] (F. Violleau). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2012.01.014
crumb (Sandhu et al., 2011a). An increase in insoluble polymeric protein was also observed (Sandhu et al., 2011b). Ozone was also applied on whole wheat grains. Low effect of ozonated water tempering on soft and hard wheat samples was shown (Ibanoglu, 2001). The OxygreenÒ ozone process for the treatment of grain, especially wheat, has been developed using gaseous ozone. A prehumidification stage increasing the moisture of the grains to approximately 15e17% has been realized. Grains are then directly ozonated with ozone quantities between 5 and 10 g O3/kg of grain. Ozone concentration in the carrier gas ranged between 80 and 140 g O3/m3 TPN, ozone pressure in the reactor was between 250 and 650 mbars and duration of the treatment was about 30e180 min. This tool has been very efficient for quasi-total destruction of coliform, aerobic mesophilic flora, mould and yeast on wheat grain surface. This process has also been efficient in the destruction of mycotoxin as ochratoxine A (Yvin et al., 2001). Another study (Desvignes et al., 2008) has shown the efficiency of the Oxygreen Process for hard wheat treatment. This process led to a significant decrease of (by 10e20%) the required energy at the breaking stage. The biochemical composition of the milling fractions has been modified by the treatment as starch damage reduction, aleurone content enrichment and increase of insoluble glutenin polymers.
F. Violleau et al. / Journal of Cereal Science 55 (2012) 392e396
Applied to soft wheat, the Oxygreen process led to changes in the flour properties (Coste et al., 2005). In order to better control the flours’ characteristics from ozonated grain, we investigated the influence of operational treatment parameters (Ozone concentration in the carrier gas, ozone pressure in the reactor and added water during the pre-humidification stage). Alveographic data (W and P/L) and protein polymerisation index are presented in this paper. 2. Materials and methods 2.1. Plant material and pre-treatment Wheat samples used in this study came from a blend of classical bread wheat (40% Apache, 20% Camp Rémy, 20% Soisson, 20% Cap Horn). Moisture was controlled by NIR measurement. A quantity of water ranging form 2e4% of dry weight was added to 10 kg of wheat grain. Initial moisture of wheat was 13% (0.2%) of total weight.
393
Table 2 Coded values of inlet ozone concentration, reactor pressure and added water during pre-humidification stage used for wheat treatment following a central composite design (3 factors 3 levels - 16 runs). Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Inlet ozone concentration ([O3])
Reactor pressure (PO3)
Added water (% H2O)
X1
X2
X3
1 1 1 1 1 0 0 0 0 0 0 1 1 1 1 1
1 1 0 1 1 1 0 0 0 0 1 1 1 0 1 1
1 1 0 1 1 0 1 0 0 1 0 1 1 0 1 1
2.2. Ozone treatment 10 kg of wheat were treated immediately in the OxygreenÒ process. This reactor was supplied with ozone produced by an ozone generator. During the treatment, the grain was agitated in the reactor using a central endless screw. Temperature was controlled (between 20 C and 27 C) by cooling water in a jacket. Pressure was controlled by an outlet valve limiting or not the gas flow velocity. A 5 g ozone treatment/kg of grain was applied for each experiment. Sixteen experiments with several combinations of ozone pressure in the reactor, ozone concentration in the ozone inlet and prehumidification rate during preparation of wheat were realized. Previous studies (Coste et al., 2005) identified these 3 mains factors and their levels were determined.
2.4. Milling 24 h after ozone treatment, 10 kg of wheat were milled in a Quadrumat Senior (Branbender, Germany) with no adjustment or modification of water content.
2.5. Alveographic measurements Alveographic measurements were realized following the NF ISO 5530-4 standard.
2.6. Total protein content (TPC) 2.3. Experimental design The experiment was designed and conducted according to an RSM central composite design (Table 1). A second-order design was employed using statistical software (Nemrod) and conducted with three levels: high, medium, and low, coded as 1, 0, 1 respectively. Independent variables were humidification rate, reactor pressure, and ozone concentration. This allows the generation of a secondorder multiple regression equation to simultaneously relate the dependent variables to all independent variables. The R2 values measure the goodness of fit of the resulting model to the experimental data (Yoo et al., 2009). The results were analysed using Statistica 7.1Ò at P < 0.05. Response surface analysis was used to estimate the model coefficient and perform a response surface regression (RSREG) procedure by the software. The relation between coded and uncoded values is presented in Table 1. Table 2 presents the correspondence between runs and coded levels.
Table 1 Variables coded and uncoded values relation. Coded level
1 0 1
Total protein content of each sample was measured on 30 mg freeze-dried and crushed wheat by elementary nitrogen analysis. Used method was based on the DUMAS method (AOAC 7024) using a NA 2000 analyzer (Fisons instruments, Italy).
2.7. Quantification and measurement of un-extractable polymeric protein (UPP) Ground grains (30 mg) were suspended in 3 mL of 0.1 M sodium phosphate buffer (pH 6.9), containing 2% (w/v) sodium dodecyl sulphate (SDS) and stirred for 2 h at 30 C. After centrifugation during 30 min at 15,900 g at 20 C, the supernatant (extractable fraction) was removed. The pellet (un-extractable fraction) containing mainly the polymeric protein (UPP) was lyophilised. The protein contents of UPP were determined by the DUMAS method using a NA 2000 analyzer (Fisons instruments, Italy), with a methionin standard. The extractable Protein (EP) fraction can be determined by using the formula EP ¼ TPC e UPP as well as the UPP/EP.
Variables uncoded level [O3] (g. O3/m3 TPN)
Reactor pressure (Bars)
Humidification rate (% H2O)
3. Results
80 90 100
0.25 0.45 0.65
2 3 4
Results are presented in Table 3. Some measured parameters showed that the treatment conditions used in this study modified technological qualities of flour.
394
F. Violleau et al. / Journal of Cereal Science 55 (2012) 392e396
Table 3 Results of alveographic analysis (W and P/L), protein quantification (UPP/EP). Run
Wa (10E4 J)
P/La
UPP/EPb
Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
200e230 253 273 261 258 231 270 234 289 251 237 256 260 243 254 262 232
z1 1.86 1.41 1.38 1.34 1.61 1.18 1.54 1.01 1.19 1.51 1.17 1.32 1.53 1.32 1.23 1.29
0.49 0.02 0.56 0.07 0.52 0.04 0.48 0.03 0.65 0.02 0.45 0.00 0.62 0.01 0.45 0.07 0.55 0.07 0.47 0.03 0.48 0.00 0.45 0.01 0.43 0.00 0.42 0.04 0.48 0.04 0.57 0.04 0.41 0.05
0.56c
0.58c
r a b c
1.00 Mean value of 5 experiments. Mean value of 2 experiments. Significant at 5% level.
3.1. Alveographic data Data in Table 3 indicate that all flours from ozonated wheat have W values similar or higher than that in the control W (200e230.10E4J). As shown in Fig. 1a, increase of inlet ozone concentration and reactor pressure have negative effects on W. Reactor pressure effect is higher than inlet ozone concentration. Effect of added water seems to be higher than inlet ozone concentration and reactor pressure (Fig. 1c). Higher W values were obtained for mid value of added water. Consequently, the maximum of W was obtained for the 0 level of added water and low levels of inlet ozone concentration and reactor pressure. As shown in Table 3, P/L of flours from ozonated wheat have higher values than control (P/L z 1). This parameter can be nearly doubled, to reach a 1.8 value (run 1). These first results showed that ozone treatment led to flour with higher tenacity and lower extensibility (according to alveographic data) than native wheat. 3.2. Protein analysis It can be seen on Table 3 that an ozone treatment modified the UPP/EP ratio. UPP/EP ratio ranged from 0.41 to 0.62 and the control
value was 0.49 (Table 3). As shown in Fig. 2, effect of reactor pressure is lower than inlet ozone concentration and added water. The increase of inlet ozone concentration and added water led to a decrease of the UPP/EP ratio. The effect of both was equivalent based on Fig. 2b. 3.3. Correlation between factors Table 3 presents the correlation obtained between alveographic parameters and the protein analysis. A significant positive correlation was obtained between W and UPP/EP, a negative one between P/L and UPP/EP. 4. Discussion Results of this study have permitted identification of the best parameters for flour modifications after ozonation of whole wheat grains. Authors treated wheat in columns (0.57 m dia. 3 m) and 25 L bin by ozone (Mendez et al., 2003). A 30 days treatment with 50 ppm ozone concentration was applied. In this latter study, ozone treatment does not significantly change the bread-making properties of hard wheat, including tolerance of the dough to overmixing, absorption of water, mixing time, dough weight, and proof height. Ozone treatment of maize had no effect on the dry and wet milling performances, giving yields similar to that of the control’s performance. Conditions of treatment can explain these differences. Firstly, in our procedure, a pre-humidification stage was used. A quantity of water ranging from 2 to 4%/dry wheat weight was added. Thus, during treatment, the ozone was then dissolved in the added water. The time of contact between the grain and the gas was increased. In that case, the ozone treatment could have an effect on grain constituents (protein, starch,.). This phenomenon is increased by the pressure in the reactor during the treatment. The higher the pressure was, the higher the ozone quantity was dissolved in the water. Thus more ozone could react with grain constituents. It is well known that flour bread-making quality is linked to the quality of gluten. Gliadin is responsible for the viscous properties of dough whereas glutenin contributes to dough strength. Authors showed that the amount of more insoluble glutenin polymers is linked to the wheat bread-making quality (MacRitchie, 1992; Weegels et al., 1996). This glutenin insolubility (due to high molecular weights) is due to subunits cross-linking through disulphide bonds. Several works show in-vitro polymerisation of High and Low molecular glutenin subunits with inorganic oxidizing
Fig. 1. W (104 J) isoresponse curves as a function of (a) inlet ozone concentration and reactor pressure, (b) inlet ozone concentration and added water and (c) reactor pressure and added water.
F. Violleau et al. / Journal of Cereal Science 55 (2012) 392e396
0.52
0.52 0.50 0.50
0.52
0.5 0.0
0.54
0.54
0.50
0.48
0.48 0.46
0.52
-0.5 -1.0 -1.0
0.54 0.56
0.52
b
0.50 0.48 0.46
0.54 0.52 0.50 0.48
-0.5 0.0 0.5 1.0 Inlet Ozone Concentration
1.0 0.5
0.46 0.48 0.50
0.0 0.52 -0.5 -1.0 -1.0
0.44 0.46 0.48 0.50 0.52
0.52
0.42 0.44 0.46 0.48 0.50
c 1.0 0.42 0.44
0.46 0.48 0.46
0.50 0.48 0.46
-0.5 0.0 0.5 1.0 Inlet Ozone Concentration
Added Water
1.0
Added Water
Reactor Pressure
a
395
0.5
0.48 0.46 0.44 0.50 0.52 0.50 0.52
0.48
0.00.54
0.50 0.52
0.44 0.46 0.48 0.48 0.50
0.46
0.50 0.52
-0.5 0.52 -1.0 -1.0
0.50
0.52
0.52 0.54 0.56
0.54 0.56
-0.5 0.0 0.5 Reactor Pressure
1.0
Fig. 2. UPP/EP isoresponse curves as a function of (a) inlet ozone concentration and reactor pressure, (b) inlet ozone concentration and added water and (c) reactor pressure and added water.
agents such as hydrogen peroxide, potassium bromate, potassium iodate (Veraverbeke et al., 2000a, 2000b) and molecular oxygen (Verbruggen et al., 2003). This polymerization is attributed to the oxidation of sulfhydryl groups. Author showed that ozone oxidizes cysteine in protein to form cystine disulfide bonds (Cataldo, 2003). Studies performed in solution showed that ozoneecysteine reaction is extremely rapid and that sulfhydryl groups is one of the most important biochemical targets for ozone oxidation (Kanofsky and Sima, 1995; Pryor and Uppu, 1993). The oxidation of sulfhydryl groups to disulfide bonds was advanced to explain dough strength increase (Sandhu et al., 2011a) and increase of un-extractable polymeric proteins (Sandhu et al., 2011b) of ozonated wheat flours. These studies are in good agreement with ours. Other amino acids could be oxidized by ozone. In fact, tyrosine is oxidised to dityrosine by ozone (Verweij et al., 1982). Some authors discovered dityrosine in wheat flour, dough, and bread (Tilley et al., 2001). These authors suggested that dityrosine could be a new kind of stabilizing cross-link in the wheat gluten structure in addition to disulphide bonds. Numerous authors (Hanft and Koehler, 2005; Rodriguez-Mateos et al., 2006; Takasaki et al., 2005) have worked on the quantification and on the role of dityrosine in wheat flour dough quality. Oxidation of tyrosine also leads to the formation of 3,4-dihydroxyphenylalanine (DOPA) (Cataldo, 2003). DOPA can form protein bound 5-S- cysteinyl-3,4- dihydroxyphenylalanine in gluten (Takasaki and Kawakishi, 1997). Then all the previous oxidation led to the protein polymerization and in the same time, the insolubility or size of glutenin polymers. Amino acid oxidation by ozone can also lead to protein depolymerisation. In fact, authors show that ozone can oxidise cysteine to cysteic acid (Berlett et al.,1996) and disulfide bonds to cysteanic acid. These reactions could explain the technological and protein modifications observed in our study. When the ozone leads to an increase of the dough strength, we can suppose that the disulfide bond formation is the main reaction. Ozone excess can also explain a decrease of some of the parameters. All these hypotheses will be validated in subsequent studies. The effect of ozone on other components, such as starch or lipids may be realised. For example, oxidative cross linkage of feruloylated arabinoxylans by hydrogen peroxide was demonstrated (Figueroa-Espinoza and Rouau, 1998; Labat et al., 2000; Schooneveld-Bergmans et al., 1999). Ozone action on these compounds will have to be taken into account. 5. Conclusion Our study showed that the OxygreenÒ wheat ozone treatment led to flour(s) with higher force and tenacity and lower extensibility
than the control. Accurate analysis of response surfaces and protein composition indicated that a drastic ozone treatment is not appropriate. It might be a competition between a protein polymerization/de-polymerization probably due to oxidation by ozone. These hypotheses will be investigated in subsequent studies. The effect of ozone on other components, such as starch and lipids will also be realized.
References Berlett, B.S., Levine, R.L., Stadtman, E.R., 1996. Comparison of the effects of ozone on the modification of amino acid residues in glutamine synthetase and bovine serum albumin. Journal of Biological Chemistry 271, 4177e4182. Cataldo, F., 2003. On the action of ozone on proteins. Polymer Degradation and Stability 82, 105e114. Coste, C., Bailli, A., Dubois, M., 2005. Method for ozone treatment of plant materials. French Patent FR 2845015 (A1). Desvignes, C., Chaurand, M., Dubois, M., Sadoudi, A., Abecassis, J., LullienPellerin, V., 2008. Changes in common wheat grain milling behavior and tissue mechanical properties following ozone treatment. Journal of Cereal Science 47, 245e251. Figueroa-Espinoza, M.C., Rouau, X., 1998. Oxidative cross-linking of pentosans by a fungal laccase and horseradish peroxidase: mechanism of linkage between feruloylated arabinoxylans. Cereal Chemistry 75, 259e265. Guzel-Seydim, Z.B., Greene, A.K., Seydim, A.C., 2004. Use of ozone in the food industry. Lebensmittel-Wissenschaft Und-Technologie-Food Science and Technology 37, 453e460. Hanft, F., Koehler, P., 2005. Quantitation of dityrosine in wheat flour and dough by liquid chromatography-tandem mass spectrometry. Journal of Agricultural and Food Chemistry 53, 2418e2423. Ibanoglu, S., 2001. Influence of tempering with ozonated water on the selected properties of wheat flour. Journal of Food Engineering 48, 345e350. Kanofsky, J.R., Sima, P.D., 1995. Reactive absorption of ozone by aqueous biomolecule solutions e Implications for the role of sulfhydryl compounds as targets for ozone. Archives of Biochemistry and Biophysics 316, 52e62. Kells, S.A., Mason, L.J., Maier, D.E., Woloshuk, C.P., 2001. Efficacy and fumigation characteristics of ozone in stored maize. Journal of Stored Products Research 37, 371e382. Labat, E., Morel, M.H., Rouau, X., 2000. Effects of laccase and ferulic acid on wheat flour doughs. Cereal Chemistry 77, 823e828. MacRitchie, F., 1992. Physicochemical properties of wheat proteins in relation to functionality. Advances in Food and Nutrition Research 36, 1e87. 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, 33e44. Pryor, W.A., Uppu, R.M., 1993. A kinetic-model for the competitive reactions of ozone with amino-acid-residues in proteins in reverse Micelles. Journal of Biological Chemistry 268, 3120e3126. Rodriguez-Mateos, A., Millar, S.J., Bhandari, D.G., Frazier, R.A., 2006. Formation of dityrosine cross-links during breadmaking. Journal of Agricultural and Food Chemistry 54, 2761e2766. Sandhu, H.P.S., Manthey, F.A., Simsek, S., 2011a. Quality of bread made from ozonated wheat (Triticum aestivum L.) flour. Journal of the Science of Food and Agriculture 91, 1576e1584. Sandhu, H.P.S., Manthey, F.A., Simsek, S., Ohm, J.-B., 2011b. Comparison between potassium bromate and ozone as flour oxidants in breadmaking. Cereal Chemistry 88, 103e108.
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Schooneveld-Bergmans, M.E.F., Dignum, M.J.W., Grabber, J.H., Beldman, G., Voragen, A.G.J., 1999. Studies on the oxidative cross-linking of feruloylated arabinoxylans from wheat flour and wheat bran. Carbohydrate Polymers 38, 309e317. Sousa, A.H., Faroni, L.R.D., Guedes, R.N.C., Totola, M.R., Urruchi, W.I., 2008. Ozone as a management alternative against phosphine-resistant insect pests of stored products. Journal of Stored Products Research 44, 379e385. Takasaki, S., Kato, Y., Murata, M., Homma, S., Kawakishi, S., 2005. Effects of peroxidase and hydrogen peroxide on the dityrosine formation and the mixing characteristics of wheat-flour dough. Bioscience Biotechnology and Biochemistry 69, 1686e1692. Takasaki, S., Kawakishi, S., 1997. Formation of protein-bound 3,4dihydroxyphenylalanine and 5-S-cysteinyl-3,4-dihydroxyphenylalanine as new cross-linkers in gluten. Journal of Agricultural and Food Chemistry 45, 3472e3475. Tilley, K.A., Benjamin, R.E., Bagorogoza, K.E., Okot-Kotber, B.M., Prakash, O., Kwen, H., 2001. Tyrosine cross-links: molecular basis of gluten structure and function. Journal of Agricultural and Food Chemistry 49, 2627e2632. Tiwari, B.K., Brennan, C.S., Curran, T., Gallagher, E., Cullen, P.J., Donnell, C.P., 2010. Application of ozone in grain processing. Journal of Cereal Science 51, 248e255. Veraverbeke, W.S., Larroque, O.R., Bekes, F., Delcour, J.A., 2000a. In vitro polymerization of wheat glutenin subunits with inorganic oxidizing agents. I.
Comparison of single-step and stepwise oxidations of high molecular weight glutenin subunits. Cereal Chemistry 77, 582e588. Veraverbeke, W.S., Larroque, O.R., Bekes, F., Delcour, J.A., 2000b. In vitro polymerization of wheat glutenin subunits with inorganic oxidizing agents. II. Stepwise oxidation of low molecular weight glutenin subunits and a mixture of high and low molecular weight glutenin subunits. Cereal Chemistry 77, 589e594. Verbruggen, I.M., Veraverbeke, W.S., Delcour, J.A., 2003. In vitro polymerisation of high and low molecular weight glutenin subunits with molecular oxygen. Journal of Cereal Science 37, 223e229. Verweij, H., Christianse, K., Vansteveninck, J., 1982. Ozone-induced formation of 0,0’-Dityrosine cross-links in proteins. Biochimica Et Biophysica Acta 701, 180e184. Weegels, P.L., Hamer, R.J., Schofield, J.D., 1996. Functional properties of wheat glutenin. Journal of Cereal Science 23, 1e18. Yoo, J., Lamsal, B.P., Haque, E., Faubion, J.M., 2009. Effect of enzymatic tempering of wheat Kernels on milling and baking performance. Cereal Chemistry 86, 122e126. 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).
Contents lists available at SciVerse ScienceDirect
Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Effect of OxygreenÒ wheat ozonation process on bread dough quality and protein solubility F. Violleau a, *, A.-G. Pernot b, O. Surel a a b
Université de Toulouse, INPT, Ecole d’Ingénieurs de Purpan, Laboratoire d’Agro-Physiologie, UPSP/DGER 115, 75 voie du TOEC, BP 57611, F-31076 Toulouse Cedex 03, France Green Technologies, Z.A.C. La Madeleine, Avenue Général Patton, 35418 Saint Malo, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 November 2010 Received in revised form 5 October 2011 Accepted 28 January 2012
OxygreenÒ ozonation process for wheat grain is efficient to modify its technological properties. Experiments have been realized to evaluate the influence of operational conditions (humidification rate, ozone pressure and ozone concentration in the inlet flow). Dough alveographic data and protein solubility have been pursued. Ozonated samples exhibited W between 231 104 and 289 104 J. An increase of W, and P/L was observed; nevertheless, ozonation treatment had to be moderated. In fact, high ozone concentration and pressure had a negative impact on dough strength. For treatment pursued with low ozone pressure and concentration, the Un-extractable Polymeric Protein/Extractable Protein ratio (UPP/EP) was higher (from 0.45 to 0.65) than that for the control (0.49). It was lower (from 0.41 to 0.57) after a harsh treatment. Our study shows that the OxygreenÒ wheat ozone treatment leads to flours with the force and the tenacity higher, and extensibility lower than the control. Protein oxidation by ozone is the main suggested phenomenon which could explain the modification of protein solubility and technological properties of flours. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Ozone Wheat Protein Oxygreen
1. Introduction Ozone has been used for years in water treatment. This powerful oxidant has made it possible to destroy micro-organisms and organic molecules. Recognized GRAS in 1997, this product has been used more and more often in the food industry (Guzel-Seydim et al., 2004). There have been numerous application areas of ozone in the industry but research has been rarely performed using ozone on cereals and cereal-based products (Tiwari et al., 2010). Ozone has been used as a fumigant to disinfest stored products (Sousa et al., 2008) as maize (Kells et al., 2001). The effects of long exposure to a high ozone concentration (50 ppm) on grain quality for end-users have been studied (Mendez et al., 2003). Treatment of grains with 50 ppm ozone for 30 days has no detrimental effect on popping volume of popcorn, fatty acid and amino acid composition of soybean, wheat and maize milling characteristics, baking characteristics of wheat, and stickiness of rice. Ozone in the gas phase was used for wheat flour treatment. This treatment led to bread with greater specific volume and whiter Abbreviations: EP, extractable protein; UPP, un-extractable polymeric protein. * Corresponding author. Tel.: þ33 5 61 15 29 78; fax: þ33 5 61 15 30 60. E-mail address: [email protected] (F. Violleau). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2012.01.014
crumb (Sandhu et al., 2011a). An increase in insoluble polymeric protein was also observed (Sandhu et al., 2011b). Ozone was also applied on whole wheat grains. Low effect of ozonated water tempering on soft and hard wheat samples was shown (Ibanoglu, 2001). The OxygreenÒ ozone process for the treatment of grain, especially wheat, has been developed using gaseous ozone. A prehumidification stage increasing the moisture of the grains to approximately 15e17% has been realized. Grains are then directly ozonated with ozone quantities between 5 and 10 g O3/kg of grain. Ozone concentration in the carrier gas ranged between 80 and 140 g O3/m3 TPN, ozone pressure in the reactor was between 250 and 650 mbars and duration of the treatment was about 30e180 min. This tool has been very efficient for quasi-total destruction of coliform, aerobic mesophilic flora, mould and yeast on wheat grain surface. This process has also been efficient in the destruction of mycotoxin as ochratoxine A (Yvin et al., 2001). Another study (Desvignes et al., 2008) has shown the efficiency of the Oxygreen Process for hard wheat treatment. This process led to a significant decrease of (by 10e20%) the required energy at the breaking stage. The biochemical composition of the milling fractions has been modified by the treatment as starch damage reduction, aleurone content enrichment and increase of insoluble glutenin polymers.
F. Violleau et al. / Journal of Cereal Science 55 (2012) 392e396
Applied to soft wheat, the Oxygreen process led to changes in the flour properties (Coste et al., 2005). In order to better control the flours’ characteristics from ozonated grain, we investigated the influence of operational treatment parameters (Ozone concentration in the carrier gas, ozone pressure in the reactor and added water during the pre-humidification stage). Alveographic data (W and P/L) and protein polymerisation index are presented in this paper. 2. Materials and methods 2.1. Plant material and pre-treatment Wheat samples used in this study came from a blend of classical bread wheat (40% Apache, 20% Camp Rémy, 20% Soisson, 20% Cap Horn). Moisture was controlled by NIR measurement. A quantity of water ranging form 2e4% of dry weight was added to 10 kg of wheat grain. Initial moisture of wheat was 13% (0.2%) of total weight.
393
Table 2 Coded values of inlet ozone concentration, reactor pressure and added water during pre-humidification stage used for wheat treatment following a central composite design (3 factors 3 levels - 16 runs). Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Inlet ozone concentration ([O3])
Reactor pressure (PO3)
Added water (% H2O)
X1
X2
X3
1 1 1 1 1 0 0 0 0 0 0 1 1 1 1 1
1 1 0 1 1 1 0 0 0 0 1 1 1 0 1 1
1 1 0 1 1 0 1 0 0 1 0 1 1 0 1 1
2.2. Ozone treatment 10 kg of wheat were treated immediately in the OxygreenÒ process. This reactor was supplied with ozone produced by an ozone generator. During the treatment, the grain was agitated in the reactor using a central endless screw. Temperature was controlled (between 20 C and 27 C) by cooling water in a jacket. Pressure was controlled by an outlet valve limiting or not the gas flow velocity. A 5 g ozone treatment/kg of grain was applied for each experiment. Sixteen experiments with several combinations of ozone pressure in the reactor, ozone concentration in the ozone inlet and prehumidification rate during preparation of wheat were realized. Previous studies (Coste et al., 2005) identified these 3 mains factors and their levels were determined.
2.4. Milling 24 h after ozone treatment, 10 kg of wheat were milled in a Quadrumat Senior (Branbender, Germany) with no adjustment or modification of water content.
2.5. Alveographic measurements Alveographic measurements were realized following the NF ISO 5530-4 standard.
2.6. Total protein content (TPC) 2.3. Experimental design The experiment was designed and conducted according to an RSM central composite design (Table 1). A second-order design was employed using statistical software (Nemrod) and conducted with three levels: high, medium, and low, coded as 1, 0, 1 respectively. Independent variables were humidification rate, reactor pressure, and ozone concentration. This allows the generation of a secondorder multiple regression equation to simultaneously relate the dependent variables to all independent variables. The R2 values measure the goodness of fit of the resulting model to the experimental data (Yoo et al., 2009). The results were analysed using Statistica 7.1Ò at P < 0.05. Response surface analysis was used to estimate the model coefficient and perform a response surface regression (RSREG) procedure by the software. The relation between coded and uncoded values is presented in Table 1. Table 2 presents the correspondence between runs and coded levels.
Table 1 Variables coded and uncoded values relation. Coded level
1 0 1
Total protein content of each sample was measured on 30 mg freeze-dried and crushed wheat by elementary nitrogen analysis. Used method was based on the DUMAS method (AOAC 7024) using a NA 2000 analyzer (Fisons instruments, Italy).
2.7. Quantification and measurement of un-extractable polymeric protein (UPP) Ground grains (30 mg) were suspended in 3 mL of 0.1 M sodium phosphate buffer (pH 6.9), containing 2% (w/v) sodium dodecyl sulphate (SDS) and stirred for 2 h at 30 C. After centrifugation during 30 min at 15,900 g at 20 C, the supernatant (extractable fraction) was removed. The pellet (un-extractable fraction) containing mainly the polymeric protein (UPP) was lyophilised. The protein contents of UPP were determined by the DUMAS method using a NA 2000 analyzer (Fisons instruments, Italy), with a methionin standard. The extractable Protein (EP) fraction can be determined by using the formula EP ¼ TPC e UPP as well as the UPP/EP.
Variables uncoded level [O3] (g. O3/m3 TPN)
Reactor pressure (Bars)
Humidification rate (% H2O)
3. Results
80 90 100
0.25 0.45 0.65
2 3 4
Results are presented in Table 3. Some measured parameters showed that the treatment conditions used in this study modified technological qualities of flour.
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Table 3 Results of alveographic analysis (W and P/L), protein quantification (UPP/EP). Run
Wa (10E4 J)
P/La
UPP/EPb
Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
200e230 253 273 261 258 231 270 234 289 251 237 256 260 243 254 262 232
z1 1.86 1.41 1.38 1.34 1.61 1.18 1.54 1.01 1.19 1.51 1.17 1.32 1.53 1.32 1.23 1.29
0.49 0.02 0.56 0.07 0.52 0.04 0.48 0.03 0.65 0.02 0.45 0.00 0.62 0.01 0.45 0.07 0.55 0.07 0.47 0.03 0.48 0.00 0.45 0.01 0.43 0.00 0.42 0.04 0.48 0.04 0.57 0.04 0.41 0.05
0.56c
0.58c
r a b c
1.00 Mean value of 5 experiments. Mean value of 2 experiments. Significant at 5% level.
3.1. Alveographic data Data in Table 3 indicate that all flours from ozonated wheat have W values similar or higher than that in the control W (200e230.10E4J). As shown in Fig. 1a, increase of inlet ozone concentration and reactor pressure have negative effects on W. Reactor pressure effect is higher than inlet ozone concentration. Effect of added water seems to be higher than inlet ozone concentration and reactor pressure (Fig. 1c). Higher W values were obtained for mid value of added water. Consequently, the maximum of W was obtained for the 0 level of added water and low levels of inlet ozone concentration and reactor pressure. As shown in Table 3, P/L of flours from ozonated wheat have higher values than control (P/L z 1). This parameter can be nearly doubled, to reach a 1.8 value (run 1). These first results showed that ozone treatment led to flour with higher tenacity and lower extensibility (according to alveographic data) than native wheat. 3.2. Protein analysis It can be seen on Table 3 that an ozone treatment modified the UPP/EP ratio. UPP/EP ratio ranged from 0.41 to 0.62 and the control
value was 0.49 (Table 3). As shown in Fig. 2, effect of reactor pressure is lower than inlet ozone concentration and added water. The increase of inlet ozone concentration and added water led to a decrease of the UPP/EP ratio. The effect of both was equivalent based on Fig. 2b. 3.3. Correlation between factors Table 3 presents the correlation obtained between alveographic parameters and the protein analysis. A significant positive correlation was obtained between W and UPP/EP, a negative one between P/L and UPP/EP. 4. Discussion Results of this study have permitted identification of the best parameters for flour modifications after ozonation of whole wheat grains. Authors treated wheat in columns (0.57 m dia. 3 m) and 25 L bin by ozone (Mendez et al., 2003). A 30 days treatment with 50 ppm ozone concentration was applied. In this latter study, ozone treatment does not significantly change the bread-making properties of hard wheat, including tolerance of the dough to overmixing, absorption of water, mixing time, dough weight, and proof height. Ozone treatment of maize had no effect on the dry and wet milling performances, giving yields similar to that of the control’s performance. Conditions of treatment can explain these differences. Firstly, in our procedure, a pre-humidification stage was used. A quantity of water ranging from 2 to 4%/dry wheat weight was added. Thus, during treatment, the ozone was then dissolved in the added water. The time of contact between the grain and the gas was increased. In that case, the ozone treatment could have an effect on grain constituents (protein, starch,.). This phenomenon is increased by the pressure in the reactor during the treatment. The higher the pressure was, the higher the ozone quantity was dissolved in the water. Thus more ozone could react with grain constituents. It is well known that flour bread-making quality is linked to the quality of gluten. Gliadin is responsible for the viscous properties of dough whereas glutenin contributes to dough strength. Authors showed that the amount of more insoluble glutenin polymers is linked to the wheat bread-making quality (MacRitchie, 1992; Weegels et al., 1996). This glutenin insolubility (due to high molecular weights) is due to subunits cross-linking through disulphide bonds. Several works show in-vitro polymerisation of High and Low molecular glutenin subunits with inorganic oxidizing
Fig. 1. W (104 J) isoresponse curves as a function of (a) inlet ozone concentration and reactor pressure, (b) inlet ozone concentration and added water and (c) reactor pressure and added water.
F. Violleau et al. / Journal of Cereal Science 55 (2012) 392e396
0.52
0.52 0.50 0.50
0.52
0.5 0.0
0.54
0.54
0.50
0.48
0.48 0.46
0.52
-0.5 -1.0 -1.0
0.54 0.56
0.52
b
0.50 0.48 0.46
0.54 0.52 0.50 0.48
-0.5 0.0 0.5 1.0 Inlet Ozone Concentration
1.0 0.5
0.46 0.48 0.50
0.0 0.52 -0.5 -1.0 -1.0
0.44 0.46 0.48 0.50 0.52
0.52
0.42 0.44 0.46 0.48 0.50
c 1.0 0.42 0.44
0.46 0.48 0.46
0.50 0.48 0.46
-0.5 0.0 0.5 1.0 Inlet Ozone Concentration
Added Water
1.0
Added Water
Reactor Pressure
a
395
0.5
0.48 0.46 0.44 0.50 0.52 0.50 0.52
0.48
0.00.54
0.50 0.52
0.44 0.46 0.48 0.48 0.50
0.46
0.50 0.52
-0.5 0.52 -1.0 -1.0
0.50
0.52
0.52 0.54 0.56
0.54 0.56
-0.5 0.0 0.5 Reactor Pressure
1.0
Fig. 2. UPP/EP isoresponse curves as a function of (a) inlet ozone concentration and reactor pressure, (b) inlet ozone concentration and added water and (c) reactor pressure and added water.
agents such as hydrogen peroxide, potassium bromate, potassium iodate (Veraverbeke et al., 2000a, 2000b) and molecular oxygen (Verbruggen et al., 2003). This polymerization is attributed to the oxidation of sulfhydryl groups. Author showed that ozone oxidizes cysteine in protein to form cystine disulfide bonds (Cataldo, 2003). Studies performed in solution showed that ozoneecysteine reaction is extremely rapid and that sulfhydryl groups is one of the most important biochemical targets for ozone oxidation (Kanofsky and Sima, 1995; Pryor and Uppu, 1993). The oxidation of sulfhydryl groups to disulfide bonds was advanced to explain dough strength increase (Sandhu et al., 2011a) and increase of un-extractable polymeric proteins (Sandhu et al., 2011b) of ozonated wheat flours. These studies are in good agreement with ours. Other amino acids could be oxidized by ozone. In fact, tyrosine is oxidised to dityrosine by ozone (Verweij et al., 1982). Some authors discovered dityrosine in wheat flour, dough, and bread (Tilley et al., 2001). These authors suggested that dityrosine could be a new kind of stabilizing cross-link in the wheat gluten structure in addition to disulphide bonds. Numerous authors (Hanft and Koehler, 2005; Rodriguez-Mateos et al., 2006; Takasaki et al., 2005) have worked on the quantification and on the role of dityrosine in wheat flour dough quality. Oxidation of tyrosine also leads to the formation of 3,4-dihydroxyphenylalanine (DOPA) (Cataldo, 2003). DOPA can form protein bound 5-S- cysteinyl-3,4- dihydroxyphenylalanine in gluten (Takasaki and Kawakishi, 1997). Then all the previous oxidation led to the protein polymerization and in the same time, the insolubility or size of glutenin polymers. Amino acid oxidation by ozone can also lead to protein depolymerisation. In fact, authors show that ozone can oxidise cysteine to cysteic acid (Berlett et al.,1996) and disulfide bonds to cysteanic acid. These reactions could explain the technological and protein modifications observed in our study. When the ozone leads to an increase of the dough strength, we can suppose that the disulfide bond formation is the main reaction. Ozone excess can also explain a decrease of some of the parameters. All these hypotheses will be validated in subsequent studies. The effect of ozone on other components, such as starch or lipids may be realised. For example, oxidative cross linkage of feruloylated arabinoxylans by hydrogen peroxide was demonstrated (Figueroa-Espinoza and Rouau, 1998; Labat et al., 2000; Schooneveld-Bergmans et al., 1999). Ozone action on these compounds will have to be taken into account. 5. Conclusion Our study showed that the OxygreenÒ wheat ozone treatment led to flour(s) with higher force and tenacity and lower extensibility
than the control. Accurate analysis of response surfaces and protein composition indicated that a drastic ozone treatment is not appropriate. It might be a competition between a protein polymerization/de-polymerization probably due to oxidation by ozone. These hypotheses will be investigated in subsequent studies. The effect of ozone on other components, such as starch and lipids will also be realized.
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