Fundamental study on the effect of

hydrostatic pressure treatment

on the bread-making performance of

oat flour


Authors: K. Huttner, Fabio Dal Bello, Elke K. Arendt

Source: European Food Research and Technology, Springer Berlin / Heidelberg. №6, 2010. P. 827-835 (Springer link)

Abstract

The effect of hydrostatic pressure (HP) has been investigated on cereal starches, proteins and flour systems, but its efficacy in cereal food processing has not been estab­lished. This study describes the use of HP as a tool to improve the bread-making performance of oat flour. For this purpose, oat batters were HP-treated at 200, 350 or 500 MPa, and the microstructure was investigated using scanning electron microscopy and bright field microscopy. Furthermore, HP-treated oat batters were added to an oat-bread recipe, replacing 10, 20 or 40% of untreated oat flour. Breads analysis revealed significantly improved bread vol­ume upon addition of 10% oat batter treated at 200 MPa. The staling rate was reduced of all breads containing oat batter treated at 200 MPa. In contrast, bread quality deterio­rated due to addition of oat batters treated at pressures >350 MPa, independent of the addition level. Overall, weakening of the protein structure, moisture redistribution and possibly changed interactions between proteins and starch were responsible for the positive efects of HP-treat­ment at 200 MPa on the bread-making performance of oat lour. Protein network formation and pre-gelatinisation of starch did not improve oat-bread quality.

Keywords

Oats • Bread • Gluten-free • Hydrostatic pressure • Rheology

Introduction

Celiac disease is one of the most common lifelong disorders, affecting about 1% of the world population [1]. Consequently, the need for high-quality gluten-free foods is becoming economically more and more important. Bread, mostly made from wheat, is an important constituent of the human diet all over the world [2]. However, bread making using grains alternative to wheat is a challenging task for cereal technologists and most commercially available gluten-free breads are characterised by poor crumb and crust characteristics, poor mouthfeel and lavour, low nutritional quality and fast staling [3-5]. Thus, different technologies have been exploited to improve the bread-making performances of gluten-free lours. Overall, studies on the improvement of gluten-free bread focus mainly on compensating for the missing gluten network by adding structural agents, such as hydrocolloids [6, 7], enzymes [8-10] and/or dairy proteins [5, 11]. Structural and functional properties of proteins and starches can also be modiied through HP processing [12-14] which suggests that HP may represent a new frontier for enhancing the quality of gluten-free bread. However, scientiic studies mainly investigated the efect of HP on pure cereal starches [15-18] or cereal proteins [19-22] and little information is available concerning the efect of HP on whole lour systems.

In this study the potential of HP-treatment for the improvement of oat-bread quality was investigated since oat lour has poor bread-making properties compared to wheat lour due to the fact that oat proteins do not possess the unique visco-elastic properties distinctive for wheat gluten [23, 24]. Yet, oats are characterised by their high-nutritional quality and pleasant aroma and taste [24, 25] which makes them an ideal raw material for the production of bread in order to satisfy the consumer demand for healthy and tasty foods. In addition, recent studies have shown that the majority of celiac disease patients can tolerate oats [26-31]. However, the status of oats in the gluten-free diet has not been clear due to conlicting results of some clinical studies [32, 33]. Nonetheless, the safe inclusion of oat products into the celiac diet has been recognised in at least three countries (UK, Finland, Canada), although some precautions and recommendations were made, such as that the celiac disease patient should consult a doctor before starting a diet containing oats [34] and that the gluten level has to be below 20 mg/kg [35].

In our recent work [36] we have shown that HP-treatment can be used to improve the functionality of oat lour. HP-treatment of oat batters at >300 MPa caused gelatinisa-tion of starch and formation of urea-insoluble protein complexes as well as disulphide bonds, which resulted in higher batter elasticity. Similar results were obtained by Ahmed et al. [37] who investigated the efect of HP on basmati rice slurries and found gelatinisation of starch and modiication of protein components, as well as increased mechanical strength of the HP-treated slurries at >350 MPa. Yet, to the best of our knowledge, the efect of HP-modiied proteins and/or starch on bread quality has not been studied until now. Consequently, the objectives of this study were to investigate if the observed changes induced by HP-treatment can improve the technological performance of oats during bread making.

Experimental


Materials

Commercial endosperm oat lour consisting of the varieties Roope, Suomi and Veli (Raisio, Finland) was used for this study. The flour was characterised by: 10.8% moisture (Approved method 44-15A [38]); 1.2% ash (Approved method 08-01 [38]); 10.1% protein (Approved method 4630 [38]); 63.0% starch (Approved method 76.13 [38]); 24.5% amylose and 75.5% amylopectin (Megazyme, Bray, Ireland). For bread making, dried baker's yeast (Pante, Puratos, Groot-Bijgaarden, Belgium), salt (Salt Union, West Point, UK), sucrose (Suicra, Carlow, Ireland) and tap water were used.


HP-treatment of oat lour

Oat batters were prepared and treated as described by Hiitt-ner et al. [36]. Briely, endosperm oat lour and tap water (24 °C) were combined at a flour-water ratio of 1:0.95 (w/w) and mixed with a Kenwood mixer (Kenwood Major Classic KM 800, UK) at speed 2 for 1 min, scraped down and mixed at speed 3 for another minute. The mixtures were packed in stomacher bags (BA6041, Seward, UK), minimising the amount of air entrapped. Packed samples were put into a vacuum bag and vacuum-packed two more times to prevent contact between pressurisation luid and the oat batters. Samples were then transferred to the pressure-treatment chamber (Stansted Fluid Power ISO-Lab 900 High-pressure Food Processor, Stansted Fluid Power Ltd., Stansted, UK). The pressurisation medium used was 10% castor oil (v/v) in ethanol. After approximately 2 min the desired pressure was reached and the time course started. The samples were treated for 10 min at 20 °C under pressures of 200, 350 or 500 MPa. The temperature of the vessel of the pressure unit was thermostatically controlled at 20 °C throughout the treatment. Due to compressive heating, increases in the temperature of the processing luid by up to a maximum of 10 °C at 500 MPa were observed. Increases in the temperature of the processing luid were transient and the set temperature §1 °C was re-attained within 2 min of the start of treatment.


Microscopy

The efect of HP-treatment on the microstructure of oat batters was investigated using bright ield microscopy and scanning electron microscopy (SEM). Samples for bright ield microscopy were prepared as described by Salmen-kallio-Marttila et al. [39]. Briely, pieces of untreated oat batter and HP-treaded oat batter (0 0.5 cm) were embedded in a 2% agar solution (immediately after HP-treat-ment). After the agar solution had solidiied, the samples were ixed using 1% glutaraldehyde in 0.1 M phosphate bufer (pH 7.0), dehydrated with ethanol, embedded in Historesin (Leica, Germany) and sectioned with a Leica microtome (HM 355, Leica Instruments GmbH, Germany). For bright ield microscopy, the sections were stained using aqueous Light green solution (1 g/L; speciically stained protein appear green) for 1 min (Gurr, BDH Ltd, Poole, England) and with 1:10 diluted Lugol's iodine solution (I2, 3 g/100 g and KI 0.67 g/100 g; specifically stained amylose and amylopectin appear blue and brown, respectively). The samples were examined with an Olympus BX-50 microscope (Tokyo, Japan). Micrographs were obtained using a SensiCam CCD camera (PCO, Kelheim, Germany). SEM was also performed on freeze-dried oat batters. Fractures of samples were mounted on circular aluminium specimen holders with double carbon tape and coated with gold in a vacuum evaporator. The specimens obtained were viewed in a JEOL scanning electron microscope (type 5510, JEOL, Tokyo, Japan) at 3 to 5 kV using 10 mm working distance.


Rheological measurements

Rheological measurements were conducted on untreated oat batter, HP-treated oat batters as well as oat-bread batter containing 0, 10, 20 or 40% of the HP-treated batters. Yeast was omitted in the bread batters used for rheological measurements. Briefly, samples were placed between the plates of a controlled stress and strain rheometer (Anton Paar MCR 301, Germany) for 30 min at 30 °C to allow stress relaxation. Excess batter was removed and the exposed edges were covered with silicon oil to avoid desiccation. The rheometer consisted of a parallel plate geometry (50-mm diameter) and the gap between the two plates was set to 2 mm. During measurements sample temperature was kept constant at 30 °C by a Peltier plate system attached to a water circulation unit. In order to ensure that all measurements were carried out within the linear visco-elastic region, oscillation stress amplitude sweeps were performed in the range of 0.003 to 100% strain. Based on these results, oscillation strain of 0.01% and oscillation stress of 1 Pa were selected which were in the linear visco-elastic region of all samples. All rheological measurements were performed in triplicate.


Batter analysis for water adjustment

Frequency sweep tests were performed at angular frequencies between 0.1 and 50 (1/s) with a target strain of 1 x 10-4 (0.01%) and the phase angle (8) was calculated by the manufacturer software (Rheoplus, Anton Paar, Germany). To evaluate the effect of HP-treatment on oat-bread quality the amount of water added to the recipe was adjusted in order to compensate for changes in bread batter consistency upon addition of HP-treated oat batters (Fig. 1). Hence, the changes in 8 as a consequence of the HP-treatments were calculated as percentage of 8 obtained for untreated batter (Table 1).

Depending on the amount of HP-treated oat batter added to the recipe and the applied pressure (10, 20 or 40% of untreated oat lour was replaced by oat lour HP-treated at 200, 350 or 500 MPa), the amount of water in the recipe was either decreased or increased according to the results obtained for 8 (Table 1). The bread recipes after water adjustment as well as the recipe of the control are presented in Table 2.


Bread batter analysis

Frequency sweep tests were also carried out on the bread batter and on batters with adjusted water levels, containing 10, 20 or 40% of oat batter HP-treated at 200, 350 or 500 MPa. Rheological properties, such as phase angle (8), complex modulus (G*), storage modulus (G') and loss modulus (G") were calculated by the manufacturer software.

Table 1 Phase angle (8) of the untreated oat batter (control) and oat batters treated at 200, 350 or 500 MPa, as well as the change in 8 of the HP treated oat batters compared to the control (%)
Mean values § standard errors of three replicates. Mean values followed by the same letter in the same column are not significantly different (p <0.05)



Bread making

The bread formulation consisted of 100% oat lour, 95% tap water, 1.8% salt, 1% sucrose and 2% dried baker's yeast.


Fig.1 Pictures of the control bread batter and bread batters prepared with 20% of oat batter theated at 200, 350 or 500 MPa? before the water adjustment


Table 2 Recipe for bread containing no HP-treated oat batter (control) and recipes of breads containing 10, 20 or 40% of oat batter HP-treated at 200, 350 or 500 MPa with adjusted water levels as % of each ingredient.

Depending on the addition level 10, 20 or 40% of untreated lour was replaced by HP-treated lour and the amount of water in the recipe was adjusted according to the results obtained for the phase angle of the HP-treated oat batters (Table 1). Briely, 1,040 g of dough was prepared for one replicate, resulting in two loaves. For bread production, dried baker's yeast was dissolved in a solution of water (30°) and sucrose and activated at 30 °C for 10 min. The remaining dry ingredients were placed in a Kenwood mixing bowl (Kenwood Chef, UK), combined with HP-treated oat batter (if indicated), and activated baker's yeast was added. Mixing was performed with a paddle tool (K beater) at level 2 for 30 s, followed by scraping of the bowl and additional mixing at level 4 for 1.5 min. The doughs were scaled to 450 g into baking tins (875-mL volume; 7-cm height; 9 x 15 cm top; 8 x 13 cm bottom) and proofed at 30 °C and 85% RH for 30 min in a proofer (Koma BV, Roermond, The Netherlands). Finally, baking was performed at 190 °C top and bottom heat for 45 min in a deck oven (MIWE, Arnstein, Germany). The oven was steamed before loading (0.3 L of water) and, on loading, steamed again by injecting 0.3 L of water. After baking, the loaves were depanned and cooled for 2 h on cooling racks at room temperature. The baking tests were repeated in triplicate for each bread type.


Bread evaluation

After 2 h cooling at room temperature, one loaf of each replicate was analysed. The remaining loaf was packaged as previously described [11] and further evaluation was performed after 5 days of storage. Loaf weight and volume (rapeseed displacement method) were determined. Loaf speciic volume (mL/g) and bake loss (%) were calculated. Subsequently, breads were sliced transversely using a slice regulator and bread knife to obtain uniform slices of 25-mm thickness. Slice height (cm) was measured in the middle of the 3 slices. Colour of crust and crumb (CIE L*a*b* colour system) were determined with a Chroma Meter (Minolta CR-300, Osaka, Japan). Bread moisture was determined according to the AACC method 44-15A [38]. All measurements were done in triplicate for each bread type.


Bread crumb analysis

Crumb properties were determined by TPA on three slices from the centre of each loaf after 2 h (day 0) of cooling and after 5 days of storage, using a universal testing machine TA-XT2I (Stable Microsystems, Surrey, UK) equipped with a 25 kg load cell and a 35-mm aluminium cylindrical probe. Pre-test speed, test speed and post-test speed were 2 mm/s, trigger force was 20 g, distance was 10 mm (40% compression) and wait time between irst and second compression cycle was 5 s. TPA crumb hardness was extracted from the curves and used as an indicator for staling. All measurements were repeated in triplicate for each bread type.


Statistical analysis

Results obtained were analysed using ANOVA and the software Statistica 7.1 (StatSoft, Tulsa, OK, USA). Fisher LSD (least signiicant diference) comparison tests were used to detect signiicant diferences at a conidence level of 95% (p <0.05).

Results

HP-treatment clearly afected the properties of oat batters which changed from cake batter-like (untreated oat batter and batter treated at 200 MPa) to gum-like (350 MPa) or almost solid (500 MPa) (data not shown).


Microscopy

Bright ield microscopy and SEM were used to investigate the efect of HP-treatment on the oat batter constituents (Fig. 2). Comparison of untreated and HP-treated oat batters revealed that proteins and starch were afected. However, changes were dependent on the applied pressure and became evident at >350 MPa. The majority of oat starch granules retained their granular structure but modiications in their surface appearance were visible, i.e. swelling and slight disintegration of some granules (Fig. 2). Additionally, treatment at pressures >350 MPa resulted in a more continuous distribution of proteins compared to the untreated oat batter (Fig. 2).


Rheological analysis of bread batters

Small amplitude oscillatory shear measurements within the linear visco-elastic region were used to study the efect of addition of HP-treated oat batters on the rheo-logical properties of bread batters. Hence, frequency sweeps were performed on oat-bread batters with adjusted water levels containing 0, 10, 20 or 40% of oat batter treated at 200, 350 or 500 MPa. G' of all bread batters was higher than G", indicating elastic-solid like behaviour and G* of all bread batters increased with increasing angular frequency, suggesting visco-elastic properties of the batters (data not shown). Results obtained for 8 are presented in Table 3, which show that 8 of bread batters prepared with 10 to 40% oat batter treated at 200 MPa and 10% oat batter treated at 350 MPa were not signiicantly diferent compared to the control. In contrast, 8 decreased signiicantly at addition levels >10% and pressures >350 MPa.


Fig.2 SEM (first row) and brieght field microscopy (second row: proteins are stained in green and starch in black) pictures of untreated oat batter (a) and oat batters theated at 200 (b) and 500 MPa (d)

Table 3 Bread batter and bread properties prepared without HP-treated oat batter (control) and with 10, 20 or 40% of oat batter treated at 200, 350 or 500 MPa.

bread samples
Fig.3 Bread made without HP-theated oat batter (control) and breads containing 10, 20 or 40% of oat batter theated of preassures of 200, 350 or 500 MPa


Bread analysis

The addition of HP-treated oat batters to an oat-bread recipe resulted in breads with varying quality. Overall, the addition of oat batters treated at 200 MPa resulted in good quality breads with appealing crumb structure and even gas cell distribution (Fig. 3). Bread analysis revealed a significant increase in the specific loaf volume when 10% of oat batter treated at 200 MPa was added, while no signiicant improvement was observed at higher addition levels (Table 3). Incorporation of oat batters treated at 350 or 500 MPa resulted in reduced bread quality with low spe-ciic loaf volumes and uneven gas cell distribution, independent of the addition level (Fig. 3; Table 3). Furthermore, incorporation of batters treated at 500 MPa into the dough was incomplete since pieces of HP-treated batter were visible in the bread crumb (Fig. 3). Bread moisture increased with increasing pressure due to the higher water level in the bread formulation (Table 3), while bake loss and colour of crust and crumb did not differ significantly (data not shown). Textural properties of the bread without HP-treated oat batter and breads containing diferent amounts of HP-treated oat batters were monitored over a 5-day storage period (Table 3). Crumb hardness values showed signii-cant diferences depending on bread type. After 2 h of cooling (day 0) no signiicant diferences were observed for breads containing 10 to 40% of oat batter treated at

200 MPa or 10 to 20% of oat batter treated at 350 MPa compared to the control. However, addition of 40% oat batter treated at 350 MPa or 10 to 40% treated at 500 MPa pressure resulted in breads with signiicantly higher crumb hardness. The staling rate of the oat-breads was established by measuring the increase in crumb hardness after 5 days of storage and an increase in crumb hardness was observed for all bread types. Yet, the increase in hardness and consequently the rate of staling was signiicantly lower for breads containing 10 to 40% oat batter treated at 200 MPa compared to the control. Crumb hardness of breads containing diferent amounts of oat batters treated at 350 or 500 MPa was not signiicantly diferent compared to the control

Discussion

The potential of HP-treatment for the improvement of oat-bread quality was investigated since it was shown [36] that HP-treatment can be used to modify the visco-elastic properties of oat batters due to alterations of both starch and proteins. Incorporation of oat batters treated at >350 MPa into an oat-bread recipe resulted in increased bread batter elasticity (Fig. 1). Thus, increased batter elasticity resulted in bread with low speciic loaf volume (data not shown). This is in agreement with our previous work on the bread-making performance of commercial oat lours which showed that high batter elasticity resulted in low bread volume [25]. The increase in batter elasticity was caused by gelatinisation oat starch and protein network formation due to HP-treatment at pressures >300MPa and >350MPa, respectively [36]. Consequently, the amount of water added to the oat-bread recipe was adjusted in order to obtain the same consistency for all bread batters. Rheological analysis after water adjustment revealed that 8 was the same for bread batters containing oat batter treated at 200 MPa, independent of the addition level as well as bread batter with 10% oat batter treated at 350 MPa, compared to the control. In contrast, a decrease in 8 was observed for bread batters containing more than 10% of oat batter treated at pressures > 350 MPa. These results indicate that the response of 8 was not proportional to the concentration of oat batters treated with high HP. Thus, the formation of a strong protein network and starch gelatinisation due to the HP-treatment changed the dough properties irreversibly.

Furthermore, oat-bread quality was afected by the addition of HP-treated oat batters. Overall, addition of 10% oat batter treated at 200 MPa resulted in signiicantly increased speciic loaf volume, while the increase was not signiicant at higher addition levels. The positive efects observed when adding oat batter treated at 200 MPa can be attributed to modiication of proteins [36]. HP-treatment at 200 MPa most likely resulted in a weakening of electrostatic and hydrophobic bonds, which are most sensitive to pressure [40] as well as in dissociation of protein aggregates into their subunits [19]. The findings are confirmed by the rheol-ogy results, which show an increase in 8 of oat batters treated at 200 MPa, therefore indicating a softening of the batters and weakening of proteins since oat starch is not afected at pressures <300 MPa. Commercial oat lours are routinely heat treated in order to inactivate enzymes which cause rancidity. Heat treatment most likely also inactivated proteolytic enzymes and thus, hydrolytic protein degradation due to activation of endogenous lours proteases under moderate pressure (200 MPa) most likely did not occur and therefore did not inluence the rheological properties of the HP-treated oat batters. Kiefer et al. [19] reported that gluten became more extensible and less strong after treatment with 200 MPa. When baking with oats, proteins probably interfere with starch by disrupting the uniformity of the starch gel during baking since the starch gel plays a central role in the bread architecture in the absence of a gluten network. Consequently, weakening of proteins at 200 MPa resulted in less interference with the starch gel during bread making. The modiications of the protein network might have also improved the foaming properties of oat proteins and consequently the bread-making performance. Celic et al. [41] stated that protein foaming properties are fundamental in determining the overall textural quality of sponge cake-like systems. Similar efects on oat batters are likely since they are closer in viscosity to sponge cake batters than wheat doughs. Furthermore, positive efects of protein degradation on oat-bread quality were also observed by Hiittner et al. [36] as well as Renzetti et al. [9]. In contrast, addition of oat batters treated at 350 or 500 MPa resulted in low speciic volumes and consequently poor bread quality, independent of the addition level. Thus, starch gelatinisa-tion due to HP-treatment did not improve the functionality of oat lour and the positive efects described for pre-gela-tinised starch, such as improvement of texture, increase in volume and/or enhanced shelf life of baked goods [42] were not observed. Moreover, strengthening of the protein network as a consequence of the formation of new non-covalent or disulphide bonds at pressures >350 MPa [36] negatively afected the bread-making properties of oat lour.

In addition, changes in the crumb properties were investigated over a 5-day storage period. Overall, a sig-niicant reduction in the rate of staling was observed for breads containing oat batters treated at 200 MPa, while breads prepared with oat batters treated at 350 or 500 MPa showed a staling rate similar to the control. These data indicate that a reduction of cross-linking and weakening of proteins during HP-treatment at 200 MPa resulted in restricted staling. In contrast, starch gelatini-sation and protein cross-linking [36] most likely led to the development of entanglements between starch and protein. Cross-linked starches make bread crumb harder because the granular structure of starch in the protein network is retained [43]. Moreover, interactions between starch and protein further increase in number and strength during staling [44]. The increase in hardness and staling rate of bread containing batters treated at 350 and 500 MPa can also be caused by the higher amounts of gelatinised starch. Gray and Bemiller [45] reported that the more gelatinised the starch, the higher the staling rate. Yet, the unfavourable properties of breads containing oat batters treated at 350 or 500 MPa might have been compensated to some extent by the augmented amount of water in the breads since crumb hardness was not significantly diferent compared to the control. Andreu et al. [46] reported, that high water content in bread can delay starch retrogradation. Nevertheless, breads containing oat batters treated at 200 MPa had the lowest moisture content and staled the least (Table 3). Consequently, these data suggest that HP-treatment of oat batters at 200 MPa not only weakened proteins, it also inluenced moisture distribution, and possibly interactions between proteins and starch, thus resulting in a decreased rate of staling of oat-bread. These properties are of particular importance, since endosperm oat lour was used in this study, which contains a high amount of starch and consequently stales rapidly.

Conclusions

Altogether the results of this study suggest that HP-treatment can be successfully applied for the improvement oat flour functionality and bread quality by selecting the right pressure and suitable addition levels. In view of the potential of HP-processing for the reduction of oat-bread staling this technique might also be beneficial for the production of other freshly baked gluten-free breads, which are predominantly starch based and therefore characterised by fast staling [5]. Overall, protein modification and changes in the moisture distribution positively inluenced oat-bread quality while starch gelatinisation and protein network formation had detrimental effects.

Acknowledgments

The authors would like to thank Hilde Wijngaard for the SEM pictures and Kaisa Poutanen and Laura Flander for the help with the bright ield microscope. This study was inancially supported by the European Commission in the Communities Sixth Framework Program, Project HEALTHGRAIN (FP6-514008). This publication reflects only author's views and the Community is not liable for any use that may be made of the information contained in this publication. Funding for this research was also provided by the Food Institutional Research Measure (Irish National Development Plan 2007-2013).

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