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Grain of high digestible, high lysine (HDHL) sorghum contains kafirins which enhance the protein network of composite dough and bread
Journal of Cereal Science 56 (2012) 352e357
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Grain of high digestible, high lysine (HDHL) sorghum contains kafirins which enhance the protein network of composite dough and bread Morgan A. Goodall a, Osvaldo H. Campanella b, Gebisa Ejeta c, Bruce R. Hamaker a, * a
Department of Food Science, Purdue University, 745 Agricultural Mall Drive, West Lafayette, IN 47907, USA Department of Agricultural and Biological Engineering, Purdue University, 745 Agricultural Mall Drive, West Lafayette, IN 47907, USA c Department of Agronomy, Purdue University, 915 West State Street, West Lafayette, IN 47907, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2011 Received in revised form 27 March 2012 Accepted 3 April 2012
The aim of this study was to determine whether protein body-free kafirins in high digestibility, highlysine (HDHL) sorghum flour can participate as viscoelastic proteins in sorghum-wheat composite dough and bread. Dough extensibility tests revealed that maximum resistance to extension (g) and time to dough breakage (sec) at 35 C for HDHL sorghum-wheat composite doughs were substantially greater (p < 0.01) than for normal sorghum-wheat composite doughs at 30 and 60% substitution levels. Functional changes in HDHL kafirin occurred upon exceeding its Tg. Normal sorghum showed a clear decrease in strain hardening at 60% substitution, whereas HDHL sorghum maintained a level similar to wheat dough. Significantly higher loaf volumes resulted for HDHL sorghum-wheat composites compared to normal sorghum-wheat composites at substitution levels above 30% and up to 56%, with the largest difference at 42%. HDHL sorghum-wheat composite bread exhibited lower hardness values, lower compressibility and higher springiness than normal sorghum-wheat composite bread. Finally, HDHL sorghum flour mixed with 18% vital wheat gluten produced viscoelastic dough while normal sorghum did not. These results clearly show that kafirin in HDHL sorghum flour contributes to the formation of an improved protein network with viscoelastic properties that leads to better quality composite doughs and breads. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Sorghum Kafirins Viscoelastic Bread making
1. Introduction Due to adverse growing conditions and high import prices, developing countries in Africa do not have good access to wheat, the primary ingredient in bread, which is consumed in most urban areas. To reduce cost, much work over the past decades has focused on composite flour technologies that replace wheat with flours made from locally grown grains and tubers. Low incorporation of alternative flours has generally been achieved as they have no functional components to aid in bread making. Sorghum is one of the grains that has been used and has certain advantages in that high quality sorghum flours are neutral in flavor and light in color. Sorghum is grown in the semi-arid regions of the world and is a major food crop in the West Africa Sahel (Burkina Faso, Mali, Niger, northern Nigeria, Senegal) and the Horn of Africa (Ethiopia, Kenya, Sudan, Uganda).
Abbreviation: High digestibility, High-lysine, HDHL. * Corresponding author. Tel.: þ1 765 494 5668; fax: þ1 765 494 7953. E-mail address: [email protected] (B.R. Hamaker). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2012.04.001
Research on partial substitution of wheat flour with sorghum has shown that it can typically be mixed with wheat at levels of 15e20% to obtain acceptable bread products (Dendy, 1970; Kim and De Ruiter, 1968; Schober et al., 2004). Higher substitution levels have a negative impact on dough rheology and decrease loaf volume. Recent research has shown that somewhat higher incorporation sorghum-wheat composite breads can be achieved by formulation changes including addition of malt and addition of gluten (Carson and Sun, 2000a; Hugo et al., 2000). Gluten-free batter type bread has been made from sorghum flour using a sourdough fermentation with hydroxypropyl methyl cellulose (Schober et al., 2007). These methods have, in some cases, allowed for incorporation of sorghum at levels near 30e40%, however issues of poor loaf volume, and poor texture and taste are still a problem. While it is generally considered that the sorghum grain storage proteins, the kafirins, lack good gluten-like properties, their functional role in bread making is also confounded by their encapsulation in rigid protein bodies that do not break apart with dough mixing, thus making them inaccessible for any possible functional role. Wheat gluten has the unique ability to form viscoelastic doughs, which trap gas during yeast fermentation and produce leavened
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bread. The development of proteins during wheat grain filling results in a continuous protein matrix within the endosperm of which the proteins are free and available to interact. The functionality of wheat proteins to produce gluten with good rheological properties is attributed to its polymer mobility, amino acid composition (high Gln, high Pro, low basic amino acids), changes in secondary structure on mixing, and disulfide-sulfhydryl interchange occurring between cysteine/cystine groups (Erickson et al., in press; Hoseney et al., 1986; Wellner et al., 2005). Gluten has low charge density that, when coupled with hydrogen bonding and hydrophobic interactions, allows for close associations between gluten polymers and provides a stabilizing force during dough development. Sorghum kafirin comparably has a higher proportion of helical conformations and a greater number of hydrophobic residues (Belton et al., 2006). It also lacks the high molecular weight glutenin proteins, which are considered as the dough component that provides suitable viscoelastic properties to developed dough (Fevzioglu et al., in press). Studies on sorghum kafirin have shown it to be analogous in many ways to maize zein. Both proteins are encapsulated in protein bodies within the endosperm and have similar chemical composition and properties (Belton et al., 2006). Kafirins and zeins are further classified by their a-, b-, and g- subunits (Lending et al., 1988; Shull et al., 1991). Internal structure of the protein body shows the main a-kafirin/zein resides at its interior, while b- and gkafirins/zeins are mainly found at its periphery. One area in which kafirin and zein differ is in their cross-linking behavior, specifically in relation to digestibility (Hamaker et al., 1987). Kafirins are considerably less digestible than other cereal proteins if consumed equally as porridges. Sorghum protein had 46% apparent protein digestibility in young children, compared to maize and wheat proteins at 73% and 81%, respectively (Maclean et al., 1981). The causal factor is an extensive cross-linking of b- and g-kafirins at the protein body periphery which decreases the ability of digestive enzymes to break down the protein body (Oria et al., 1995). This negative nutritional implication of sorghum led to the use of reducing agents, processing changes, and selective breeding programs for the development of sorghum with increased digestibility (Hamaker et al., 1987; Maclean et al., 1983; Tesso et al., 2008). A high digestibility sorghum genotype was identified in already existing high-lysine sorghum germplasm at Purdue University that has alterations in the protein body shape which result in accessible a-kafirin protein (Ejeta et al., 1987; Oria et al., 2000; Weaver et al., 1998). In the high digestibility, high lysine (HDHL) sorghum, the normal spherical protein body shape is changed to altered folded morphology due to a shift in location of g-kafirin from the periphery to the interior of the body (Oria et al., 2000). Most of the major easy-to-digest a-kafirin protein is thus exposed and results in digestibility improvement. At room temperature, gluten forms a viscoelastic protein network upon addition of water and mixing. The protein undergoes a glass transition whereby amorphous proteins change their physical state from a glassy to rubbery state, and is dependent upon the glass transition temperature, Tg, and associated processing temperature, as well as the amount of plasticizer (water) present in the mixture. Thus, exceeding the Tg of the protein is an important requirement for mixing of dough. Gluten with 16% or higher moisture content has a Tg below room temperature (Hoseney et al., 1986). On the other hand, zein is reported as having a Tg above room temperature at about 28 C with 20% or higher moisture content. Kafirin is believed to have a similar Tg property (Lawton, 1992; Oom et al., 2008). Accordingly, isolated (protein-body free) zein and kafirin can be mobilized at temperatures above their Tg and have been shown to participate in viscoelastic dough development (Bugusu et al., 2001; Lawton, 1992; Oom et al., 2008; Schober et al.,
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2008). Up to this point, functionality of kafirin or zein in composite doughs has only been achieved using isolated protein, because in flours they are naturally contained within protein bodies. The increased availability of the HDHL sorghum kafirins to digestive enzymes led us to believe that kafirins from this flour may also be able to participate in the development of viscoelastic protein networks in dough. If so, then HDHL sorghum grain may offer the potential for high incorporation in composite wheat flour leavened products through functioning of the protein. Moreover, such sorghum varieties adapted to the environments of Africa would permit farmers to produce sorghum with improved function in composite flours that could be sold into urban markets and reduce reliance on imported wheat. 2. Materials and methods 2.1. Sorghum flour Normal sorghum, P721N, and HDHL sorghum (cv. PHD-024789), grains were decorticated to remove approximately 10% of their weight, were pin milled (Alpine American Corp., Natick, MA), and then passed through the reduction roll of a roller mill. To separate the remaining large particles, the flour was passed through a 54GG sieve. 2.2. Composite dough and bread preparation Sorghum flour was combined with wheat flour (Gold Medal Bread Flour, Minneapolis, MN) in the following proportions just before mixing: 30% and 60% sorghum (flour weight basis) for dough rheology testing, 20e60% sorghum at 2% increments for bread volume testing, and 30% and 42% sorghum for the bread compression test. Dough samples were created using the same ingredients: 2% yeast (Fleischmann’s Active Dry Yeast, San Francisco, CA), 2% salt (Morton International, Inc., Chicago, IL), and 67 2% water, depending on flour moisture content, except for doughs with added vital wheat gluten. Tests with gluten addition were made with 100% normal and HDHL sorghum flours, 18% vital wheat gluten (Sigma Aldrich, St. Louis, MO) and 67% water. Soybean oil (2%) was added to dough intended for baking. Ingredients were kept at room temperature, except for the water which was kept at 35 C for Tg related tests where mixing was done in a 35 C temperature-controlled room. All doughs were mixed using a pin-type mixer (National Mfg. Co., Lincoln, NE) with 10, 35, and 100 g capacities. Mixing times were obtained for each formula change using a mixograph (National Mfg. Co., Lincoln, NE). After mixing, doughs were either used for rheological testing or molded into a loaf pan for baking. For baking, fermentation was done in a temperature (35 C) and humidity (85% RH) controlled proofing cabinet (InterMetro Industries Co., Wilkes-Barre, PA) for 45e60 min, whereupon the loaf was baked in a rotary electric oven (National Mfg. Co., Lincoln, NE) at 218 C for 30 min. 2.3. Dough rheology Dough extensibility was tested using the TA.HDplus Texture Analyzer (Stable Micro Systems Ltd., Surrey, UK) at room temperature and in a 35 C temperature-controlled room. A sheet deforming device was used consisting of two plates containing a 5.5 mm diameter circular aperture in the middle, similar to that used by Bugusu et al. (2001). Dough (35 g) was mixed, sheeted and placed between the set of plates. A 31.75 mm diameter cylindrical probe was used to deform the dough sample in a planar extensional direction at a speed of 3 mm/s. Force (g) was measured over time (sec), and the area (m2) under the curve and peak height (g) was
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Table 1 Dough extensibility measurements for composite flours containing normal and HDHL sorghum.a,b Sample
Area (m2) ratio at RT
Area (m2) ratio at 35 C
Peak (g) ratio at RT
Peak (g) ratio at 35 C
Time (sec) to breakage at RT
Time (sec) to breakage at 35 C
100% Wheat 30% HDHL Sorghum 30% Normal Sorghum 60% HDHL Sorghum 60% Normal Sorghum
1 0.42aA 0.30b 0.23c 0.14d
1 0.56aB 0.33b 0.23c 0.10d
1 0.74aA 0.57b 0.80cA 0.42dA
1 0.65aB 0.57b 0.54bB 0.37cB
13.78 0.22aA 8.12 0.06bA 8.29 0.14b 4.41 0.06cA 4.77 0.14c
14.73 0.09aB 12.54 0.22bB 8.65 0.06c 6.34 0.08dB 4.79 0.19e
a Means followed by different lowercase letters within the same column are significantly different using One Way ANOVA, p < 0.01. Only means followed by different uppercase bolded letters are significantly different between temperatures RT and 35 C of that specific two sample set. b Room temperature is abbreviated as RT.
normalized using values of wheat flour dough measured by the same rheological variables. The time to dough breakage was determined based on the observation that dough breakage occurs after the maximum resistance, approximately at one half the peak height. Force, area, and time values were recorded and statistical analysis performed. Biaxial extension was tested using the Texture Analyzer at room temperature and 35 C. This method uses a set of lubricated plates (2.54 mm diameter), one stationary on the stage and the other affixed to a probe. The probe deformed at a strain rate of 20%/sec up to 80%/sec. Force (g) was measured and converted to extensional viscosity (hext) and stress (s) following the procedure of Campanella and Peleg (2001). Strain rate was used to convert time (s) to strain (%). The linear portion of the stress versus strain plot was used to calculate regression lines and the slopes were used for statistical analysis. 2.4. Baked loaf volume test Baked loaves were allowed to cool for 1 h before measuring their mass (g) and volume (cm3). Volume was measured by the rapeseed displacement method (AACC Approved Method 10-05.01). 2.5. Bread compression Baked loaves were allowed to cool for 1 h before obtaining measurements using the Texture Analyzer. A 2 cm thick bread slice was taken from the middle of the loaf and a 3 cm diameter circle was cut from the crumb center to acquire a 2 3 cm cylinder. The bread cylinder was placed on a level stage and a 25.4 mm diameter probe was used to compress the sample with a strain rate of 10%/sec up to 80%/sec. Force (kg) was measured over time (s). The bread sample was removed after compression and measured with a caliper.
resistance dough can exert against an expanding gas bubble may be inferred by the measured peak height. High values of force are desirable because they relate to the ability of dough to expand and retain gas generated through yeast fermentation. The time to dough breakage shows how much extension or strain the dough can withstand before collapsing or tearing. The protein network developed during dough mixing must have the ability to resist expanding gas bubbles during fermentation so that a sufficient increase in volume is achieved. Area, peak, and time at 35 C for 30 and 60% HDHL sorghumwheat composite doughs were notably and significantly greater (p < 0.01) than for normal sorghum composites at both substitution levels (Table 1 and Fig. 1). This shows that the HDHL sorghumwheat composite doughs were more extensible and exerted greater extensional force for a longer time period compared to normal sorghum-wheat composite doughs. The HDHL sorghumwheat composite extensibility profiles had a similar shape to the wheat control, but these doughs were not as extensible (peak height) or of the same dough strength. As the amount of sorghum increased in the composite flours, the area, peak, and time values decreased. This illustrates that high levels of sorghum flour negatively impacts dough rheological properties, even with functional proteins present. Significant improvements in all parameters were observed between 30% HDHL sorghum-wheat composite doughs processed at room temperature and 35 C, while 30% normal sorghum-wheat composite doughs displayed no differences due to temperature. This indicates that functional changes in HDHL dough were caused
2.6. Statistical analysis All tests were performed in triplicate. One-way and two-way analysis of variance tests were used to test for significant differences among samples within and between temperatures, sorghum type, or sorghum amount at a p < 0.01. Tukey’s range test was used to differentiate statistical differences among sample means (Kutner, 2005). 3. Results and discussion 3.1. Dough extensibility Dough extensibility is an important characteristic in determining the optimum development of dough, as well as the thermal expansion of bread during baking. Measurements of peak height and time to dough breakage provide valuable information about the performance of dough during fermentation. The maximum
Fig. 1. Extensibility curves measured by planar extensional test for 30 and 60% sorghum-wheat composite doughs compared to a 100% wheat control (measured at 35 C).
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by the mobilization of kafirin upon exceeding its Tg [believed to be similar to the Tg of maize zein (Lawton, 1992). The same trend was observed for parameters of the 60% HDHL substitution doughs, though not statistically significant for area under the curve. Perhaps this is due to the incomplete representation that calculation of area provides. For example, at room temperature, the 60% HDHL sorghum-wheat composite dough extensibility profile had a large peak height and a small width (low time value). At 35 C, the graph had a small peak height and a large width. The integration of these measurements at their respective temperatures yielded approximately the same value for area regardless of having two distinctly different graph shapes. 3.2. Biaxial extension Biaxial extension provides information which can be related to a dough’s ability to withstand expanding gas cells during fermentation and oven rise (Dobraszczyk and Morgenstern, 2003). Characteristics of extensional viscosity and strain hardening can be inferred from this test (Fig. 2a and b). The capacity of dough to exert more than proportionally higher stresses at increasing strain values
Fig. 3. Bread loaf volumes for 20e60% normal and HDHL sorghum substitutions.
is indicative of strain hardening properties that are assumed to be necessary for gas retention. Strain hardening provides stability against gas cell rupture and aids in slowing disproportionation or the overgrowth of one gas cell at the expense of another (van Vliet, 2008). Given that this test was conducted at a constant strain rate, the slope of each sample from Fig. 2a can be used as a value for comparison of strain hardening of the different doughs (Kokelaar et al., 1996). HDHL and normal sorghum-wheat composite dough values were not significantly different from wheat dough at the 30% substitution level, indicating that strain hardening properties of the composite doughs were not affected at low levels of sorghum substitution (Fig. 2a). However, at the 60% substitution level, strain hardening for the normal sorghum-wheat composite dough was significantly lower than that of wheat flour dough or the HDHL composite. HDHL sorghum-wheat composite dough produced a slope slightly lower than that of dough prepared with wheat flour, indicating greater retention of its strain hardening properties compared to the normal sorghum-wheat composite dough. The up or down shift in the plots at a 60% substitution level may be better
Fig. 2. Biaxial extension test represented by (a) stress, s, as a function of strain and (b) extensional viscosity, hext, as a function of strain. Tests completed for 30% and 60% sorghum-wheat composite doughs and a 100% wheat control. Values on graph (a) are calculated slopes for each stress-strain curve.
Fig. 4. Effect of sorghum substitution on bread compression.
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Fig. 5. Normal sorghum (left) and HDHL sorghum (right) flour doughs with 18% vital wheat gluten addition.
explained by extensional viscosity values (Fig. 2b). HDHL sorghum displayed higher viscosity at an increased substitution level while normal sorghum decreased at increasing substitution levels. Either too low or too high extensional viscosity can negatively impact the ability of dough to retain gas and expand. It may signify low elasticity or high viscosity, both which have the potential to result in either gas cell wall rupture, coalescence, or the ultimate loss of gas produced during fermentation (Kokelaar et al., 1996).
can be defined as the ability to be stretched and return to its original shape. It is related to the elasticity of the crumb which is affected by its protein network. HDHL sorghum-wheat composite bread samples recovered shape and volume and had no apparent change in sample dimensions after compression, indicating a high degree of springiness. Normal sorghum-wheat composite bread samples did not recover shape and volume indicating low springiness. This supports the notion that the HDHL sorghum-wheat composite bread had more viscoelastic protein network present.
3.3. Bread volume It is accepted that viscoelastic doughs have a unique ability to retain gas during fermentation and create high loaf volumes upon baking (Kokelaar et al., 1996). Bread volume data revealed significantly greater loaf volumes for HDHL sorghum-wheat composites compared to normal sorghum-wheat composites at substitution levels above 30% and up to 56%, with the largest difference at 42% sorghum (Fig. 3). There were no differences between the HDHL and normal sorghum-wheat composites loaf volumes at low and high levels of sorghum incorporation. At very low levels of sorghum incorporation, the reason might be that the wheat gluten remains the dominant protein in the system. At high incorporation levels, gluten fibril formation would likely be hindered due to low gluten concentration levels and lack of sufficient viscoelasticity of the sorghum proteins to capture gas and produce high loaf volumes. The biaxial extension data suggested that the high concentration HDHL sorghum-wheat composite dough has strong elastic properties compared to that of the wheat dough which would negatively impact gas cell expansion. 3.4. Bread compression test A compression test was conducted to compare bread crumb hardness, an important textural property in bread quality evaluation. The hardness of bread samples infers performance of the protein network to desirable sensory characteristics. At the 30% substitution level, there were no differences observed in bread hardness between HDHL and normal sorghum-wheat composites (Fig. 4). Increasing the level of HDHL sorghum substitution did not change hardness values, unlike for the normal sorghum-wheat composite that produced a harder crumb. Testing was done at this substitution level because it was the point at which there was the largest difference in loaf volumes between HDHL and normal sorghum-wheat composites. Both samples exhibited crumb hardness well above that of 100% wheat. The dimensions (diameter and height) of the bread samples were measured before and after compression testing in order to qualitatively explain springiness. The springiness of a bread sample
3.5. Gluten addition The ability of kafirin in HDHL flour to function as a viscoelastic protein was further tested by adding incremental levels of vital wheat gluten protein. A dramatic difference in behavior of the two sorghum flours was observed where HDHL kafirin in its freed form formed a dough-like material at 18% vital wheat gluten incorporation, while normal sorghum flour did not produce a dough even at higher levels (Fig. 5). It has been previously reported that exogenous gluten cannot form the viscoelastic network necessary for bread making in a sorghum flour system due to the lack of interaction between exogenous wheat gluten and endogenous sorghum proteins (Carson and Sun, 2000a,b). 4. Conclusions HDHL sorghum, which has freed kafirins available for interaction in the flour form, resulted in much improved viscoelastic doughs and bread crumb texture when the dough was treated above its Tg. While not as good as wheat gluten proteins, the HDHL sorghum kafirins, in a high incorporation composite flour system, were shown to have the ability to form improved protein networks that entrap gas, while normal sorghum-wheat composite flours performed poorly. This was demonstrated through dough extensibility and baked loaf volume tests. Thus, HDHL kafirins participated with wheat gluten in sorghum-wheat composite dough to improve its viscoelastic properties while normal sorghum did not. At higher sorghum flour incorporation levels, it appears that elastic properties of kafirin are not sufficiently viscoelastic as those of wheat gluten to produce good dough and bread properties. In part, this is likely due to inherently different properties of the kafirin proteins, though it may also be related to other components within the sorghum flour, such as phenolic compounds, that could negatively affect dough and bread making properties. If the functionally of the HDHL flours can be further enhanced, then there is potential for high incorporation of sorghum in composite breads while maintaining quality similar to wheat products.
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Grain of high digestible, high lysine (HDHL) sorghum contains kafirins which enhance the protein network of composite dough and bread Morgan A. Goodall a, Osvaldo H. Campanella b, Gebisa Ejeta c, Bruce R. Hamaker a, * a
Department of Food Science, Purdue University, 745 Agricultural Mall Drive, West Lafayette, IN 47907, USA Department of Agricultural and Biological Engineering, Purdue University, 745 Agricultural Mall Drive, West Lafayette, IN 47907, USA c Department of Agronomy, Purdue University, 915 West State Street, West Lafayette, IN 47907, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2011 Received in revised form 27 March 2012 Accepted 3 April 2012
The aim of this study was to determine whether protein body-free kafirins in high digestibility, highlysine (HDHL) sorghum flour can participate as viscoelastic proteins in sorghum-wheat composite dough and bread. Dough extensibility tests revealed that maximum resistance to extension (g) and time to dough breakage (sec) at 35 C for HDHL sorghum-wheat composite doughs were substantially greater (p < 0.01) than for normal sorghum-wheat composite doughs at 30 and 60% substitution levels. Functional changes in HDHL kafirin occurred upon exceeding its Tg. Normal sorghum showed a clear decrease in strain hardening at 60% substitution, whereas HDHL sorghum maintained a level similar to wheat dough. Significantly higher loaf volumes resulted for HDHL sorghum-wheat composites compared to normal sorghum-wheat composites at substitution levels above 30% and up to 56%, with the largest difference at 42%. HDHL sorghum-wheat composite bread exhibited lower hardness values, lower compressibility and higher springiness than normal sorghum-wheat composite bread. Finally, HDHL sorghum flour mixed with 18% vital wheat gluten produced viscoelastic dough while normal sorghum did not. These results clearly show that kafirin in HDHL sorghum flour contributes to the formation of an improved protein network with viscoelastic properties that leads to better quality composite doughs and breads. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Sorghum Kafirins Viscoelastic Bread making
1. Introduction Due to adverse growing conditions and high import prices, developing countries in Africa do not have good access to wheat, the primary ingredient in bread, which is consumed in most urban areas. To reduce cost, much work over the past decades has focused on composite flour technologies that replace wheat with flours made from locally grown grains and tubers. Low incorporation of alternative flours has generally been achieved as they have no functional components to aid in bread making. Sorghum is one of the grains that has been used and has certain advantages in that high quality sorghum flours are neutral in flavor and light in color. Sorghum is grown in the semi-arid regions of the world and is a major food crop in the West Africa Sahel (Burkina Faso, Mali, Niger, northern Nigeria, Senegal) and the Horn of Africa (Ethiopia, Kenya, Sudan, Uganda).
Abbreviation: High digestibility, High-lysine, HDHL. * Corresponding author. Tel.: þ1 765 494 5668; fax: þ1 765 494 7953. E-mail address: [email protected] (B.R. Hamaker). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2012.04.001
Research on partial substitution of wheat flour with sorghum has shown that it can typically be mixed with wheat at levels of 15e20% to obtain acceptable bread products (Dendy, 1970; Kim and De Ruiter, 1968; Schober et al., 2004). Higher substitution levels have a negative impact on dough rheology and decrease loaf volume. Recent research has shown that somewhat higher incorporation sorghum-wheat composite breads can be achieved by formulation changes including addition of malt and addition of gluten (Carson and Sun, 2000a; Hugo et al., 2000). Gluten-free batter type bread has been made from sorghum flour using a sourdough fermentation with hydroxypropyl methyl cellulose (Schober et al., 2007). These methods have, in some cases, allowed for incorporation of sorghum at levels near 30e40%, however issues of poor loaf volume, and poor texture and taste are still a problem. While it is generally considered that the sorghum grain storage proteins, the kafirins, lack good gluten-like properties, their functional role in bread making is also confounded by their encapsulation in rigid protein bodies that do not break apart with dough mixing, thus making them inaccessible for any possible functional role. Wheat gluten has the unique ability to form viscoelastic doughs, which trap gas during yeast fermentation and produce leavened
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bread. The development of proteins during wheat grain filling results in a continuous protein matrix within the endosperm of which the proteins are free and available to interact. The functionality of wheat proteins to produce gluten with good rheological properties is attributed to its polymer mobility, amino acid composition (high Gln, high Pro, low basic amino acids), changes in secondary structure on mixing, and disulfide-sulfhydryl interchange occurring between cysteine/cystine groups (Erickson et al., in press; Hoseney et al., 1986; Wellner et al., 2005). Gluten has low charge density that, when coupled with hydrogen bonding and hydrophobic interactions, allows for close associations between gluten polymers and provides a stabilizing force during dough development. Sorghum kafirin comparably has a higher proportion of helical conformations and a greater number of hydrophobic residues (Belton et al., 2006). It also lacks the high molecular weight glutenin proteins, which are considered as the dough component that provides suitable viscoelastic properties to developed dough (Fevzioglu et al., in press). Studies on sorghum kafirin have shown it to be analogous in many ways to maize zein. Both proteins are encapsulated in protein bodies within the endosperm and have similar chemical composition and properties (Belton et al., 2006). Kafirins and zeins are further classified by their a-, b-, and g- subunits (Lending et al., 1988; Shull et al., 1991). Internal structure of the protein body shows the main a-kafirin/zein resides at its interior, while b- and gkafirins/zeins are mainly found at its periphery. One area in which kafirin and zein differ is in their cross-linking behavior, specifically in relation to digestibility (Hamaker et al., 1987). Kafirins are considerably less digestible than other cereal proteins if consumed equally as porridges. Sorghum protein had 46% apparent protein digestibility in young children, compared to maize and wheat proteins at 73% and 81%, respectively (Maclean et al., 1981). The causal factor is an extensive cross-linking of b- and g-kafirins at the protein body periphery which decreases the ability of digestive enzymes to break down the protein body (Oria et al., 1995). This negative nutritional implication of sorghum led to the use of reducing agents, processing changes, and selective breeding programs for the development of sorghum with increased digestibility (Hamaker et al., 1987; Maclean et al., 1983; Tesso et al., 2008). A high digestibility sorghum genotype was identified in already existing high-lysine sorghum germplasm at Purdue University that has alterations in the protein body shape which result in accessible a-kafirin protein (Ejeta et al., 1987; Oria et al., 2000; Weaver et al., 1998). In the high digestibility, high lysine (HDHL) sorghum, the normal spherical protein body shape is changed to altered folded morphology due to a shift in location of g-kafirin from the periphery to the interior of the body (Oria et al., 2000). Most of the major easy-to-digest a-kafirin protein is thus exposed and results in digestibility improvement. At room temperature, gluten forms a viscoelastic protein network upon addition of water and mixing. The protein undergoes a glass transition whereby amorphous proteins change their physical state from a glassy to rubbery state, and is dependent upon the glass transition temperature, Tg, and associated processing temperature, as well as the amount of plasticizer (water) present in the mixture. Thus, exceeding the Tg of the protein is an important requirement for mixing of dough. Gluten with 16% or higher moisture content has a Tg below room temperature (Hoseney et al., 1986). On the other hand, zein is reported as having a Tg above room temperature at about 28 C with 20% or higher moisture content. Kafirin is believed to have a similar Tg property (Lawton, 1992; Oom et al., 2008). Accordingly, isolated (protein-body free) zein and kafirin can be mobilized at temperatures above their Tg and have been shown to participate in viscoelastic dough development (Bugusu et al., 2001; Lawton, 1992; Oom et al., 2008; Schober et al.,
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2008). Up to this point, functionality of kafirin or zein in composite doughs has only been achieved using isolated protein, because in flours they are naturally contained within protein bodies. The increased availability of the HDHL sorghum kafirins to digestive enzymes led us to believe that kafirins from this flour may also be able to participate in the development of viscoelastic protein networks in dough. If so, then HDHL sorghum grain may offer the potential for high incorporation in composite wheat flour leavened products through functioning of the protein. Moreover, such sorghum varieties adapted to the environments of Africa would permit farmers to produce sorghum with improved function in composite flours that could be sold into urban markets and reduce reliance on imported wheat. 2. Materials and methods 2.1. Sorghum flour Normal sorghum, P721N, and HDHL sorghum (cv. PHD-024789), grains were decorticated to remove approximately 10% of their weight, were pin milled (Alpine American Corp., Natick, MA), and then passed through the reduction roll of a roller mill. To separate the remaining large particles, the flour was passed through a 54GG sieve. 2.2. Composite dough and bread preparation Sorghum flour was combined with wheat flour (Gold Medal Bread Flour, Minneapolis, MN) in the following proportions just before mixing: 30% and 60% sorghum (flour weight basis) for dough rheology testing, 20e60% sorghum at 2% increments for bread volume testing, and 30% and 42% sorghum for the bread compression test. Dough samples were created using the same ingredients: 2% yeast (Fleischmann’s Active Dry Yeast, San Francisco, CA), 2% salt (Morton International, Inc., Chicago, IL), and 67 2% water, depending on flour moisture content, except for doughs with added vital wheat gluten. Tests with gluten addition were made with 100% normal and HDHL sorghum flours, 18% vital wheat gluten (Sigma Aldrich, St. Louis, MO) and 67% water. Soybean oil (2%) was added to dough intended for baking. Ingredients were kept at room temperature, except for the water which was kept at 35 C for Tg related tests where mixing was done in a 35 C temperature-controlled room. All doughs were mixed using a pin-type mixer (National Mfg. Co., Lincoln, NE) with 10, 35, and 100 g capacities. Mixing times were obtained for each formula change using a mixograph (National Mfg. Co., Lincoln, NE). After mixing, doughs were either used for rheological testing or molded into a loaf pan for baking. For baking, fermentation was done in a temperature (35 C) and humidity (85% RH) controlled proofing cabinet (InterMetro Industries Co., Wilkes-Barre, PA) for 45e60 min, whereupon the loaf was baked in a rotary electric oven (National Mfg. Co., Lincoln, NE) at 218 C for 30 min. 2.3. Dough rheology Dough extensibility was tested using the TA.HDplus Texture Analyzer (Stable Micro Systems Ltd., Surrey, UK) at room temperature and in a 35 C temperature-controlled room. A sheet deforming device was used consisting of two plates containing a 5.5 mm diameter circular aperture in the middle, similar to that used by Bugusu et al. (2001). Dough (35 g) was mixed, sheeted and placed between the set of plates. A 31.75 mm diameter cylindrical probe was used to deform the dough sample in a planar extensional direction at a speed of 3 mm/s. Force (g) was measured over time (sec), and the area (m2) under the curve and peak height (g) was
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Table 1 Dough extensibility measurements for composite flours containing normal and HDHL sorghum.a,b Sample
Area (m2) ratio at RT
Area (m2) ratio at 35 C
Peak (g) ratio at RT
Peak (g) ratio at 35 C
Time (sec) to breakage at RT
Time (sec) to breakage at 35 C
100% Wheat 30% HDHL Sorghum 30% Normal Sorghum 60% HDHL Sorghum 60% Normal Sorghum
1 0.42aA 0.30b 0.23c 0.14d
1 0.56aB 0.33b 0.23c 0.10d
1 0.74aA 0.57b 0.80cA 0.42dA
1 0.65aB 0.57b 0.54bB 0.37cB
13.78 0.22aA 8.12 0.06bA 8.29 0.14b 4.41 0.06cA 4.77 0.14c
14.73 0.09aB 12.54 0.22bB 8.65 0.06c 6.34 0.08dB 4.79 0.19e
a Means followed by different lowercase letters within the same column are significantly different using One Way ANOVA, p < 0.01. Only means followed by different uppercase bolded letters are significantly different between temperatures RT and 35 C of that specific two sample set. b Room temperature is abbreviated as RT.
normalized using values of wheat flour dough measured by the same rheological variables. The time to dough breakage was determined based on the observation that dough breakage occurs after the maximum resistance, approximately at one half the peak height. Force, area, and time values were recorded and statistical analysis performed. Biaxial extension was tested using the Texture Analyzer at room temperature and 35 C. This method uses a set of lubricated plates (2.54 mm diameter), one stationary on the stage and the other affixed to a probe. The probe deformed at a strain rate of 20%/sec up to 80%/sec. Force (g) was measured and converted to extensional viscosity (hext) and stress (s) following the procedure of Campanella and Peleg (2001). Strain rate was used to convert time (s) to strain (%). The linear portion of the stress versus strain plot was used to calculate regression lines and the slopes were used for statistical analysis. 2.4. Baked loaf volume test Baked loaves were allowed to cool for 1 h before measuring their mass (g) and volume (cm3). Volume was measured by the rapeseed displacement method (AACC Approved Method 10-05.01). 2.5. Bread compression Baked loaves were allowed to cool for 1 h before obtaining measurements using the Texture Analyzer. A 2 cm thick bread slice was taken from the middle of the loaf and a 3 cm diameter circle was cut from the crumb center to acquire a 2 3 cm cylinder. The bread cylinder was placed on a level stage and a 25.4 mm diameter probe was used to compress the sample with a strain rate of 10%/sec up to 80%/sec. Force (kg) was measured over time (s). The bread sample was removed after compression and measured with a caliper.
resistance dough can exert against an expanding gas bubble may be inferred by the measured peak height. High values of force are desirable because they relate to the ability of dough to expand and retain gas generated through yeast fermentation. The time to dough breakage shows how much extension or strain the dough can withstand before collapsing or tearing. The protein network developed during dough mixing must have the ability to resist expanding gas bubbles during fermentation so that a sufficient increase in volume is achieved. Area, peak, and time at 35 C for 30 and 60% HDHL sorghumwheat composite doughs were notably and significantly greater (p < 0.01) than for normal sorghum composites at both substitution levels (Table 1 and Fig. 1). This shows that the HDHL sorghumwheat composite doughs were more extensible and exerted greater extensional force for a longer time period compared to normal sorghum-wheat composite doughs. The HDHL sorghumwheat composite extensibility profiles had a similar shape to the wheat control, but these doughs were not as extensible (peak height) or of the same dough strength. As the amount of sorghum increased in the composite flours, the area, peak, and time values decreased. This illustrates that high levels of sorghum flour negatively impacts dough rheological properties, even with functional proteins present. Significant improvements in all parameters were observed between 30% HDHL sorghum-wheat composite doughs processed at room temperature and 35 C, while 30% normal sorghum-wheat composite doughs displayed no differences due to temperature. This indicates that functional changes in HDHL dough were caused
2.6. Statistical analysis All tests were performed in triplicate. One-way and two-way analysis of variance tests were used to test for significant differences among samples within and between temperatures, sorghum type, or sorghum amount at a p < 0.01. Tukey’s range test was used to differentiate statistical differences among sample means (Kutner, 2005). 3. Results and discussion 3.1. Dough extensibility Dough extensibility is an important characteristic in determining the optimum development of dough, as well as the thermal expansion of bread during baking. Measurements of peak height and time to dough breakage provide valuable information about the performance of dough during fermentation. The maximum
Fig. 1. Extensibility curves measured by planar extensional test for 30 and 60% sorghum-wheat composite doughs compared to a 100% wheat control (measured at 35 C).
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by the mobilization of kafirin upon exceeding its Tg [believed to be similar to the Tg of maize zein (Lawton, 1992). The same trend was observed for parameters of the 60% HDHL substitution doughs, though not statistically significant for area under the curve. Perhaps this is due to the incomplete representation that calculation of area provides. For example, at room temperature, the 60% HDHL sorghum-wheat composite dough extensibility profile had a large peak height and a small width (low time value). At 35 C, the graph had a small peak height and a large width. The integration of these measurements at their respective temperatures yielded approximately the same value for area regardless of having two distinctly different graph shapes. 3.2. Biaxial extension Biaxial extension provides information which can be related to a dough’s ability to withstand expanding gas cells during fermentation and oven rise (Dobraszczyk and Morgenstern, 2003). Characteristics of extensional viscosity and strain hardening can be inferred from this test (Fig. 2a and b). The capacity of dough to exert more than proportionally higher stresses at increasing strain values
Fig. 3. Bread loaf volumes for 20e60% normal and HDHL sorghum substitutions.
is indicative of strain hardening properties that are assumed to be necessary for gas retention. Strain hardening provides stability against gas cell rupture and aids in slowing disproportionation or the overgrowth of one gas cell at the expense of another (van Vliet, 2008). Given that this test was conducted at a constant strain rate, the slope of each sample from Fig. 2a can be used as a value for comparison of strain hardening of the different doughs (Kokelaar et al., 1996). HDHL and normal sorghum-wheat composite dough values were not significantly different from wheat dough at the 30% substitution level, indicating that strain hardening properties of the composite doughs were not affected at low levels of sorghum substitution (Fig. 2a). However, at the 60% substitution level, strain hardening for the normal sorghum-wheat composite dough was significantly lower than that of wheat flour dough or the HDHL composite. HDHL sorghum-wheat composite dough produced a slope slightly lower than that of dough prepared with wheat flour, indicating greater retention of its strain hardening properties compared to the normal sorghum-wheat composite dough. The up or down shift in the plots at a 60% substitution level may be better
Fig. 2. Biaxial extension test represented by (a) stress, s, as a function of strain and (b) extensional viscosity, hext, as a function of strain. Tests completed for 30% and 60% sorghum-wheat composite doughs and a 100% wheat control. Values on graph (a) are calculated slopes for each stress-strain curve.
Fig. 4. Effect of sorghum substitution on bread compression.
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Fig. 5. Normal sorghum (left) and HDHL sorghum (right) flour doughs with 18% vital wheat gluten addition.
explained by extensional viscosity values (Fig. 2b). HDHL sorghum displayed higher viscosity at an increased substitution level while normal sorghum decreased at increasing substitution levels. Either too low or too high extensional viscosity can negatively impact the ability of dough to retain gas and expand. It may signify low elasticity or high viscosity, both which have the potential to result in either gas cell wall rupture, coalescence, or the ultimate loss of gas produced during fermentation (Kokelaar et al., 1996).
can be defined as the ability to be stretched and return to its original shape. It is related to the elasticity of the crumb which is affected by its protein network. HDHL sorghum-wheat composite bread samples recovered shape and volume and had no apparent change in sample dimensions after compression, indicating a high degree of springiness. Normal sorghum-wheat composite bread samples did not recover shape and volume indicating low springiness. This supports the notion that the HDHL sorghum-wheat composite bread had more viscoelastic protein network present.
3.3. Bread volume It is accepted that viscoelastic doughs have a unique ability to retain gas during fermentation and create high loaf volumes upon baking (Kokelaar et al., 1996). Bread volume data revealed significantly greater loaf volumes for HDHL sorghum-wheat composites compared to normal sorghum-wheat composites at substitution levels above 30% and up to 56%, with the largest difference at 42% sorghum (Fig. 3). There were no differences between the HDHL and normal sorghum-wheat composites loaf volumes at low and high levels of sorghum incorporation. At very low levels of sorghum incorporation, the reason might be that the wheat gluten remains the dominant protein in the system. At high incorporation levels, gluten fibril formation would likely be hindered due to low gluten concentration levels and lack of sufficient viscoelasticity of the sorghum proteins to capture gas and produce high loaf volumes. The biaxial extension data suggested that the high concentration HDHL sorghum-wheat composite dough has strong elastic properties compared to that of the wheat dough which would negatively impact gas cell expansion. 3.4. Bread compression test A compression test was conducted to compare bread crumb hardness, an important textural property in bread quality evaluation. The hardness of bread samples infers performance of the protein network to desirable sensory characteristics. At the 30% substitution level, there were no differences observed in bread hardness between HDHL and normal sorghum-wheat composites (Fig. 4). Increasing the level of HDHL sorghum substitution did not change hardness values, unlike for the normal sorghum-wheat composite that produced a harder crumb. Testing was done at this substitution level because it was the point at which there was the largest difference in loaf volumes between HDHL and normal sorghum-wheat composites. Both samples exhibited crumb hardness well above that of 100% wheat. The dimensions (diameter and height) of the bread samples were measured before and after compression testing in order to qualitatively explain springiness. The springiness of a bread sample
3.5. Gluten addition The ability of kafirin in HDHL flour to function as a viscoelastic protein was further tested by adding incremental levels of vital wheat gluten protein. A dramatic difference in behavior of the two sorghum flours was observed where HDHL kafirin in its freed form formed a dough-like material at 18% vital wheat gluten incorporation, while normal sorghum flour did not produce a dough even at higher levels (Fig. 5). It has been previously reported that exogenous gluten cannot form the viscoelastic network necessary for bread making in a sorghum flour system due to the lack of interaction between exogenous wheat gluten and endogenous sorghum proteins (Carson and Sun, 2000a,b). 4. Conclusions HDHL sorghum, which has freed kafirins available for interaction in the flour form, resulted in much improved viscoelastic doughs and bread crumb texture when the dough was treated above its Tg. While not as good as wheat gluten proteins, the HDHL sorghum kafirins, in a high incorporation composite flour system, were shown to have the ability to form improved protein networks that entrap gas, while normal sorghum-wheat composite flours performed poorly. This was demonstrated through dough extensibility and baked loaf volume tests. Thus, HDHL kafirins participated with wheat gluten in sorghum-wheat composite dough to improve its viscoelastic properties while normal sorghum did not. At higher sorghum flour incorporation levels, it appears that elastic properties of kafirin are not sufficiently viscoelastic as those of wheat gluten to produce good dough and bread properties. In part, this is likely due to inherently different properties of the kafirin proteins, though it may also be related to other components within the sorghum flour, such as phenolic compounds, that could negatively affect dough and bread making properties. If the functionally of the HDHL flours can be further enhanced, then there is potential for high incorporation of sorghum in composite breads while maintaining quality similar to wheat products.
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