Mixolab: A New Approach to Rheology

CHAPTER 1

The Mixolab A. Dubat CHOPIN Technologies, Laboratoire d’Applications, Villeneuve la Garenne, France 1. PRESENTATION 1.1 History The Mixolab is a modern device developed for the quality control of cereals. The instrument measures dough and flour quality by exposing a sample to predetermined heating and cooling cycles while placing the sample under a strain field. Data are collected as a set of stress-strain plots analyzed via an algorithm for multigraph data structure analysis. The method and analysis of the results from the measurement are based on the same principles used by the Pétrinex dough-testing equipment.

This measurement was initially envisioned in the early 1900s by van Stock, a Rotterdam miller (German patent 293078, dated 22/7/1914). Victor-Lambert Buys developed dough-processing machinery and rheology instrumentation during the 1940s and 1950s, culminating in the Pétrinex (French patents 918303A, 923252A, 923253A, 937227A, 987206A, 987207A, 1119928A, 1148944A, 1260716A). In the 1970s, Duranel (1970) and Bussiere et al (1972), among others, concluded that the Pétrinex was suitable as a quality control instrument based on the good repeatability of the results from its measurements. In 2000, the Multigraphe was created (Sinnaeve 2000). The instrument was then improved and redesigned by CHOPIN Technologies, and the final version was introduced in 2004 as the Mixolab (Dubat 2004b).

The Mixolab offers enhanced functionality over existing devices because of the geometry of the mixing blades and mixing bowl and the variable speed and temperature-testing options. It is possible to incorporate temperature cycles, warming the dough to 90°C (194°F) and subsequently cooling the sample. The user can, in a single test, determine the water-absorption capacity, mixing stability, gelatinization peak and temperature, amylase activity, and starch retrogradation. Wide measurement potentialities are therefore possible on various cereals (M. C. Tulbek, personal communication; Manthey et al 2007; Piguel et al 2007; Tulbek and Hall 2007), breads (e.g., the French baguette, by determination of flours according to their final use; Boizeau et al 2007), noodles (L. Cato, personal communication; L. Cato and M. C. Gianibelli, personal communication), and cakes (Koksel et al 2007). The Mixolab can also be used for ingredient assessment (Bollain and Collar 2005, Collar et al 2007) and for investigating the effects of additives, such as hydrocolloids (Rosell et al 2007) and proteins (Bonet et al 2006).

Furthermore, the Mixolab is capable of analyzing ground whole grains in addition to flours, so it can be used for whole-cereal processing (Sinnaeve 2000, Lenartz et al 2006). The instrument has also proved its utility in the analysis of other cereals, such as durum wheat (Moscaritolo et al 2008).

The operation of the Mixolab is extremely simple. The user chooses an existing protocol (from among the protocols included with the Mixolab software or those created by a user of that particular system) and follows the instructions on the screen. The desired absorption and sample moisture are programmatically defined by the user, which in turn defines the quantity of flour needed for the test.

The water injection nozzle is then placed above the mixer, and as mixing starts, the Mixolab automatically delivers the necessary quantity of water.

The Mixolab relies on the principle of conservation of mass. In the standard Mixolab procedure, the default value of dough weight is set to 75 g, but it can be set to 30–110 g, depending on the tested product and the user needs. The default value for mixing speed is 80 rpm, but it can be set to 30–250 rpm. Temperature and heat-

Fig. 1.1. The Mixolab.

1.2 Device Description The Mixolab is shown in Figure 1.1. The mixer bowl (Fig. 1.2) is designed for a 50-g sample size. The sample can be in the form of flour or ground cereal. To facilitate cleaning, the mixer bowl can be fully dismantled with ease (Fig. 1.3).

Heating resistances warm up the device to 90°C (194°F), and the cooling is controlled through water flow (i.e., open with tap water or closed with a water chiller). The mixer bowl temperature (and thus the dough temperature) is constantly recorded (using a patented system) to ensure thorough analysis of the quality of the tested sample.

Fig. 1.2. The Mixolab mixer bowl.

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ing/cooling rates are similarly modular, thereby yielding endless possibilities for adapting the Mixolab protocol to any end user’s needs. The instrumental settings are shown in Table 1.1.

With the operating software provided, the instrument comprises three parts, which are described in the following sections: • the Mixolab Standard (for research or research and development purposes), • the Mixolab Profiler (based on the Mixolab Standard, for quality control needs), and • the Mixolab Simulator (to simulate Farinograph-equivalent results).

2. THE MIXOLAB STANDARD In the two years following the launch of the Mixolab, the quality of its data collection (repeatability, reproducibility, and integrity) and the variability among instruments and trained users were carefully validated through a collaborative study led by the International Association for Cereal Science and Technology (ICC). The tests were performed in 13 laboratories by trained users from various countries with two different sample matrixes: flour and whole wheat. The results of that ring test (Table 1.2) led to development of the new ICC Standard 173. Results included method performance for torque and dough temperature measurement.

The Mixolab showed excellent repeatability and reproducibility, with most parameters showing a standard deviation lower than 5%.

The same methodology was approved by AACC International in 2010 as Method 54-60.01 (AACC International, no date) and is also accepted by the Association Française de Normalisation, the French national organization for standardization. The device and the method

were widely described by various authors (Dubat 2004a, Rosell et al 2007). The dough consistency was measured as torque (Nm) of the dough during mixing at constant speed, and the dough was subjected to a series of temperature cycles (30 to 90 to 50°C; 86 to 194 to 122°F). An example of a typical Mixolab curve is shown in Figure 1.4 (i.e., the heavy curve).

Water is added to reach the first maximum consistency of 1.1 Nm. This provides information on the water-absorption potential. As the test continues, it provides information about dough rheology during mixing (phase 1), the strengthening of gluten (phase 2), the starch gelatinization (phase 3), the amylase activity (phase 4), and the starch retrogradation (phase 5). The Mixolab serves as a complete tool for analyzing dough behavior, which depends on composition, ingredient quality, and interactions. In fact, it reflects the complexity of the dough system, and this complexity is important to consider when analyzing each part of the Mixolab curve (Fig. 1.5).

2.1 Water Sorption Water sorption is the first element used to assess flour quality. In baking terminology, the water sorption corresponds to the quantity of water required (in liters) for 100 kg of flour to reach the desired dough consistency. In industrial processes, the dough behavior must be consistent, for end-product quality assurance and to avoid production stops. It is of the utmost importance to know the optimum flour hydration and to understand the meaning of this value. The water sorption is influenced by five main parameters: 1) the flour moisture content, 2) the quality and the content of proteins, 3) the native starch, 4) the damaged starch, and 5) the fiber content (pentosans).

The drier the flour, the greater the amount of water that must be added; also, a wheat protein can absorb slightly more water than its own weight. An empirical method states that the absorption capacity increases 1% (w/w) for each 1% of additional protein (Sluimer 2005).

Some studies have also determined that the quality of the gluten, in addition to the quantity, impacts the water-absorption capacity (Cauvain and Young 1998). The undamaged (native) starch of the flours absorbs only 40% of its weight of water (Sluimer 2005). However, the native starch strongly influences the absorption process.

Indeed, approximately 60–70% of the flour is starch, which offers a large contact interface. Therefore, the native starch holds more than 20% of the water (surface absorption), as shown in Table 1.3. Because of the high pressure imparted by the cylinders during rotation, the starch granule is essentially damaged. This damage leads to an increase of the water-sorption capacity of the granules up to three times their weight. The fiber content (pentosans and arabinoxylans) also affects water sorption, with the fibers absorbing up to 10 times their weight of water (Hamer and Hoseney 1998, Sluimer 2005).

TABLE 1.2 Mixolab Ring Test Performance Fig. 1.3. The Mixolab mixer bowl dismantled.

Type TABLE 1.1 Instrumental Settings Defined in Mixolab Software Setting

Mixolab Standard

Mixolab Simulator

Dough mass Target torque Mixing speed Tank temperature Temperature, first plateau Duration, first plateau Heating rate Temperature, second plateau Duration, second plateau Cooling rate Temperature, third plateau Duration, third plateau Total analysis time

75 g 1.1 Nm 80 rpm 86°F (30°C) 86°F (30°C) 8 min 39°F/min (4°C/min) 194°F (90°C) 7 min 39°F/min (4°C/min) 122°F (50°C) 5 min 45 min

75 g 1.1 Nm 80 rpm 86°F (30°C) 86°F (30°C) 30 min … … … … … …

30 min

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WA C2 C3 C4 C5 Stability

Constant Constant Constant Constant Constant Variable

Time to T1

Variable

Dough T°1 Dough T°2 Dough T°3 Dough T°4 Dough T°5

Constant Constant Constant Constant Constant

a

Sr a

SR b

51.6–63.4 (%) 0.37–0.63 (nm) 1.59–2.27 (nm) 0.95–2.12 (nm) 1.46–3.73 (nm) 4.69–11.42 (min/100) 0.99–7.36 (min/100) 29.7–33.9 (°C) 52.2–57.7 (°C) 75.2–86.2 (°C) 83.5–88.7 (°C) 58.1–60.6 (°C)

nd 0.01 0.02 0.03 0.08 …

0.9 0.03 0.08 0.09 0.19 …

nd 3 1 2 3 …

2 5 4 5 7 …

…

…

…

…

0.57 0.65 0.78 0.77 0.74

0.97 1.59 2.06 2.03 2.72

2 1 1 1 1

3 3 2 2 5

Sr = Repeatability standard deviation.

SR = Reproducibility standard deviation.

CVr = Repeatability variation coefficient. d CV = Reproducibility standard variation coefficient.

R b c

CVrc CVRd (%) (%)

Range

Hydration impacts many parameters, but most importantly, it affects the mechanical properties, the dough yield (economical aspect), and the end-product quality (Hamer and Hoseney 1998). It has also been proven that high hydration decreases the protein and starch interactions (Hamer and Hoseney 1998).

Most of the time, increasing water absorption leads to more complete gelatinization, better oven rising, softer crumb, and lower retro-

gradation. These are the reasons that water-sorption capacity is so critical for breadmaking (Sluimer 2005).

2.2 The Mixing Stage Under quiescent conditions at room temperature and typical atmospheric pressure, the mixing of flour and water is limited to surface absorption. The structural changes at the molecular level that

Fig. 1.4. A typical Mixolab curve (heavy line). T = temperature.

Fig. 1.5. Main changes that occur during the Mixolab test.

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are necessary to form dough can be realized only under the presence of shear introduced through a mixing stage. In addition to dough homogenization, a major objective of the mixing process is to yield extensible dough (Sluimer 1998).The mixing process also develops the gluten, so this stage is also called gluten development.

Gliadin and glutenin are the major constituents of gluten. Gliadins are low-molecular-weight protein molecules that confer extensibility to dough. Glutenins are high-molecular-weight protein molecules that strengthen the dough (Sluimer 2005). The mixing process increases the interaction of enzymes and substrate while incorporating air bubbles, which will become the alveols in the crumb. A dough that exhibits high resistance to mixing allows for a high level of air incorporation.

At the beginning of the dough-mixing process, the proteins hydrate and begin to expand. The increase in protein interactions leads to development of a viscoelastic gluten network. During this stage, the protein thiol groups play an essential role by creating disulfide bridges between and among the chains (Sluimer 2005). During the mixing process, these interactions are transient, breaking and reforming (Feillet 2000). The disulfide bridges are critical for the formation of structure during breadmaking. During baking, more disulfide bridges are created because of the thermal processing; this bonding sets a permanent protein network matrix (Hamer and Hoseney 1998).

These links bring a certain viscosity to the dough. The dough behavior is the result of the viscosity, elasticity, plasticity, stickiness, and relaxing, and it changes during mixing. At the beginning of the mixing process, the dough is not cohesive and breaks easily. Gradually, as the gluten develops, the dough becomes more cohesive and stronger. During mixing, the dough’s resistance to mixing develops until it reaches a peak value, after which the protein network breaks and this resistance decreases (Sluimer 2005).

TABLE 1.3 Water Absorption Breakdown Between Various Flour Componentsa

Component Protein Native starch Damaged starch Pentosans a

Water Absorption Component Component for Each Quantity in 100 g Absorption for Component (g) of Flour (g) 100 g of Flour 1.3 0.4 2.0 7.0

12 57 8 2

15.6 22.8 16.0 14.0

Adapted from Stauffer (2007).

Fig. 1.6. Temperature influence on protein behavior.

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The dough behavior during mixing cannot be explained by its protein components only. Starch also affects the behavior of the dough during mixing, although it is less influential than protein (Cauvain and Young 1998, Cauvain 2003).

2.3 Gluten Strength An increase in dough temperature brings a decrease in viscosity (Sluimer 2005). The hydrophobic- or hydrogen-bonded network of glutenin molecules changes continuously. The junctions in the glutenin network are relatively low-energy interactions and can easily be broken, causing the network structure to be transient. The kinetics of network formation and breakage increase in rate with an increase in temperature. The dough viscosity decreases fivefold with an increase in temperature from 20 to 60°C (68 to 140°F) (Cauvain and Young 1998). This is caused by the kinetics of molecular and network relaxation occurring at a higher rate than the kinetics of orientation of the molecules that form the network and by the formation of elastically active network junctions. This results in an overall decrease in modulus and thus less-elastic dough.

Given the assumption of affiliated deformation, the strain imposed on a bulk material with a continuous network is transmitted down evenly throughout the bulk, and the strain at the molecular level is equal to the strain imposed on the bulk. Although each extension is only a nanometer in size, when applied to thousands (or millions) of linkages, it can support a significant level of stress as these molecules become oriented. As macromolecular orientation increases, stress is supported by the covalent linkages along the main molecular chains, and resistance to further deformation increases, despite a decrease in the number of elastically active network junctions. This increase in elasticity results in an increase in modulus. Thus, the dough resists further deformation in the direction of orientation, and apparent viscosity increases. Once a certain level of molecular orientation has been achieved, the loss in concentration of network junctions becomes so great that the network loses connectivity and the molecules slide past each other, with the only resistance to deformation being relatively weak frictional forces between the molecules. As the temperature increases, the kinetics of molecular relaxation dominate the kinetics of molecular orientation. (This occurs at temperatures below those at which covalent network junctions form.) If the rate of deformation is held constant under these thermal conditions, the orientation of the molecules decreases with increasing temperature, and the elastic contribution of covalent bonds along the main macromolecular chains becomes less significant, resulting in lower viscosity.

Dough is a viscoelastic material, and its stress response to imposed strain is a combination of viscous and elastic components.

When no further strain is imposed on the dough, which corresponds to a resting stage, then stress begins to relax because of the elastic component. During this stress relaxation, the cross-link density (i.e., the concentration of network junctions) begins to increase. However, the destruction of the network that occurs during imposition of strain is thermodynamically irreversible because of the presence of a viscous component of stress response, and these linkages cannot return to their original conformation or density. As the elastic component becomes more dominant relative to the viscous component of stress response, the network is reformed over time to become more similar to the original network structure.

This statement has been confirmed on the Mixolab by several tests conducted by the CHOPIN Technologies application laboratory. A first test was done while maintaining the dough at 30°C (86°F); the resulting curve (Fig. 1.6) shows typical mixing behavior. The test was repeated but with the temperature increased to 50°C (122°F) and then decreased to 30°C (86°F). A direct relationship can be observed between the temperature increase and the consistency decrease.

When dough is not heated above 50°C (122°F) and then cooled, it recovers to the same consistency as the nonheated dough because of its dominant elastic component. This shows that the phenomena during the first phase of heating are dependent on reversible linkages between the gluten chains. The hydrophobic linkages are in the same energy range as the hydrogen linkages (Table 1.4); however, their energy increases with the temperature. This phenomenon can strengthen the stability during the first phase of baking. Hamer and Hoseney (1998) discuss the importance of the phenomena resulting from the dough temperature increase.

2.4 The Viscosification Stage When the temperature reaches more than 50–60°C (122–140°F; or, for Feillet [2000], 55–65°C [131–149°F]), the dough viscosity increases rapidly, as the starch gelatinizes and the proteins polymerize (Pomeranz 1988). This phase is one of the most documented and studied in the cereal chemistry literature. Pomeranz (1988) described the phenomena during starch gelatinization as “one of the most important transformations of the starch for the foodstuff functionalities.”

TABLE 1.4 Atomic and Molecular Interactions and Their Energy and Mobilitya Interaction Type

Energy (kcal/mol)

Mobility

30–100 10–100 2–5 1–4 0.5

Negligible Moderate Strong Strong Strong

Covalent Ionic Hydrogen Hydrophobic Van der Wall a

Reprinted from Hamer and Hoseney (1998).

Sluimer (2005) showed that starch gelatinization begins at approximately 54–63°C (129–145°F). Cauvain (2003) stated a temperature of 60°C (140°F). For Feillet (2000), the bread-making ability of wheat starch appears even better when the gelatinization temperature is higher. This statement is based on the observation that the increase in bread volume in the oven stops when the starch gelatinizes. Sluimer (2005) stated that the dough becomes a “crumb” at approximately 65°C (149°F). Gelatinization corresponds to the destruction of a crystalline phase and the occurrence of the glass transition (Alexander and Zobel 1992, BeMiller 2007). Pomeranz (1988) specified that wheat starch has two volume increases. The first occurs at approximately 60–70°C (140–158°F) and corresponds to the breaking of the weak or easily accessible linkages. The second occurs at approximately 80–90°C (176–194°F) and involves stronger and less-accessible linkages, most likely resulting from the highly branched amylopectin structures. Feillet (2000) noticed that the gelatinization temperature increases as the moisture content decreases.

In a batter or any liquid environment, the starch gelatinizes entirely and loses its structure. If less water is present (e.g., in bread dough), gelatinization is incomplete and some granules of starch remain in the crumb (Sluimer 2005). This finding agrees with that of Cauvain and Young (1998), who stated that, in a water-starved composition, moisture is transferred from the protein to the starch during cooking. This information is quite important, as it explains some large test differences in gelatinization according to the process used in a diluted environment, such as a batter versus a dough. Hoseney et al (1978) stated that starch modification during the cooking process relies not only on the temperature but also on the water activity, which is controlled by the recipe and the other ingredients (e.g., fat and sugar).

Most of the time, the properties of starch are analyzed by removing it from its natural environment (i.e., wheat or flour). Large differences are apparent between the analyses done on dough and on its starch extract. These differences are caused by the protein-starch interactions in the dough. The dough also contains other components, such as lipids and ionic species, that influence the starch behavior (Cauvain 2003).

The two phenomena that control bread crumb formation are starch gelatinization in the dough and protein coagulation. During baking, the gelatinized starch and the proteins compete for water. In bread, the gelatinized starch network structure is predominant. In the absence of starch, crumb formation is impossible. However, too much starch hydrolysis leads to a sticky and unstable crumb. The role of protein in crumb formation is less important. It was shown previously that crumb formation can occur in the absence of (wheat) proteins (Sluimer 2005).

An important aspect for crust formation is for the crust to have a “crisp” property. This property relies mainly on the viscosity of the starch phase in the dough. At a high viscosity, cracks are more likely to form on the crust during cooling (Cauvain and Young 1998). It has

TABLE 1.5 Optimum and Inactivation Temperatures of Various Enzymesa

Enzyme

Location

Product(s)

α-Amylases Cereal

Starch α→(1,4)

Oligosaccharides, dextrins … …

Fungal Bacterial β-Amylase Cereal Glucoamylase Proteases Lypoxygenase Catalase a Reprinted

… …

Starch α→(1,4) Starch α→(1,4) Starch α→(1,6) Proteins Free fatty acids 2 H2O2

Maltose, dextrins Glucose …

Peptides, amino acids Hydroperoxides O2 + 2 H2O

Optimum Temperature (°C)

Inactivation Temperature (°C) Beginning

Ending

Dextrins/ Oligosaccharides (%)

3.5 13 14

60–65 55 70

65 55 70

80–90 80 >100

55 55–60

55 65

75 80

… …

60 40–50 …

65 60–65 …

80 … …

… … …

from Kruger et al (1987).

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been observed that during the cooking process, the true framework of the structure migrates from the gluten to the starch (Sluimer 2005). It is therefore mandatory to check the starch evolution in dough systems to predict its behavior in the manufacturing process.

2.5 The Amylasic Resistance The amylase activity has traditionally been measured with the Hagberg falling number, the Rapid Visco Analyser, or the Amylograph. Two major amylases exist in wheat: α-amylases and βamylases. The α-amylases transform the starch to glucose (dextrins), which are then transformed to maltose by the β-amylases (Feillet 2000). The released maltose is utilized for yeast fermentation. In typical wheat, the level of β-amylases is sufficient for breadmaking; however, the α-amylases act as a limiting agent. Often, millers and bakers add α-amylase or malt to adjust the amylase activity. The potential for the enzyme to move into contact with the substrate depends on the viscosity of the batter or dough and the mechanical (mixing) actions, which improve the exchanges of enzyme and substrate. The endogenous wheat α-amylase inactivates at approximately 70–85°C (158–185°F) and has maximum activity at approximately 60–70°C (140–158°F). For fungal amylase, inactivation occurs at approximately 60–70°C (140–158°F), and for bacterial amylase, inactivation occurs at temperatures greater than 90°C (194°F) (Feillet 2000, Sluimer 2005) (Table 1.5).

The damaged starch is quickly hydrolyzed by the amylases. These specific amylases are used for measuring the level of starch damage by means of an enzymatic method (Feillet 2000, Cauvain 2003). The reaction is as follows (Kruger et al 1987): Starch damage + H2O + amylases → Dextrin + maltose + glucose Baking temperature significantly influences the crumb structure.

The period of time between starch gelatinization and amylase inactivation is critical. Fungal amylase, which has a low inactivation temperature, most likely acts to hydrolyze the starch during the dough phase (Pomeranz 1988).

The acceptable level of amylase activity depends on the breadmaking process. Most flat breads—such as Moroccan bread, chapati bread (from Pakistan), and numerous Indian breads (excluding the Egyptian baladi and the Iranian baradi)—are tolerant to high amylase activity. However, the Japanese angel cake is quite intolerant (Pomeranz 1988).

2.6 The Retrogradation Stage Bread is composed of approximately 50% starch, 40% water, and 7% proteins (Cauvain and Young 1998). When it is stored, some changes in texture and springiness occur. This phenomenon, known as staling, results partly from the recrystallization of the gelatinized starch, or retrogradation (caused, in part, by changes in the protein network) (Sluimer 2005).

Starch plays a critical role in bread staling. When bread cools after baking, the starch molecules begin to agglutinate (i.e., nucleation of the crystalline phase occurs), affecting the firmness of the crumb.

Amylopectin crystallization is the primary explanation for crumb hardening during storage. The rate of crystallization depends on the temperature at which the bread is stored. The rate is low at 25–50°C (77–122°F) and high at 0°C (32°F). With frozen dough, no recrystallization occurs.

Whistler and BeMiller (1997) found that, for baked products, when enough water is available for starch gelatinization, amylose retrogradation occurs during the cooling phase of the product and the recrystallization kinetics of the amylopectin are slower. The presence of lipids also influences the rate of retrogradation. Polar lipids impact the gelatinization process and inhibit recrystallization. The type of lipid present has an important impact on the reaction; emulsifiers and fats added to the dough do not have the same impact on retrogradation. The removal of lipids from oats speeds up the retrogradation (Kragh 2003).

Gluten is also important in the retrogradation process because of its interaction with starch. Various authors have found that wheat flours with high protein content and quality show weak retrogradation. Others have concluded that the gluten in low-quality flours

Fig. 1.7. Difference between the predicted value from the Mixolab and the actual value for bread volume (upper graph) and dough scoring (lower graph). The zero line corresponds to no difference between the values.

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