Influence of the Heating Rate on the Pasting Properties of Various Flours
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1 564 DOI /star Starch/Stärke 57 (2005) Manuela Mariotti Marta Zardi Mara Lucisano Maria Ambrogina Pagani DiSTAM (Department of Food Science and Microbiology), University of Milan, Milan, Italy Influence of the Heating Rate on the Pasting Properties of Various Flours Viscoamylographic tests were carried out on six commercial flour samples, three wheat flours (WF), two semolinas (S) and one rice flour (RF), using the Brabender Micro Visco-Amylo-Graph (MVA). The slurries were subjected to a definite temperature profile (307C-957C, 957C630 min, 957C-507C, 507C630 min), stirring at 250 min 21 and using a 300 cm?g f cartridge and recording the viscosity (in Brabender Units, BU) as a function of temperature and time. The aim of this work was to evaluate the influence of different heating rates (1.5, 3.0, 5.0, 7.5, 10.07C/min) on the pasting properties of the various flours. The peak viscosity of WFs and Ss increased when high heating rates were applied, while the RF showed similar pasting properties independently of the heating rate. These behaviours were mainly ascribed to the different molecular organisation of the starch granules, responsible of different swelling and gelatinising extents, and also to a different kinetic of alpha-amylase inactivation according to the heating rate applied. The key role of the alpha-amylase activity in controlling the pasting viscosity of the different samples was demonstrated by the viscoamylographic test performed in the presence of silver nitrate as enzyme inhibitor. Keywords: Viscoamylograph; Pasting properties; Heating rate; Alpha-amylase activity; Light microscopy Research Paper 1 Introduction The most common way to fingerprint starch is via its pasting profile [1]: a starch suspension is subjected to a defined heating and cooling program and the viscosity is recorded as a function of temperature and time. This curve can be seen as a reflection of the structural changes of the granules that occur during starch gelatinisation and retrogradation [2] and of endogenous alphaamylase activity. Another widely used means of observing gelatinisation is via microscopic evaluation of starch: it is possible to use a light microscope equipped with a polariser to quantify the loss of birefringence of a starch sample during heating in excess water; this phenomenon is often used as an indicator of gelatinisation [2]. Moreover, light microscopy is used to identify the source of a particular starch, because the size and shape of the granules are the characteristic of their botanic origin. As regards pasting profiles, the Brabender Visco-Amylograph (BVA) has been widely used to measure the viscosity profile of starches for screening and quality control and to provide product specifications. In the BVA, a granular starch suspension is subjected to a defined heating and cooling program and the viscosity (measured Correspondence: Manuela Mariotti, DiSTAM (Department of Food Science and Microbiology), University of Milan, Via G. Celoria 2, Milan, Italy. Phone: , Fax: , manuela.mariotti@unimi.it. in arbitrary units Brabender Units, BU or torque, cm?g f ) is recorded as a function of temperature and time. The BVA requires a large sample size and the heating/ cooling rate is equal to 1.57C/min, which means a very long analysis time (ca. 2 h) [3]. That is why many efforts have been made to generate alternatives for the BVA. Indeed, in recent years new instruments for the evaluation of pasting properties of starch have been put on the market. The Rapid Visco Analyser (RVA) and, more recently, the Micro Visco-Amylo-Graph (MVA) are two clear instances of this technological development. Both require short analysis time and small sample size and provide for a large number of heating/cooling rates to apply to the slurries (from 1.57Cto10or127C/min), but they also have essential differences: the rotation speed and the spindle geometries, the heat transfer apparatus and the sample temperature measuring system [4, 5]. A number of studies have been reported on the pasting behaviour of starch suspensions determined with RVA or BVA [6 8]. A comparative study between BVA and RVA showed that BVA, because of its higher discriminating power, remained the first choice when starch samples with only slightly different properties in quality control are compared [3]. Batey and Curtin [9] studied how the viscosity parameters were affected in wheat starch, wheat flour and maize starch when the RVA operating conditions were changed (heating times, holding times, cooling times, etc.). More recently, Dong Soon and Jane [10]
2 Starch/Stärke 57 (2005) Influence of Heating Rate on Pasting Properties 565 compared the pasting properties of selected starches using MVA and RVA under the same conditions and concluded that the differences in pasting properties measured by using the two instruments could result from the different geometries of their spindle. The influence of the heating/cooling rate on the pasting properties of flour or starch samples using the MVA has not yet been studied. This instrument is a further development of the well-known viscograph and amylograph. The MVA is a rotating viscosimeter for measuring viscosity as a function of time and temperature. For the test, a water/starch or flour suspension is submitted to controlled heating and cooling conditions. The resulting torque is transformed into an electric signal which is recorded on-line and evaluated by means of a special software: the instrument needs to be operated by a computer, which controls the test procedure and records the evaluation. The MVA is designed in particular for food industries for measuring the gelatinisation properties of starch and products containing starch (modified starch, flour, pregelatinised starch, thickening agents, oat flakes): the measurement under defined temperature programs supplies a complete profile of the viscosity behaviour in the different production and application steps [5]. The aim of this study was to evaluate the influence of the heating rate on the pasting properties of flours characterised by different botanical origin, chemical composition and technological properties using the MVA. To better appreciate what happens to starch granules at the different heating rates, the evolution of their structure during the test was monitored by means of light and polarised light microscopy. 2 Materials and Methods 2.1 Materials Six samples of commercial flours were used: three wheat flours, two semolina and one rice flour. Wheat flour samples were selected according to their alveographic characteristics (WF1, W = J; WF2, W = J; WF3, W = J). The commercial semolina samples were coded S1 and S2 and the rice flour sample was identified as RF. 2.2 Methods Moisture content The moisture content of the flour was determined according to the official standard method AACC 44 15A [11]. Results (n = 2) are expressed as percentage Protein content The total nitrogen content of the different flours was determined according to the official standard method AOAC [12]. The protein content was calculated adopting 6.25 as a conversion factor, except for wheat flour (5.70). Results (n = 2) are given as g/100g dry basis (d.b.) Total starch content The total starch content of the samples was determined enzymatically using the Total Starch Assay Kit by Megazyme International Ireland Ltd. (Wicklow, Ireland). Results (n = 4) are given as g/100g d.b Damaged starch content Starch damage was determined enzymatically using the Starch Damage Assay Kit by Megazyme International Ireland Ltd. (Wicklow, Ireland). Results (n = 4) are expressed as g/100g d.b. and as a percentage of the total starch content of the sample Alpha-amylase activity Alpha-amylase activity was measured using the Alpha Amylase Assay Procedure by Megazyme International Ireland Ltd. (Wicklow, Ireland). In this method, 1 International Unit (IU) of alpha-amylase activity is defined as the amount of enzyme required to release 1 mmol of glucose reducing-sugar equivalents per minute under defined conditions of temperature and ph. Results (n = 4) are expressed as IU/g d.b Viscoamylographic tests The pasting profiles of flours were carried out in duplicate with a Brabender Micro Visco-Amylo-Graph (MVA) (Brabender OHG, Duisburg, Germany). Fifteen grams of the sample were dispersed in 100 ml of distilled water, scaling both flour and water weight on 14% flour moisture basis. The suspensions were subjected (stirring at 250 min 21 and using a 300 cm?g f cartridge) to the following standard temperature profile: heating from 307C upto 957C, holding at 957C for 30 min, cooling from 957C to 507C and holding at 507C for 30 min. Five different heating/cooling rates were applied, so as to cover the whole range of the MVA temperature rates capacity: 1.5, 3.0, 5.0, 7.5 and 10.07C/min. Total running test times were, respectively: 133 min 20 s, 96 min 40 s, 82 min, 74 min 40 s and 71 min. The following indices were considered:
3 566 M. Mariotti et al. Starch/Stärke 57 (2005) beginning of gelatinisation (temperature, 7C, at which an initial increase in viscosity occurs), peak viscosity (maximum paste viscosity, BU, achieved during the heating cycle) and breakdown (index of viscosity decrease, BU, during the first holding period corresponding to the peak viscosity minus the final viscosity at 957C). The viscoamylographic tests were also carried out in the presence of an alpha-amylase inhibitor. In this case, 1 mm aqueous AgNO 3 solution was used instead of distilled water [13 15] Light microscopy The slurries, prepared as described for the MVA test, were heated from 307C up to three different temperatures (50, 60 and 707C) and held at each temperature 1 min before withdrawal. The two extreme MVA heating rates were applied, i.e. 1.5 and 10.07C/min. A small drop of the material was taken from the slurry, placed on a microscope slide and immediately examined with a light microscope (Nikon Eclipse ME 600 Microscope, Nikon Corporation, Tokyo, Japan). Observations were taken using both normal light, to observe the progressive swelling and breaking of starch granules, and polarised light. Images were taken with a Nikon Japan Camera TV Lens 0.66directly interfaced with and controlled by a personal computer via suitable software (LUCIA Sc. Means, Version 4.51). 3 Results and Discussion 3.1 Flour characterization Moisture, protein, total and damaged starch content and the alpha-amylase activity of the samples are listed in Tab. 1. The protein content was high in WFs and Ss while it was quite low (7.5% d.b.), as expected, in the RF. The total starch content ranged between 73.9% d.b. (S1) and 79.7% d.b. (WF2). The damaged starch amount was influenced both by the kernel hardness and by the milling conditions. In fact, the highest starch damage was observed in the RF, which is obtained by a milling process involving impact breakers. Among wheat samples, the higher percentage of damaged starch was found in the hard variety flour (WF1). The alpha-amylase activity, enzymatically measured, varied widely (from 0.10 to 0.82 IU/g d.b.). In particular, the three WFs ranged from 0.61 to 0.82 IU/g d.b.; the two Ss showed a lower activity (S1 = 0.30 IU/g d.b., S2 = 0.50 IU/g d.b.); the lowest value, very closed to the detectable limit of the method, was observed in the RF (0.10 IU/g d.b.). 3.2 MVA analysis The pasting properties of flours can be influenced by several factors, such as starch granule size and structure, amylose/amylopectin ratio and molecular weight [2]. Large starch granules, for instance, tend to build up higher viscosity, but the situation can be tricky because the physical size of the granules makes starch more sensitive to shear. Beside starch characteristics, the pasting properties are strongly affected, as well known, by native alpha-amylase activity [1]. In Tab. 1, the pasting temperature and peak viscosity of WFs, Ss and RF are shown with regard to the heating rate. No influence of the heating rate was recorded at the first steps of gelatinisation as the pasting temperatures of the different samples were very similar. For wheat samples, the peak viscosity increased with the rise of the heating rate: WF showed the highest differences between the peak viscosities at the two extreme heating rates (1.57C/min and 107C/min); in the case of the Ss samples the gap was lower. Only the RF sample did not exhibit substantial variations related to the heating rate. Tab. 1. Raw material characterization (average 6 s.d.). Sample Moisture [%] Protein [g/ 100g d.b.] Total starch (TS) [g/100g d.b.] Damaged starch (DS) [g/100g d.b.] DS/TS [%] Alpha-amylase activity [IU/g d.b.] WF WF WF S S RF
4 Starch/Stärke 57 (2005) Influence of Heating Rate on Pasting Properties 567 The different behaviour of the three groups of samples (WFs, Ss, RF), recorded at the five heating rates, can be ascribed to many factors which were simultaneously involved in the progress of the MVA test. Certainly, the presence of endogenous alpha-amylase strongly affected the pasting profile of the flour sample. The action of alpha-amylase rapidly decreases the size of the starch molecules and thereby reduces the viscosity of a starch slurry [14]. The higher the heating rate, the shorter the time during which the endogenous alphaamylase can act on starch granules and the higher the peak viscosity. This phenomenon is clearly pointed out in Tab. 2: in particular WFs, which were characterized by the higher alpha-amylase activity (Tab. 1), exhibited a lowering of the peak viscosity when the heating rate applied to the slurry decreased. On the contrary, in the RF sample, in which the alpha-amylase activity was practically absent, this parameter reached nearly the same values at each heating rate. At the same time, the size distribution of starch granules also seemed to influence paste viscosity. As the size distribution of wheat starch granules is bimodal, the phenomena of swelling, pasting and gelatinisation take place in a wide range of temperatures [16]. Therefore, the small granules are still intact while the larger are already disrupted, especially when the changes in temperature are slower and the running time is longer. In this last case, the larger granules are highly swelled and more easily hydrolysed by the enzymes. Thus, the peak viscosity of the WF and S slurries was less and less pronounced at the lower heating rates. The different heating rates also strongly affected the sample viscosity during the remaining part of the viscoamylographic test (Fig. 1). In fact, when samples showed low viscosity during the heating phase, a similar behaviour at the beginning and the end of the cooling period was also observed. Thus, alpha-amylase activity modifies starch in a way that decreases the viscosity after cooling, i.e. the retrogradation tendency. It has been suggested that the outer branches of the amylopectin are hydrolysed and thus made unavailable for forming large amylopectin crystals [17]. These small crystallites do not form a three-dimensional network capable of promoting an important increase in viscosity during cooling [18]. The previously discussed phenomena are also related to starch damage promoted by shear. All MVA tests were carried out at a speed rate of 250 min 21. The instrument has a particular spindle geometry, whose mechanical effects on the slurry must be taken into account. In general, the longer the time of the analysis, the higher the mechanical damage. Moreover, the larger the size of the granule the higher its swelling and the more probable its disruption. The different extent of the mechanical damage can also be related to the fine molecular structure of the starch of the various flour samples, which can determine its swelling functionality. The WF commercial samples, in fact, showed the widest variations in peak viscosity (Tab. 2) and breakdown (Fig. 2), according to the increase of the heating rate applied to the slurry. The S samples were less influenced by the speed of temperature change (especially as regards their breakdown index), probably as a consequence of the more compact structure of durum wheat. 3.3 MVA analysis in the presence of AgNO 3 In order to demonstrate that the alpha-amylase action strongly influences the viscosity of starch the viscoamylographic tests were also conducted in the presence of AgNO 3, an efficacious alpha-amylase inhibitor. Crosbie et al. [15] found that 1 mm AgNO 3 was efficient in nullifying the effect of alpha-amylase on RVA peak viscosity tests on wholemeal. Tab. 2. Micro Visco-Amylo-Graph (MVA) pasting temperature and peak viscosity of the various flours suspended in distilled water, at the five different heating rates applied to the slurries: 1.5, 3.0, 5.0, 7.5 and 10.07C/min. Sample Pasting temperature [7C] Peak viscosity [BU] WF WF WF S S RF
5 568 M. Mariotti et al. Starch/Stärke 57 (2005) Fig. 1. Comparison between the viscoamylographic profiles of the various slurries at three different heating rates: 1.5, 3.0, 10.07C/min. The increase in the pasting viscosity of WF and S in the presence of the alpha-amylase inhibitor is particularly evident when the heating rate applied is low (Fig. 3). In particular, the peak viscosity of WF1 increased more than 100% its value at the lower heating rate and only 27 37% when the heating rate was 10.07C/min, a condition associated to a short period for alpha-amylase action. On the contrary, rice flour (RF), in which the alpha-amylase activity was quite absent, exhibited essentially the same MVA pasting profiles independently of the medium in which the sample was suspended.
6 Starch/Stärke 57 (2005) Influence of Heating Rate on Pasting Properties 569 Statistically significant correlation (p,0.05) was found at both heating rates between the increase of peak viscosity due to the enzyme inactivation with AgNO 3 and the alphaamylase activity (Fig. 4). Fig. 2. MVA breakdown index of the various slurries at the five different heating rates applied: 1.5, 3.0, 5.0, 7.5 and 10.07C. A slight increase in peak viscosity was observed in the presence of the alpha-amylase inhibitor when the heating rate applied to the slurries was raised from 1.5 to 107C/mm (Fig. 3). In this last case, the phenomenon can be connected to a less severe mechanical damage of starch. It can be noticed that the phenomenon is more evident in the WFs, less Fig. 3. MVA pasting profiles of the different flours suspended in distilled water or in 1 mm aqueous AgNO 3, at two different heating/cooling rates: 1.57C/min (on the left) and 10.07C/min (on the right).
7 570 M. Mariotti et al. Starch/Stärke 57 (2005) Microscopic observations The mechanisms proposed in order to explain the differences in viscosity profiles can be better understood using microscopic observations with normal and polarised light. The illustrations in Figs. 5, 6 and 7 were obtained from WF1, S1 and RF slurries heated from 307C up to three different temperatures (50, 60 and 707C), applying the two extreme MVA heating rates, 1.5 and 10.07C/min. Fig. 4. Correlation between the increase of peak viscosity, due to the enzyme inactivation with AgNO 3 and the alpha-amylase content of the various flours, at two heating rates: 1.57C/min (r) and 10.07C/min (d). evident in the Ss and absent in the RF, due to the small size and the particular organization of the rice starch granules. As is known, wheat starch granules are biconvex discs with a fairly regular circular outline and they are generally referred to as large (25 35 mm) and small granules (2 8 mm) [16]. The granules of rice are very small, being mostly 3 mm to 5 mm diameter with a polygonal shape; compound grains comprising several granules have often been observed and present an overall angular outline [16]. These features are clearly pointed out in Figs. 5 (a, c) (for WF1), 6 (a, c) (for S1) and 7 (a, c) (for RF). The structure of the starch granule is crystalline, with the amylose and amylopectin organising in such a way as to form radially oriented crystals [18]. This crystalline Fig. 5. Normal and polarised light observations of WF1 slurries after heating from 307C up to 50, 60 and 707C at two heating rates (1.5, 10.07C/min).
8 Starch/Stärke 57 (2005) Influence of Heating Rate on Pasting Properties 571 Fig. 6. Normal and polarised light observations of S2 slurries after heating from 307C up to 50, 60 and 707C at two heating rates (1.5, 10.07C/min). Fig. 7. Normal and polarised light observation of RF slurries after heating from 307C up to 50, 60 and 707C at two heating rates (1.5, 10.07C/min).
9 572 M. Mariotti et al. Starch/Stärke 57 (2005) arrangement is what gives rise to the phenomenon of birefringence. The presence of a Maltese cross can be easily observed in Figs. 5 (b, d) (for WF1), 6 (b, d) (for S1), 7 (b, d) (for RF). Its disappearance as a result of the temperature increase is evident, as shown in Figs. 5 (f, h) (for WF1), 6 (f, h) (for S1), 7 (f, h) (for RF). Starch granules lost their birefringence in temperatures higher than 607C and were no longer recognisable. As regards the influence of the two extreme MVA heating rates on the pasting properties of the different samples, it is interesting to observe the light microscopy images reported in Figs WF exhibited a consistent swelling power in comparison to the other samples, and this phenomenon was also strongly influenced by the heating rate applied to the slurry. From the series of images taken at 607C (Fig. 5: e, g) and at 707C (Fig. 5: i, k) it can be observed that the same temperature reached with the lowest heating rate promoted a greater breaking of starch granules. This can be ascribed to the effect both of a prolonged shear and of a prolonged action of alpha-amylase (correspondent to the lowest heating rate) on swollen granules. These phenomena are particularly evident in Figs. 5 (i) and 5 (k): in the first image, starch granules are swelled and partially broken, while in the second one they are swelled and still entire. These variations are present, but less marked, in the case of the S sample (Fig. 6: e, g; i, k). No clear differences between the images taken at 60 or 707C can be seen in the case of RF (Fig. 7: e, g; i, k). 4 Conclusions As a flour or a starch slurry is heated in a viscoamylograph, typically under a constant rate of shear, the increase in viscosity is measured as torque on the spindle and a curve (the viscosity profile) is recorded. This curve can be considered a reflection of the granular changes that occur during starch gelatinisation and retrogradation. It is well known that different starches generate different viscosity profiles. But the same flour or the same starch can also give rise to different curves if it is subjected to different operating conditions. Generally, the more slowly a starch/water dispersion is heated the thinner it will be at the identical endpoint temperature. This effect is closely related to the presence of alpha-amylase that decreases the size of starch molecules with an intensity that is proportional to the time elapsed to reach the temperature of enzyme inactivation at the different heating rates. The possibility offered by new instruments such as the Rapid Visco Analyser and the Micro Visco-Amylo-Graph of applying different heating/cooling rates enables one to study the effect of both gelatinisation and the alphaamylase action adopting operating conditions that better simulate the temperature profile of a real process. References [1] H. N. Dengate: Swelling, pasting and gelling of wheat starch, in Advances in Cereal Science and Technology, Vol. VI (Ed. Y. Pomeranz) American Association of Cereal Chemists, St. Paul, MN, 1984, [2] D. J. Thomas, W. A. Atwell: Starches, American Association of Cereal Chemists, Eeagan Press Handbook Series, St. Paul, MN, USA, [3] H. J. Thiewes, P. A. M. Steeneken: Comparison of the Brabender Viskograph and the Rapid Visco-Analyser. Statistical evaluation of the pasting profile. Starch/Stärke 1997, 49, [4] Newport Scientific, RVA Series Instruction Manual, Newport Scientific Pty. Ltd., Warriewood, Australia, [5] Brabender OHG, Micro Visco-Amylo-Graph Instruction Manual, Brabender OHG, Duisburg, Germany [6] C. E. Walker, A. S. Ross, C. W. Wrigley, G. J. McMaster: Accelerated Starch-Paste Characterization with the Rapid Visco-Analyzer. Cereal Foods World 1988, 33(6), [7] L. B. Deffenbaugh, C. E. Walzer: Comparison of starch pasting properties in the Brabender Viscoamylograph and Rapid Visco-Analyser. Cereal Chem. 1989, 66, [8] P. Limpisut, V. K. Jindal: Comparison of rice flour pasting properties using Brabender Viscoamylograph and Rapid Visco-Analyser for evaluating cooked rice texture. Starch/ Stärke 2002, 54, [9] I. L. Batey, B. M. Curtin: Effects on pasting viscosity of starch and flour from different operating conditions for the Rapid Visco Analyser. Cereal Chem. 2000, 77, [10] S. Dong Soon, J.-l. Jane: Comparison of starch pasting properties at various cooking conditions using the Micro Visco-Amylo-Graph and the Rapid Visco Analyser. Cereal Chem. 2003, 80(6), [11] AACC: Approved Methods of the AACC, 8th ed., American Association of Cereal Chemists, St Paul, MN, [12] AOAC: Official Methods of Analysis, 16th ed., Association of Official Analytical Chemists, Gaithersburg, MD, [13] G. B. Crosbie, W. J. Lambe: The application of the flour swelling volume test for potential noodle quality to wheat breeding lines affected by sprouting. J. Cereal Sc. 1993, 18, [14] L. S. Collado, H. Corke: Accurate estimation of sweetpotato amylase activity by flour viscosity analysis. J. Agric. Food Chem. 1999, 47, [15] G. B. Crosbie, A. S. Ross, T. Moro, P. C. Chiu: Starch and protein quality requirements of Japanese alkaline noodles (ramen). Cereal Chem. 1999, 76, [16] G. E. Moss: The microscopy of starch, in Examination and Analysis of Starch and Starch Products, (Ed. J. A. Radley) Applied Science Publishers Ltd., Ripple Road, Barking, Essex, England, 1976, [17] K. M. Krag, C. H. Poulsen: Functionality of Bakery Enzymes for Shelf Life Extension. AACC Annual Meeting, Minneapolis, MN, [18] D. A. V. Dendy, B. J. Dobraszczyk: Cereals and Cereal Products. Chemistry and Technology, Aspen Publication, Gaithersborg, MD, (Received: April 24, 2005) (Revised: July 5, 2005) (Accepted: July 7, 2005)
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