Application of the Rapid Visco Analyser as a Rheological Tool for the Characterisation of Mash Viscosity as Affected by the Level

of Barley Adjunct
Declan L. Goode1, Eric A. Wiltschko 1, Helge M. Ulmer 1 and Elke K. Arendt 2,3
ABSTRACT

J. Inst. Brew. 111(2), 165175, 2005 The application of the Rapid Visco Analyser (RVA) as a laboratory scale rheological tool for the characterisation of mash viscosity is the subject of this study. Studies were conducted to simulate an industrial mashing process, taking into account temperature /time, grist loads, adjunct amounts and enzyme levels. The RVA was used to characterise the effects of different ratios of malt : barley adjunct. The method was found to have the ability of not only detecting the major viscosity changes which occur during starch gelatinisation /liquefaction processes, but also the minor viscosity changes which were found to occur during the proteolytic and saccharification steps. Clear correlations were found between the level of barley adjunct and the output rheological data points of the peak viscosity at 50C (PV50, R2 = 0.9931), the rate of viscosity breakdown at 50C (BR50, R2 = 0.9522), the peak viscosity prior to gelatinisation (PVG, R2 = 0.9988), the area recorded under the gelatinisation curve (PGA, R2 = 0.9928) and the peak viscosity breakdown rate (VBR, R2 = 0.9783). The developed RVA rheological method is a useful tool for characterising grain quality (adjunct level) with regard to macromolecular viscosity compounds and the grains endogenous enzymatic capabilities. Key words: Barley adjunct, mashing, rheology, viscosity.

INTRODUCTION
Different instruments which operate on the basis of rheological viscosimetric measurements have been proposed for possible applications in the determination of grain quality in the brewing industry. These have included the amylograph, which has been used to assess the quality and degree of modification of a finely ground malt sample 20,21,27,28, the falling number apparatus, which has been used to determine, starch degradation during germination 22, the rapid visco-analyser (RVA), which has been used as a tool for monitoring the overall quality of malted
1 Department

of Food and Nutritional Sciences, National Food Biotechnology Centre, National University of Ireland, University College Cork, Ireland. 2 Department of Food and Nutritional Sciences, National University of Ireland, University College Cork, Ireland. 3 Corresponding author. E-mail: e.arendt@ucc.ie
Publication no. G-2005-0816-281 2005 The Institute of Brewing & Distilling

barley 3,610 and wheat adjunct additions 3, and the Rheoswing RSD 1-1 which has been used to characterise the quality of malt 26 and to show correlations between laboratory congress mashing results and rheological curves 16. However, none of these methods have become widely used for analysis in the malting, brewing and distilling industries. The most recently developed rheological measurement system for grain characterisation was developed by the present authors 1113. In that system a Bohlin CS-50 rheometer, together with a specially designed star-shaped paddle rotor was used. In comparison to the previously mentioned rheological methods, the Bohlin mash rheological method was designed to simulate more closely the operating conditions of an industrial mashing process by taking into account temperature /time, grist loads, adjunct amounts and enzyme levels. Validation of the method was carried out by carrying out model studies using purified /non purified substrates and enzyme types and dosage levels of known specification 13. The method was found to have the ability of not only detecting the major viscosity changes which occur during starch gelatinisation /liquefaction processes but also the minor viscosity changes which were found to occur during the proteolytic and saccharifaction mashing steps. In this method the effects on viscosity trends due to the grains chemical constituents and its endogenous enzyme components could be clearly visualised 13. When the method was applied to study the degree of grain modification, clear correlations were established between the rheological data and the simulated degree of modification of the grist. This could then be related back to the endogenous enzymatic activities of the grains, together with their levels of under-hydrolysed grain components 12. The aim of the present study was to assess whether the RVA is capable of determining a mash rheological profile. The RVA was used together with the previously developed mash rheological methods of Goode et al.1113 to assess the impact that barley adjunct addition has on rheological profiles obtained during mashing.

MATERIALS AND METHODS

Barley Unmalted and malted barley (Optic variety) (Table I) were obtained from The Malting Company of Ireland
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(Cork, Ireland). Distilled water was used as the mashing liquor. Grain analysis /enzyme activity determination The malted barley and unmalted barley grains were characterised using the standard methods of the European Brewery Convention (EBC) 5 (Table I). The total starch content of the grain was measured using the Megazyme Total Starch Assay Kit (International Association for Cereal Science and Technology (ICC) method 168 17) (Bray, Co. Wicklow, Ireland). Enzyme activity of the grain (-amylase activity, amylase activity, -glucanase activity) was determined using the following methods. The -amylase activity was measured using the Megazyme -amylase assay procedure (ceralpha method) using amylase HR reagent (ICC method 303 17). One unit of activity is defined as the amount of enzyme, that is required, in the presence of excess thermostable -glucosidase, to release one micromole of p-nitrophenol from BPNPG7 (non-reducing-end blocked p-nitrophenyl maltoheptaoside) in one minute under the defined assay conditions. It is termed a Ceralpha Unit. The -amylase activity was measured using the Megazyme -amylase assay procedure (betamyl method, RACI standard method). One unit of activity is defined as the amount of enzyme, that is required, in the presence of excess -glucosidase, to release one micromole of -nitrophenol from PNPG5 (p-nitrophenyl--D-maltopentaose) in one minute under the defined assay conditions. It is termed a Betamyl Unit. The -glucanase activity was measured using the Megazyme -glucanase assay procedure (azo-barley glucan (dyed substrate) method, RACI standard method). One unit of activity is equivalent to one International Unit of enzyme activity. This equals one mole of glucose reducing sugar equivalent released per minute at 30C and pH 4.6. Rapid Visco Analyser (RVA) The RVA (Fig. 1) was developed by the Bread Research Institute of Australia, in collaboration with the Wheat Research Unit of CSIRO 9. In this study, all rheological investigations were carried out using the RVA Super3 (Newport Scientific, Warriewood, Australia). The shear stress was measured with appliance of a constant shear rate. The RVA is a controlled shear rate instrument, in that it applies a constant shear rate (rpm) and then measures the resultant torque (force, shear stress). Torque and displacement are then converted to rheological format by means of the measuring system constants. Data can be produced in both a tabular and graphical format. Measuring system The RVA is a Searle type viscometer, with a stationary bowl and a combined stirring and sensing element suspended concentrically. Non-laminar or turbulent flow at high speeds prohibits absolute viscosity measurements, an effect which is exacerbated by the mixing-paddle design of the sensor element. However, it has been shown to correlate quite closely with similar viscosity data, determined with the amylograph 9. The RVA system, consists of a one166 JOURNAL OF THE INSTITUTE OF BREWING

Table I. Specifications of malt and barley used in the study. Malted barley Variety Harvest Moisture (%) Nitrogen (%) Total soluble nitrogen (%) Free amino nitrogen (mg/l) Total starch (% wet) -Glucan (%) Extract recovery (%) pH Colour (EBC) Viscosity (mPas) Filterability Apparent fermentability -Amylase (ceralpha units /g of wet grain) -Amylase (betamyl units /g of wet grain) -Glucanase (glucanase units /g of dry grain) n.d. = not determined. Optic Ireland 2002 4.01 0.053 1.400 0.036 0.058 0.002 107.73 1.96 n.a. 0.126 0.005 79.97 0.058 6.00 0.005 6.40 0.092 1.60 0.021 Normal 82.267 0.24 91.3 1.92 514.02 4.22 267.15 9.10 Unmalted barley Optic Ireland 2002 12.61 0.039 1.511 0.014 n.d. n.d. 70.275 0.346 3.35 0.042 n.d. n.d. n.d. n.d. n.d. n.d. 0.067 0.003 644.76 35.46 21.05 0.66

way plastic stirrer and aluminium sample canister. Before initiating a sample measurement, the plastic stirrer was placed in the torque measuring arm of the RVA and zeroed at 160 rpm against air. After pre-weighing the required water and sample weights into the aluminium sample canisters, the plastic stirrer was placed in the sample cup and jogged up and down for a few seconds to ensure sample mixing and to prevent sample clumping. The sample canister was then immediately placed in the hot block of the RVA, the plastic stirrer was re-attached to the instrument and the pre-programmed rheological profile was initiated. For all samples, an initial 10 s period of mixing at a constant shear rate of 960 rpm was carried out. For the remainder of the rheological measurement duration a constant shear rate of 160 rpm was applied. The shear stress was measured under a constant shear rate. In all investigations measurement points were taken every 0.5 s. Temperature profile In all experiments the temperature profile during the viscosity-measuring phase was kept constant. The temperature profile was as follows: 50C 30 min, 62C 40 min, 72C 20 min and 78C 5 min. The heating rate was 1C/min. The total time of mashing was 123 min. This temperature profile was chosen to simulate an upward infusion-mashing programme as is typically used during a brewery mashing programme. Experimental design Mash compositions consisting of unmalted barley (wet weight) at levels of 0, 20, 40, 60, 80, 85, 90, 95 and 100% together with malted barley (wet weight) were subjected to rheological measurement. Unmalted and malted barley were milled using a Bhler Miag laboratory scale disc mill (Bhler GmbH, Braunschweig, Germany) set at a fine grind setting of 0.20 mm. All samples were milled on the same day as the analysis was carried out. In all experiments 20.160 g 0.001 g of distilled water (50C) was weighed into the measuring cup. The sample (7.840 g 0.001 g (dry wt)) was then added to give a liquor to grist

Fig. 1. Basic design and operation of the RVA mash rheological profiling system.

ratio of 2.57:1. The sample was vigorously mixed with the plastic stirrer to prevent clumping. Rheological measurements were immediately performed. Interpretation of results Rheological measurement data were stored in the dedicated RVA software programme Thermocline for Windows (Newport Scientific, Warriewood, Australia). Using this data recording and analysis system, rheological profiles could be made. The profiles were then analysed and the chosen results transferred to a Microsoft Excel worksheet where further graphical representation and statistical analysis (ANOVA) of the data could be made. Experimental procedure Each experiment was repeated at least three times. The tabular results quoted are the mean values of the repeated experiments together with standard deviations. The graphical results shown represent one sample measurement. All viscosity data is given as mPa.s.

main enzymatic reactions occurring during mashing are the hydrolysis of proteins into peptides and free amino acids, the degradation of -glucan chains, the hydrolysis of pentosans (arabinan, xylans) and the breakdown of gelatinised starch into both fermentable (glucose, maltose and maltotriose) and unfermentable carbohydrates (DP 4) 24. These conversions occur due to the action of proteolytic, pentosanolytic, glucanolytic and amylolytic enzyme groupings respectively 26. To cover the optimal temperature range of each enzyme group 24, it was decided to simulate an industrial time /temperature curve which would typically be used in brew-house processing; a first rest (50C 30 min) for proteolysis, -glucan hydrolysis and pentosonase activity, a rest for the action of -amylase (62C 40 min), a rest for activity of -amylase (72C 20 min) followed by a temperature rest to promote enzyme inactivation (78C 5 min). The heating rate between the holding times was 1C/min. What was visualised during an RVA rheological analysis? A typical rheological profile (recorded during our trials) showing the recorded mashing conditions of time / temperature together with the chosen rheological measuring points is shown in Figure 2a. With reference to Goode et al.12 it can be observed that curve shapes obtained in these studies were similar to those obtained with the previously developed Bohlin mash rheological method.
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RESULTS AND DISCUSSION

It had been previously found with the Bohlin mash rheological method 1113 that much valuable information could be extracted from rheological curves, when the endogenous grain enzyme systems were optimised. The four

Fig. 2a. Graphical representation of a typical rheological profile, showing the main parts of the profile that are expanded in Figs. 2b, c and d.

Fig. 2b. Rheological representation of the 50C 30 min mash stand, A = start viscosity at 50C (SV50), B = peak viscosity at 50C (PV50), C = viscosity after 30 min at 50C (V50, 30), D = rate of viscosity breakdown at 50C (BR50).

The first step is mashing at 50C (Fig. 2b). At this mashing temperature the proteolytic enzymes (endo-proteinases and exo-peptidases (carboxypeptidase, amino peptidase and dipeptidase)) together with the endo--1,4-glucanase and pentosanases are reported to be active 24. During this step, the viscosity increased from the initial viscosity at 50C (SV50) to a maximum peak viscosity at 50C (PV50). It can be hypothesised that this first maximum is most probably primarily associated with solubilization of high molecular weight -glucans and mixing of the grain
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with mashing liquor, resulting in hydration and swelling of the grain components. Dextrins, arabinoxylans and glucans are known to contribute greatly to the viscosity of wort and beer. Whilst dextrins are found to be the primary determinant of viscosity, the impact of both arabinoxylan and -glucan have been found to be more pronounced at lower concentrations 25. The viscosity was then observed to decrease from PV50 to the viscosity recorded after 30 min mashing at 50C (V50, 30). This decrease (happening at a constant rate) is most likely due to the combined

Fig. 2c. Mash stands incorporating the temperatures for gelatinisation and liquefaction, E = gelatinisation temperature (GT) and the time at GT (TG), F = peak viscosity after gelatinisation (PVG), G = area under the gelatinisation peak with subtraction (PGAsubt), H = subtracted peak area, I = viscosity at breakdown (VB), J = rate of breakdown of viscosity from PVG to VB (VBR).

Fig. 2d. Mash stands at 62C 40 min, 72C 20 min and 78C 5 min, J = start viscosity at 62C (SV62), K = end viscosity at 62C (VE62), L = rate of viscosity breakdown at 62C (BR62), M = start viscosity at 72C (SV72), N = end viscosity at 72C (VE72), O = rate of viscosity breakdown at 72C (BR72), P = end viscosity at 78C (VE78).

polymer degrading effects of glucanolytic, proteolytic and xylanolytic enzymes which have optimum temperature activities at circa 50C 24. With an increase in mash temperature from 50C to 63C the viscosity was seen to increase rapidly (Fig. 2c). This temperature of viscosity increase is noted as being the point of the gelatinisation of the barley starch. During gelatinisation the starch granules take up warm water;

they soak and swell, causing a rapid increase in viscosity 18. Because the starch granules swell due to water absorption as they are heated, their volume fraction increases and reaches a maximum peak value (PVG). The PVG viscosity parameter is reflective of the concentration of barley starch and the level of amylase. Amylolytic enzymes (- and -amylase and debranching enzyme) can therefore act on this accessible starch substrate, thereby
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Fig. 3. Rheological profiles of grist compositions representing various ratios of malt : barley adjunct over the course of the 50C 30 min mash stand. (Top line = 5% malt; bottom line = 100% malt.)

decreasing the volume fraction of the granules in the suspension. Heating from 50 to 62C causes deactivation of the proteases and -glucanases 24. The rate of viscosity breakdown (VBR) following formation of the PVG gives an indication of the digestibility of the gelatinised starch. The mash was held at 62C (Fig. 2d). Further enzymatic breakdown of the gelatinised starch is occurring due to the combined action of the - and -amylases 18. Due to the action of -amylase, maltose production is continuing. The viscosity of the mash was thereby seen to reduce from the viscosity recorded at the start of the 62C hold (SV62) to the viscosity recorded at the end of the 62C hold (VE62). This viscosity decrease can be attributed mainly to the endo-hydrolysing actions of the -amylase and to a lesser extent to the exo-hydrolysing actions of amylase. The mash temperature was then raised to 72C. At this temperature, it is reported that -amylase is less active 24. Enzyme inactivation is an interaction between time and temperature. With an increase in time at temperatures 72C, -amylase is inactivated. The -amylases further break down the gelatinised starch and high molecular weight dextrins into low molecular weight dextrins and glucose 18. The viscosity of the mash was observed to reduce from the viscosity recorded at the start of the 72C stand (SV72) to the viscosity recorded at the end of the 72C stand (VE72). The temperature was then raised to 78C where enzyme inactivation is reported to occur and the final viscosity at 78C was attained (VE78). Application of the RVA rheological method to assess the rheological behaviour of mashes containing increasing levels of barley adjunct In order to simulate the effects of different ratios of malt:barley adjunct, together with different quantities of their endogenous hydrolytic enzymes, the RVA rheological method was applied to mashing systems consisting of unmalted barley together with malted barley at various proportions.
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Fig. 3 shows the effects of barley adjunct on the rheological curve when the proportion of malted barley was increased in the mashing system. As the % malt was increased, the observed trend (Fig. 3, Table II) was a decrease in SV50, PV50, V50,30 and BR50. As the percentage malt was increased from 5100%, the PV50 was seen to decrease from 1053 185 mPas to 199 10 mPas. Likewise, as the level of malt was increased, BR50 decreased (18.65 4.42 mPas to 0.24 0.61 mPas) (Table II). The higher data values are most likely related to the higher levels of undegraded macromolecular substances (i.e. -glucan, arabinoxylans and proteins) and lower amounts of endogenous enzymes at the higher levels of barley adjunct. Clear exponential correlations could be made regarding, the % malt from 5% to 100%, with regard to PV50 (y = 1122.2e0.0182x, R2 = 0.9931) and BR50 (y = 31.159e0.0415x, R2 = 0.9522). The results clearly confer with those previously shown in the Bohlin mash rheological studies 1113, in that the mash viscosity curve at 50C was dependent on the level of added adjunct/undermodified grain (P < 0.001). This can be attributed mainly to the higher levels of water-holding capacity of under degraded macromolecular substances (i.e. -glucan, arabinoxylan(s) and proteins). It was also found that lack of Table II. Rheological data of mashes containing increasing levels of unmalted barley adjunct over the course of the 50C 30 min mash stand. Malt (%) 0 5 10 15 20 40 60 80 100 SV50 (mPas) 1193 129 1015 180 950 137 842 62 727 46 491 64 354 18 232 18 176 17 PV50 (mPas) 1053 185 979 132 873 68 745 33 525 67 363 6 246 22 199 10 BR50 (mPas /min) 24 3 19 4 20 3 16 1 14 2 71 41 21 01

Full explanations of the abbreviations of the rheological parameters are outlined in Fig. 2b.

Fig. 4. Rheological profiles of grist compositions representing various ratios of malt : barley adjunct over the course of the gelatinisation /liquefaction mashing step. (Top line = 5% malt; bottom line = 100% malt.)

Table III. Rheological data of mashes containing increasing levels of unmalted barley adjunct over the course of gelatinisation/liquefaction. Malt (%) 0 5 10 15 20 40 60 80 100 GT (C) 58.9 0.1 58.9 0.3 58.6 0.7 59.2 0.2 58.6 0.9 59.4 0.2 59.6 0.0 59.6 0.1 59.7 0.1 TG (min) 39.08 0.12 39.09 0.29 38.78 0.68 39.32 0.17 38.72 0.84 39.56 0.14 39.78 0.09 39.72 0.08 39.94 0.06 VG (mPas) 530 28 488 9 388 14 400 27 313 21 308 19 285 28 229 7 211 12 PVG (mPas) 9930 44 5579 293 3556 132 2699 134 2106 52 1299 50 950 16 748 14 628 25 Time at PVG (min) 43.01 0.03 43.19 0.05 42.89 0.13 42.84 0.17 42.71 0.05 42.55 0.07 42.52 0.14 42.35 0.13 42.21 0.13

Full explanations of the abbreviations of the rheological parameters are outlined in Fig. 2c.

glucan hydrolysing enzymes, caused increased viscosity, presumably due to the combination of molecular asymmetry and high molecular weight of -glucan. Whilst it was shown that viscosity breakdown at 50C (BR50) could be attributed in part to glucanolytic enzymes 12, it can also be assumed that proteolytic and arabanolytic enzymes play important roles in this degradation process. However, it had been previously shown 14 in laboratory mashing experiments that little of the proteolytic breakdown (that resulted in solubilised nitrogen) could be attributed to the endogenous malt enzymes acting on the unmalted barley substrate 14. With regard to starch gelatinisation and amylolytic breakdown (Fig. 4), as the % malt was increased, the trends observed (Table III) were a significant (P < 0.001) decrease in the rheological parameters recorded immediately prior to primary gelatinisation and the parameters recorded after gelatinisation such as: PVG, time at PVG, total area under the peak curve (PGA), area under the peak curve with subtraction (PGAsubt), the rate of viscosity breakdown from PVG to the breakdown point (VB) and the rate of viscosity breakdown from PVG to the VE62. The primary gelatinisation temperature (GT) was

found to be significantly dependent on the level of barley adjunct (P < 0.05). It tended to rise as the percentage of malt in the grist was increased (mean value of 58.8C over the malt range of 020%; 59.7C at 100% malt). The time at gelatinisation (TG) increased, whilst the time to PVG reduced, as the percentage of malt was increased. The PVG decreased as the level of malt was increased (9930 44 mPas at 0% malt; 628 25 mPas at 100% malt) (Tables III, IV). When best-fit curves were applied to the data versus the % malted barley, clear exponential correlations could be made regarding the % malt from 5% to 100%, with regard to PVG (y = 18994x0.7342, R2 = 0.9988), PGA (y = 83438x0.6475, R2 = 0.9928), PGAsubt (y = 88052x0.8866, R2 = 0.9967) and the VBR (y = 1657.8x0.7901, R2 = 0.9783). The results clearly confer with those previously shown in the Bohlin mash rheological studies 11,12. In the studies 13, it was clearly shown that the level of amylolytic breakdown following starch gelatinisation determined the PVG. The VBR, was not only dependent on the level of amylase but also on the availability of under-hydrolysed substrate. A higher PVG (higher level of available substrate) resulted in a higher VBR. Therefore, both PVG and the VBR can be accurately repVOL. 111, NO. 2, 2005 171

Fig. 5. Rheological profiles of grist compositions representing various ratios of malt : barley adjunct following gelatinisation, at the mash stands of 62C 40 min, 72C 20 min and 78C 5 min. R = Secondary Gelatinisation Points.

Table IV. Rheological data of mashes containing increasing levels of unmalted barley adjunct over the course of liquefaction. Malt (%) 0 5 10 15 20 40 60 80 100 PGA 165306 8084 29091 276 19222 2230 14085 1164 11985 606 7839 676 6343 776 4324 63 4404 495 PGAsubtr 83578 5508 19494 575 11877 1047 7924 496 6705 267 3508 192 2360 128 1725 22 1424 42 VBR (mPas /min) 134.28 6.81 421.88 82.00 257.45 35.26 238.19 63.80 153.30 17.40 93.10 5.19 61.30 12.50 60.70 1.40 36.70 10.40 VBR62* (mPas /min) 73.70 4.23 46.30 1.53 33.94 1.78 26.67 0.98 15.00 0.50 10.20 0.35 8.11 0.29 6.61 0.37

* = VBR62 refers to the viscosity breakdown rate [(VPG VE62)/Time]. Full explanations of the abbreviations of the rheological parameters are outlined in Figs. 2c and 2d.

resented by the PGA and the PGAsubt. PGAsubt pertains to the viscosity increases due to starch gelatinisation and liquefaction processes, without taking into consideration the background under-lying viscosities associated with the non-starch chemical composition of the sample. It had previously been shown that ungelatinised starch alone was a minor component of the viscosity immediately recorded prior to gelatinisation 13. That inherent viscosity could therefore, be largely attributed to the remaining barley components of -glucans, arabinoxylans, proteins etc, whilst the higher viscosities after gelatinisation could be mainly attributed to low levels of amylolytic enzymes. With regard to the temperature stands at 63C and 72C (Fig. 5), as the % malt was increased, the trends observed (Tables IV, V) were a decrease in SV62, VE62, BR62, and VE72. These rheological data points were found to be significantly dependent on the level of unmalted barley (P < 0.001). When best-fit curves were applied to the data versus the % malted barley, clear correlations could be made regarding the % malt from 5% to 100% with regard to viscosity breakdown rate from PVG to VE62 (y = 297.34x0.8187, R2 = 0.9975) and the VE62 (y
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= 2111.3x0.4813, R2 = 0.9901). The result clearly shows, the methods capability of detecting the hydrolysing abilities of the combined effects of - and -amylase in reducing the molecular sizes of gelatinised starch and the resultant breakdown of the amylose, amylopectin structures resulting in high, medium and low molecular weight dexTable V. Rheological data of mashes containing increasing levels of unmalted barley adjunct over the course of saccharification and mash-off. Malt (%) 0 5 10 15 20 40 60 80 100 Drop-off rate* (mPas /min) 7239 1044 5097 267 3257 106 2395 143 1898 72 1098 49 739 7 597 36 476 30 BR72 (mPas /min) 1.14 0.66 1.78 0.39 1.80 0.75 1.74 0.45 1.84 0.42 1.26 0.21 1.18 0.36 0.98 0.20 VE78 (mPas) 2691 1002 482 42 299 43 304 10 208 22 201 4 211 19 152 25 153 8

*The drop-off rate refers to [(PGA VE78)/Time]. Full explanations of the abbreviations of the rheological parameters are outlined in Fig. 2d.

trins. However, no significant variability in results were found with regard to the BR72 due to the level of barley adjunct. This would suggest the inability of the method to show the sugar-hydrolysing effects of the -amylase, in converting high molecular weight dextrins to medium and low molecular weight sugars. The VE78 was found to weakly correlate with level of barley adjunct (y = 705.62x0.3323, R2 = 0.8960). The mash viscosity at this point is reflective of both the quality and degree of modification of the grist material, the effectiveness of the mashing programme and the unsolubilised grain components /particles and the wort soluble proteins (high and low molecular weight), -glucans and sugars. From previous studies 14 it has been shown that as the ratio of malt: barley adjunct is increased, the resultant worts are higher in total levels of extract. This extract is composed of lower levels of high molecular weight -glucan, increased levels of fermentable sugars, glucose and maltotriose and unfermentable middle molecular weight dextrins (DP4 DP7) together with higher levels of total soluble nitrogen (high and low molecular weight nitrogen fractions). In addition, it is composed of lower levels of high molecular weight dextrins (DP 8). At the lower levels of grist modification, the higher levels of high molecular weight dextrins (DP 8) and high molecular weight -glucans of the wort together with undegraded arabinoxylans would contribute greatly to the mash viscosity 25, whilst the residual undegraded un-solubilised viscosity creating macromolecular components of the grain particles would also contribute greatly to the mash viscosity. In addition, when one looks more closely at Fig. 5, occasional increases in viscosities are noted. This was also noted in other rheological studies 12,13,15. These increases are more noticeable at the higher levels of unmalted barley adjunct. It has already been hypothesised that these peaks represent secondary gelatinisation points 12,13. The gelatinisation temperature of starch is influenced not only by the type of starch but also by the size and structure of its starch granules 23. It is well reported in the case of bimodal barley starches, that the small starch granules have higher temperatures of gelatinisation 19. Barley starch granules are reported to be of two different granular sizes 23. The large and smaller granules are referred to as A- and B-type granules respectively. Reports show that there is a wide variation in the granule size distribution of barley starch. In general small granules (B-type) are taken to be those less than 6 m in diameter (more often 24 m in diameter). These constitute 8090% of the total number of starch granules but generally only 1015% of the total starch weight. Large granules (A-type) range in size from 10 to 30 m but generally lie in the 1520 m range. They constitute a small proportion (1020%) of the total number of starch granules but a high proportion (85 90%) of the total weight of starch 23. In most cases, raw unmalted barley has higher levels of small starch granules, than malted barley, since during the malting process the small granules are preferentially degraded 2. The reported negative impacts of small starch granules are that due to higher temperatures of gelatinisation they are less digestible during mashing. It is difficult for amylases to attack these granules during mashing as they are surrounded by more protein matrix in the endosperm cells 4.

In addition, they can impede wort filtration by cross-linking with other polymers 1. It had been previously shown in the Bohlin mash rheological studies 13 that, the increases in viscosity, due to the secondary gelatinisation processes, were found to closely correlate with the level of added amylase. It was reported, that if mash amylase levels were high enough, then these secondary starch viscosity peaks would not be observed under the Bohlin mash rheological method 13. In agreement with the previously developed Bohlin mash rheological method 1113, the developed RVA rheological method gives an in-sight into the various biochemical and physical processes that are happening during mash processing of these malt:barley adjunct combinations. Indications of the levels of endogenous enzymes within the systems and their abilities to breakdown the macromolecular viscosity causing substances are very evident. This method provides a very good indication of the intrinsic mash-hydrolytic processes of proteolysis, -glucan breakdown and starch conversion. Rather than just gaining information regarding the chemical and physical properties of the grains or their wort soluble end products, the method more closely follows the grain processabilities during mash conversion processes. Because the time /temperature conditions closely represent industrial type processes, in comparison to other RVA studies where the grains endogenous enzyme systems were not optimised (such as Glennie Holmes 610), it can give the brewer closer indications as to how the addition of different levels of barley adjunct could perform in an industrial brewing process. In addition the results from these experiments have demonstrated the potential of the RVA mash rheological methods for many other applications. These may include the assessment of the application of commercial enzyme preparations when brewing with high adjunct ratios, the assessment of different barley, malt genotypes for brewing purposes. Likewise, with further development, the RVA mash rheological method could provide a very useful tool for indirectly monitoring the biochemical and physical transformations that are taking place during the malting process. Green malt samples can be taken during the various stages of the malting process and subjected to the developed rheological method. Enzyme development and modification processes during barley germination, together with enzyme preservation /inactivation during kilning regimes can thus be followed. The RVA mash rheological method could thus provide the maltster with an increased physical knowledge of their processes and thus act as a tool to identify potential problems and enable critical decisions to be made. This study has also provided us with the possibility of making general comparisons between the RVA and the previously mentioned Bohlin method as mash rheological determination instruments. With reference to a previous publication by the current authors 1113 where the Bohlin mash rheological method was also successfully utilized for looking at different ratios of barley adjunct, close comparisons could be made between the results of those methods and the results of the current study. Therefore, both methods can be deemed suitable for determining mash rheological profiles. The main advantages of the deVOL. 111, NO. 2, 2005 173

veloped RVA mash rheological method, over the previously mentioned Bohlin mash rheological method, are that in comparison to the Bohlin method (where results were quoted as arbitrary units), the RVA viscometer is fully designed and calibrated to give readouts in standard viscosity units (mPa.s). The RVA was also judged by the current authors to be less sensitive to localised disturbances in the laboratory environment such as bench vibrations, air draughts etc. It was also judged to be more userfriendly, than the developed Bohlin method, in that oneway plastic paddles and disposable aluminium canisters, together with an easy to operate dedicated software programme are employed. Therefore, samples can be processed more quickly, with less risk of sample cross contamination. Perhaps the most notable advantage is that, the RVA is considerably cheaper to purchase. Therefore, transfer of the developed methods to the malting/brewing/distilling industries, would not be hampered by excessive costs and initial capital investment.

CONCLUSIONS
This study has shown that the RVA is an instrument capable of carrying out a mash rheological analysis. An RVA rheological profile can provide the brewer with a detailed picture of the processes that are taking place during mashing. The RVA has the ability of not only detecting the major viscosity changes which occur during starch gelatinisation /liquefaction processes, but also the minor viscosity changes which were found to occur during the mash proteolytic and saccharification steps. Clear correlations were found between the rheological data over the course of the 50C, 62C, 72C, 78C mash stands and the controlled amount of barley adjunct. Increasing the level of raw barley adjunct resulted in an increase in viscosities over the course of the 50C, 62C, 72C and 78C mash stands. This was indicative of the viscosity causing un-modified structural components of barley adjunct together with the fully-modified structural components of the malted barley, interacting with the endogenous viscosity hydrolysing malt enzymes. RVA mash rheological profiling can thus offer an insight into how raw materials interact. Increased preknowledge of such processes may provide the maltster / brewer /distiller with more functional information regarding raw materials and brew-house processing conditions.
ACKNOWLEDGEMENTS

The authors would like to acknowledge that this project was partly financed by Enterprise Ireland and the Food Industry Research Measure (FIRM) under the National Development Plan, 20002006 with co-financing by the European Commission under the European Regional Development Fund. In addition, we would like to gratefully acknowledge the part funding of the Rapid Visco-Analyser by Irish Distillers Ltd, Pernod Ricard Group, Midleton, Co. Cork, Ireland.
REFERENCES 1. Barrett, J., Clapperton, J.F., Divers, D.M. and Rennie, H., Factors affecting wort separation. Journal of the Institute of Brewing, 1973, 79, 407413.

2. Bathgate, G.N. and Palmer, G.H., The in vivo and in vitro degradation of barley and malt starch granules. Journal of the Institute of Brewing, 1973, 79, 402406. 3. Broadhead, A.L., Brosnan, J.M. and Bringhurst, T.A., The role of the Rapid Visco Analyser (RVA) in minimizing distillery processing problems. In: Distilled Spirits: Tradition and Innovation. J.H. Bryce and G.G. Stewart (Eds.). Publisher: Iowa State, 2004, Chapt. 13, pp. 8995. 4. Ellis, R.P., The use of high amylase barley for the production of whiskey malt. Journal of the Institute of Brewing, 1976, 82, 280281. 5. European Brewery Convention. Analytica EBC, 5th ed. Verlag Hans Carl: Nrnberg, Germany, 1998. 6. Glennie Holmes, M.R., Studies on barley and malt with the rapid viscoanalyser: (I) The effects of the variation in physical and chemical parameters. Journal of Institute of Brewing, 1995, 101, 1518. 7. Glennie Holmes, M.R., Studies on barley and malt with the rapid viscoanalyser: (II) The effects of modification on viscograms. Journal of Institute of Brewing, 1995, 101, 1928. 8. Glennie Holmes, M.R., Studies on barley and malt with the rapid viscoanalyser: (III) The prediction of malting potential from viscograms. Journal of the Institute of Brewing, 1995, 101, 2932. 9. Glennie Holmes, M.R., Studies on barley and malt with the rapid viscoanalyser: (IV) Amyloclasis, amylolysis and cytoclasis of malt and malting quality. Journal of the Institute of Brewing, 1995, 101, 3338. 10. Glennie Holmes, M.R., Uses for the rapid viscoanalyser in a brewery. Tech. Q. Master Brew. Assoc. Am., 1995, 32, 7275. 11. Goode, D.L., Rapp, L., Schober, T.J., Ulmer, H.M. and Arendt, E.K., Rheological studies simulating the brewery mashing process. Proceedings of the World Brewing Congress, San Diego, California, USA, Oral Presentation 97. 12. Goode, D.L., Rapp, L., Schober, T.J., Ulmer, H.M. and Arendt, E.K., Development of a new rheological laboratory method for mash systems its application in the characterization of grain modification levels. Journal of the American Society of Brewing Chemists, 2005, 63(2), 7686. 13. Goode, D.L., Ulmer, H.M, and Arendt, E.K., Model studies to understand the effects of amylase addition and pH adjustment on the rheological behaviour of simulated brewery mashes. Journal of the Institute of Brewing, 2005, 111(2), 153164. 14. Goode, D.L., Wijngaard, H.H., and Arendt, E.K., Mashing with unmalted barley impact of malted barley and commercial enzyme (Bacillus subtilis) additions. Tech. Q. Master Brew. Assoc. Am., 2005, 42, 184198. 15. Hermann, J. and Sommer, K., Development of a device to determine the rheology of mash. Tech. Q. Master Brew. Assoc. Am., 2001, 38, 5558. 16. Hoog, D., Senge, B. and Annemuller, G., Rheologische Kontrolle von Labormaischen. Brauwelt, 1997, 37, 16061610. 17. ICC Standards Standard methods of the International Association for Cereal Chemistry (ICC). Hans Kock Buch- und Offsetdruck GmbH, Germany. 18. Kunze, W., Technology Brewing and Malting, 2nd edition, VLB: Berlin, Germany, 1999. 19. Lindeboom, N., Chang, P.R. and Tyler, R.T., Analytical, biochemical and physicochemical aspects of starch granule size, with emphasis on small granule starches: a review. Starch / Strke, 2004, 56, 8999. 20. Lloyd, W.J.W., Evaluation of malt in a malting company. Journal of the Institute of Brewing, 1978, 84, 7778. 21. Lloyd, W.J.W., The relevance of amylogram characteristics in malt evaluation. Tech. Q. Master Brew. Assoc. Am., 1979, 16, 6067. 22. Lorenz, K. and Kulp, K., Sprouting of cereal grains Effects on starch characteristics. Starch, 1981, 33, 183187. 23. MacGregor A.W. and Fincher, G.B., Carbohydrates of the barley grain. In: Barley: Chemistry and Technology. A.W. MacGregor and Bhatty, R.S., Eds. American Association of Cereal Chemists, Inc: St. Paul, Minnesota, USA, 1993, pp. 73130.

174

JOURNAL OF THE INSTITUTE OF BREWING

24. Narziss, L., Die Technologie der Wurzebereitung, 7th edition, Ferdinand Enke Verlag: Stuttgart, Germany, 1992. 25. Sadosky, P., Schwarz, P.B. and Horsley, R.D., Effect of arabinoxylans, -glucans, and dextrins on the viscosity and membrane filterability of a beer model solution. Journal of the American Society of Brewing Chemists, 2002, 60, 153162. 26. Senge, B., Schwarzlos, B., Blochwitz, R. and Annemuller, G., Rheological examination of mashing in the brewery process. Applied Rheology, 1996, 6, 1118.

27. Woodward, J.D. and Oliver, W.B., Varietal features affecting mashing performance. Proceedings of the European Brewery Convention Congress, Zurich, 1989, pp. 299306. 28. Yoshida, T. and Yamada, K., Assessment of malt quality with the Brabender Viscograph. Journal of the Institute of Brewing, 1970, 76, 455464.

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