Effects of Mashing Parameters on Mash β-glucan, FAN and Soluble Extract Levels

Transcription

1 Effects of Mashing Parameters on Mash β-glucan, FAN and Soluble Extract Levels F. Kühbeck 1,3, T. Dickel 1, M. Krottenthaler 1, W. Back 1, M. Mitzscherling 2, A. Delgado 2 and T. Becker 2 ABSTRACT J. Inst. Brew. 111(3), , 2005 To gain further technological knowledge of mashing, pilot scale mashing trials were carried out varying mashing programme (upward/isothermal mashing), milling procedure, grist:liquor ratio, time of mash stands, and grist modification level (well and poorly modified malt). During mashing β-glucan, free amino nitrogen (FAN) and extract contents were analysed as key indicators for cytolysis, proteolysis, and amylolysis, respectively. The malt modification was of major impact for the β-glucan release in contrast to a variation of milling procedure and of grist:liquor ratio. Extended stands lead to increased final values only for poorly modified malt. Similarly, FAN release was predetermined by malt modification while variation of milling and of grist:liquor ratio was not relevant in contrast to stand extension. None of the variations applied influenced extract yield as long as gelatinization temperature was reached. Greatest gains occurred around 57 C. In conclusion, wort quality is critically determined by malt modification. Mashing with well modified malt in combination with short stands should result in a mash of low β-glucan and sufficient FAN level without losing extract yield. However, for poorly modified malt the variation of mashing parameters has an impact on the key indicators in which cytolysis plays the dominating role. Key words: β-glucan, extract, FAN, grist modification, mashing, milling. INTRODUCTION Mashing is the first biochemical process step of brewing and completes the enzymatic degradation procedures started during malting. The mashing process is of highest technological relevance for all following processes of wort and beer manufacturing. Besides, it is time consuming and therefore cost-intensive. 17 The three biochemical basic processes taking place during mashing and investigated in this work were cytolysis, proteolysis and amylolysis, which are indicated by β-glucan, FAN and extract concentration in mash, respectively. 1 Lehrstuhl für Technologie der Brauerei I, Technische Universität München, Weihenstephaner Steig 20, D Freising, Germany. 2 Lehrstuhl für Fluidmechanik und Prozeßautomation, Technische Universität München, Weihenstephaner Steig 23, D Freising, Germany. 3 Corresponding author. Florian.Kuehbeck@wzw.tum.de Publication no. G The Institute of Brewing & Distilling In 1967 Erdal and Gjertsen presented mashing trials, in which extracts of malt prepared at low temperatures were free of β-glucans. 8 The β-glucans were not dissolved until starch was gelatinized at about 60 C. From a mashing-in temperature of 55 C or above the β-glucan content of the resulting wort increased with a peak at 65 C, where no effect of endo-β-glucanases could be detected due to its complete inactivation at 65 C. They concluded β-glucans present in wort do not occur as soluble compounds in malt, but had to derive from enzymatic degradation of hemi-celluloses during mashing. 8 Narziß and Litzenburger investigated the relation of malt quality, mashing intensity and gum content. 13 For well modified malt they found an increasing extract content from beginning of gelatinization with increasing activity of amylases. The latter had a maximum at 65 C in isothermal mashing trials. The β-glucanases had highest activities at low temperatures of around 35 C, while at higher temperatures, particularly from 55 C up, the activities diminished drastically or were not detectable any more due to heat inactivation. When upward mashing was applied the extract content suddenly rose by heating from 50 to 65 C. At 65 C the final value was nearly reached. The β-glucan content was affected by extract release and enzymatic activities, similarly to the isothermal mashing trials. The highest values of β-glucan were observed when mashing-in at a temperature of 65 C and were not reduced at any further stage of the mashing process. 13 On the other hand, for poorly modified malt Narziß and Litzenburger found maximal extract concentration at 60 C for isothermal mashing. The β-glucanases activities were much lower compared to the well modified malt with their maximum being at C. Up to a temperature of 55 C the β-glucan level had its maximum after 30 min of mashing which decreased due to degradation by β- glucanase activity as mashing continued. However, the degradation reduced with increasing temperatures exceeding 55 C. At 65 C and particularly at 70 C the extraction of β-glucans was clearly delayed. When upward mashing was applied, the extract content showed a retarded rise at low temperatures due to lower amylase activities. Due to insufficient malt modification this could be also observed for β-glucanases. The final β-glucan contents were around six times the content compared to well modified malt. 13 Einsiedler et al. developed kinetic models of biochemical and technological processes during mashing. 5 7 According to the authors increasing grist fineness resulted in a greater surface and therefore, the released amino acids 316 JOURNAL OF THE INSTITUTE OF BREWING

2 were dissolved more quickly. When isothermal mashing at 10 C was applied, hardly any release of amino acids could be detected due to low enzyme activities. At temperatures of 35 and 50 C the enzymatic activities were higher causing significant differences in the amino acid release. Here, a better malt modification and a finer grist composition led to a more intensive proteolysis and therefore, higher amino acid concentrations in the mash, respectively. Within this trial the well modified malt applied as fine grist had the highest absolute concentrations in amino acids. However, at 50 C or above, these differences became smaller towards the end of mashing at a total mashing time of 60 min. The kinetic model based on the enzymatic catalysis and the enzyme inactivation gave a calculated proteolysis optimum of 47 C for the well modified malt and 52 C for the poorly modified malt without observing a difference for fine and coarse grist. Finally, it was concluded that the increase of amino acid concentration due to proteolytic activity during mashing was rather small compared to the amino acid concentration present in the mash at the end of mashing, which was only around 20% higher after 60 min of mashing. Therefore, the desired concentration of mg/l was available even without the proteolytic activity during mashing. 5 This is in agreement with Riis who found only about 1% of the potential proteolytic activity under mashing conditions compared to the activity during malting. 15 In terms of amylolysis, Einsiedler et al. 6 did not observe an influence of malt modification on reaction kinetics. Similarly, the grist fineness played only a minor role in amylolysis. Thus, finer grist did not cause a faster release of low molecular sugars, because the enzymatic reaction sites did not increase due to fine milling. However, the activities of α-amylase and β-amylase were halved in poorly modified malt. In the mashing trials only minor swelling and gelatinization took place during the 50 C stand causing less starch degradation. When heated to 62 C the starch content decreased quickly and further heating to 70 C completed starch degradation. The enzyme activity had its maximum at 50 C, decreasing with higher temperatures. Despite the gradual inactivation at increasing temperature, the hydrolysis of starch reached its maximum at around C for kinetic reasons. 6 In terms of cytolysis, low mashing temperatures were reported to cause only low β-glucan concentrations. Only at C or above did the β-glucan concentration increase. 7 Erdal and Gjertsen evaluated this increase as a result of starch gelatinization rather than an enzyme effect due to the inactivation of malt enzymes above 60 C. 8 On the other hand, Bamforth et al. 3 attributed the increase in β-glucan concentration to enzymatic release of β-glucan from the cell walls by β-glucan solubilase. The latter had an optimum activity at 60 C and was still active at 73 C. More recent investigations suggest this enzyme is heatstable and is not derived from microflora growing on barley. Feruloyl esterase activity seems to play a role in β- glucan solubilization. 4 The released β-glucan was further degraded to glucan dextrins and oligosaccharides, particularly by endo-β-1,3-glucanase and endo-β-1,4-glucanase. Since β-glucanase is very heat sensitive its activity was almost halved at 50 C and no activity was found after 15 min of mashing from temperatures of 65 C or above. 7 In agreement with Bamforth et al. β-glucan solubilase was very heat stable and developed activity at least up to 50 C, lost activity only slightly at 65 C and had still not been completely inactivated at 72 C. Insufficient malt modification led to a significant lower enzyme activity, however no difference was detected in the activities of coarse and fine grist. Besides, mashing temperature played an important role for the β-glucan release: while at 50 C only small amounts of β-glucan were released within 60 min, at 72 C a steep increase was observed within 10 min. Here, the β-glucan content of the fine grists was 1.5 to 3- fold compared to coarse grist. Additionally, the poorly modified malts exhibited a much higher β-glucan content compared to the well modified malt. 7 Only 15 min after mashing-in at isothermal 55 C, β-glucanase activity dropped by 75% compared to 35 C, while β-glucan solubilase activity was close to its maximum activity. Considering fine grist, not only the enzymatic release but also the further degradation of β-glucan per unit time was increased due to accelerated diffusion and enzymatic activity. Therefore, more β-glucan was degraded at low temperatures and thus, was not available to increase the final β-glucan contents in wort. Conclusion of the investigations of Einsiedler et al. were that the grist fineness did not make a difference in terms of starch degradation, but did in terms of β-glucan and protein degradation. More β- glucan and amino acids were released from finer grist within the first min of isothermal mashing due to a higher number of reaction sites available in fine grist. While an intensified proteolysis may be desirable to increase the amino acid concentrations, fine grinding increased β-glucan content as well, which was not appreciated at the time. 7 Therefore, applying finer grist does not seem to be an optimum measure to achieve shortening of mashing procedure for poorly modified malts. Schneider 16 investigated the influence of the grist fineness on the mashing process. The finer the grist the higher was the degradation rate of high molecular substances, which was particularly true for protein degradation. When fine grist was used, FAN concentrations were high already at the beginning of mashing. For coarser lauter tun grist the same level was reached only at the end of the protein stand. The extract content was increased right from the beginning of mashing, but the difference between fine and coarse became smaller as mashing proceeded. Therefore, he concluded finer grist is primarily capable of shortening mashing rather than increasing yield. The increase of the grist:liquor ratio was limited by interference of α-amylase activity. However, when using fine grist, the increased mass transport and exchange due to finer grinding were increased, so that almost no product inhibition could be observed, even with a grist:liquor ratio of 1: In terms of β-glucan, hardly any difference could be detected for different fineness grades, when a grist:liquor ratio of 1:3.5 4 was applied. For higher concentrated mashes (ratio 1:2.5 3) finer grists caused lower β-glucan contents. 16 These days brewers carry out mashing by exposing the mash to temperature stands for fixed durations. The disadvantage of an inflexible procedure becomes apparent, when varying malt qualities have to be processed: for well modified malts shorter stands may be sufficient to reach the pre-determined targets. On the other hand, when VOL. 111, NO. 3,

3 Table I. Analysis of malts according to MEBAK 14 and classification of analytes as modification indicators (d. m. = dry matter; min = minute; C = cytolytic modification indicator; A = amylolytic modification indicator; P = proteolytic modification indicator) Analyte Modification indicator MEBAK no. Well modified malt Poorly modified malt water content % 7.0% extract as is A % 76.1% extract in d. m. A % 81.9% fine-coarse difference C % 5.6% friability C % 73.9% glassiness C % 5% ph VZ 45 C % 28.2% saccharification time A min min protein in d. m % 11.6% soluble N P mg/l 579 mg/l Kolbach index P % 31.2% viscosity 12% (w/w) C mpa*s 2.72 mpa*s viscosity 8.6% (w/w) C mpa*s 2.00 mpa*s poorly modified malt has to be processed by the same mashing method, the technological targets might not be reached due to stands shorter than required. The aim of this research project was to obtain technological basic data of mashing, since cytolytic degradation has a major impact on beer filterability, for which β-glucan is a key indicator. Furthermore, proteolysis determines the FAN content in mash and as a result affects yeast nutrition. Finally, amylolysis affects extract concentration and thus brewhouse yield, which is of great commercial interest. Therefore, brewing trials were carried out based on an infusion mashing method (upward mashing) under variation of mashing parameters, which are: mashing programme (standard upward infusion/ isothermal mashing), milling procedure (roller/hammer milling), grist:liquor ratio (1:4/1:5), time of mash stands (20/30/40 min) and grist modification level (0/30/50/70/100% of poorly modified malt). MATERIALS AND METHODS Raw materials A well modified malt and a poorly modified malt (both Weyermann, Bamberg/Germany) were used for the trials. The poorly modified malt (barley variety Scarlett) is characterized by good amylolytic, but relatively low cytolytic and proteolytic modification, while the well modified malt (barley variety Scarlett) shows good amylolytic, cytolytic and proteolytic numbers (Table I). The mash water was Table II. Analysis of mashing water according to MEBAK 14. Analyte MEBAK no. Mash water calcium hardness mmol /L magnesium hardness mmol /L total hardness mmol /L carbonate hardness mmol /L non-carbonate hardness calculated according 1.00 mmol /L to m value ml p value ml residual alkalinity mmol /L obtained from Bayerische Versuchs- und Lehrbrauerei, Weihenstephan/Germany. Its water quality (Table II) relates to conventional brewing water for pale lager beers. 9 Mashing trial design All mashing trials were carried out in the pilot brewery of Lehrstuhl für Technologie der Brauerei I, Weihenstephan/Germany. For milling purposes either a tworoller mill (Künzel, Kulmbach/Germany) with a milling gap of 0.8 mm (coarse grist) or a hammer mill (Werkhuizen Schepens, Dendermolde/Belgium) with a mesh size of 2 mm (fine grist) was applied. The grist grading analysis according to MEBAK method is shown in Table III. The pilot brewhouse consists of brew water reservoir, mash kettle, lauter tun, brew kettle, whirlpool and plate heat exchanger (Kamm, Ottershausen/Germany). The mash kettle has a volume of approximately 60 L, is equipped with a stirrer, and is steam heated with an automated temperature control for controlling mash stands and allows temperature increases of 1 C/min. A 10-kg portion of malt was milled and mashed in by adding a respective volume of mash water previously heated to mashing-in temperature. The mash trials were carried out according to the experimental design presented in Table IV. The standard mashing procedure (no. 1 in Table IV) was repeated nine times, while all other mashing procedures were carried out once. For standard mashing procedure a grist modification of 30:70 (poorly: well modified malt) was chosen since this ratio represents an average quality of commercially available malts. Table III. Grist grading for roller and hammer milling according to MEBAK 10 (well modified malt; dry milling). Fraction Recommended value for lauter tun grist [%] Roller milling [%] Hammer milling [%] husks coarse grits < fine grits I fine grits II grit flour powder dust < JOURNAL OF THE INSTITUTE OF BREWING

4 Table IV. Experimental design of mash trials (T = temperature). Mash no. / variation Milling Grist modification [poorly : well modified malt] Mash stand T [ C] Mash stand [min each] Total mash time [min] Grist:liquor ratio [kg:liters] 1 / standard coarse 30:70 45 /62 / :4 2 / isothermal 60 C coarse 30: :4 3 / isothermal 20 C coarse 30: :4 4 / milling coarse 100:0 45 /62 / :4 5 / milling fine 100:0 45 /62 / :4 6 / grist:liquor ratio coarse 30:70 45 /62 / :4 7 / grist:liquor ratio coarse 30:70 45 /62 / :5 8 / mash stand coarse 100:0 45 /62 / :4 9 / mash stand coarse 100:0 45 /62 / :4 10 / mash stand coarse 100:0 45 /62 / :4 11 / mash stand coarse 0: /62 / :4 12 / mash stand coarse 0: /62 / :4 13 / mash stand coarse 0: /62 / :4 14 / grist modification coarse 0: /62 / :4 15 / grist modification coarse 30:70 45 /62 / :4 16 / grist modification coarse 50:50 45 /62 / :4 17 / grist modification coarse 70:30 45 /62 / :4 18 / grist modification coarse 100:0 45 /62 / :4 Sampling and analyzing During mashing, samples of approximately 400 ml volume were taken at intervals of 10 min (except during the 62 C stand with sampling intervals of 5 min) from the stirred kettle and were cooled to approximately 10 C in a cold water bath within 10 min to stop further enzyme activity. Right after mashing was completed the mashes were paper filtered (order no , Machery-Nagel MN 514 1/4, Düren/Germany) and analyzed for β-glucan, FAN and extract. Fluorimetric detection of high molecular β-glucan (molecular weight >10,000 Daltons) was carried out according to MEBAK method using Skalar San Plus Segmented/Continuous Flow Analyser SA 4000 coupled to Fluorimeter FL6300-D (Skalar Analytical B.V., De Breda/The Netherlands). The latter was also used to detect FAN applying the photometric method with Ninhydrin according to MEBAK method The extract content of the mash samples was detected by a density meter according to the oscillating U-tube principle (Anton Paar GmbH, Graz/Austria), MEBAK method Each sample was analysed in duplicate. RESULTS AND DISCUSSION Variation of mashing programmes (standard upward infusion versus isothermal mashing (20 C; 60 C); Table IV, no. 1 3) When carrying out a standard upward infusion mashing method the β-glucan concentration showed a constant and low concentration during the 45 C mash stand (Fig. 1). When the temperature exceeded approximately 57 C the concentration suddenly increased and reached a saturation level of around 500 mg/l at the end of mashing for a poorly to well modified malt ratio of 30:70. Due to the nature of the method the analytical error is quite big. Comparing the standard mashing method with isothermal mashing methods at 20 and 60 C, an increase of β-glucan concentration could be observed right after mashing-in at 60 C because of gelatinization and enzymic β-glucan solubilization, while no increase of β-glucan was detected when mashing isothermally at 20 C (Fig. 1). The β-glucan concentrations are a result of the activity of endo-β-glucanases (endo-β-1,3-glucanases and particularly endo-β-1,4- glucanases) as well as β-glucan solubilases. In the range below 57 C β-glucan solubilases release β-glucan which is further degraded by the endo-β-glucanases. 2,11,18 Once the temperature reached the inactivation temperature of endo-β-glucanases (approximately 55 C) their ability to degrade β-glucan was gradually lost confirming earlier results. 13 At the same time β-glucan solubilase still released β-glucan up to a temperature of 73 C without further degradation causing an increase in concentration confirming the findings of Einsiedler 7 and Bamforth et al. 3. Our conclusions were confirmed by the isothermal mashings at 20 and 60 C. Since the inactivation temperature of the endo-β-glucanases was never reached at the 20 C mash, the β-glucan content was constant and low during the entire mashing process, which was already observed earlier. 7 In contrast, when mashing isothermally at 60 C with the endo-β-glucanases already inactivated, a permanent release of β-glucan with low or no degradation was observed from mashing-in with final concentrations of approximately 450 mg/l. This basically confirms the finding of constant and high β-glucan concentrations at isothermal mashing (65 C) reported. 13 Beside enzyme activities the increasing levels of β-glucan observed at temperatures above 57 C may additionally be connected to starch gelatinization and saccharification as suggested by Erdal and Gjertsen 8. The confidence intervals were quite large as soon as the β-glucan concentration increased from the basic level. Applying standard upward mashing, the FAN concentration started at a high concentration of around 24 mg/l at mashing-in and increased up to a temperature of approximately 55 C with no significant change until the end of mashing (Fig. 2). The final level was approximately 33 mg/100 ml. These observations parallel earlier findings for proteolytic activity 15 and amino acids 5. When isothermal mashing at 20 and 60 C was applied (Fig. 2) the FAN levels were lower compared to upward mashing, but the difference decreased towards the end of mashing with VOL. 111, NO. 3,

5 Fig. 1. β-glucan concentration in mash during mashing: standard upward mashing (45/62/70 C) vs. isothermal mashing (20 and 60 C) (conditions: roller milling, 30% poorly modified malt, stands: 30 min (upward mashing), grist:liquor ratio 1:4; error bars: 95% of confidence level, n = 9). Fig. 2. FAN concentration in mash during mashing: standard upward mashing (45/62/70 C) vs. isothermal mashing (20 and 60 C) (conditions: roller milling, 30% poorly modified malt, stands: 30 min (upward mashing), grist:liquor ratio 1:4; error bars: 95% of confidence level, n = 9). final values of mg/100 ml. The lower levels at 20 C isothermal mashing may be due to low enzyme activities, similar to earlier findings for mashing trials at 10 C. 5 On the other hand, when the calculated optimum temperature of proteolysis was around 50 C for poorly modified malt, 5 isothermal mashing at 60 C led to a lower FAN release due to heat inactivation. The confidence intervals for FAN were around 10% of the respective value and were fairly constant throughout all samples. The extract concentrations of all standard mashing trials were very uniform with a sharp increase when temperature reached approximately 57 C (Fig. 3) which is completed at the 70 C stand with a final value of about 17% (w/w). This may be due to the gelatinization and saccharification and is in agreement with former observations presented above. 6,13 While isothermal mashing at 60 C allowed most of the extract to be released during the first third of mashing (approximately 14.5% (w/w), 30 min after mashing-in), mashing at 20 C caused hardly any release of extract (5% (w/w), Fig. 3) due to lack of gelatinization. Therefore, in terms of amylolysis it is important to reach temperatures of 55 C or higher to gain sufficient 320 JOURNAL OF THE INSTITUTE OF BREWING

6 Fig. 3. Extract concentration in mash during mashing: standard upward mashing (45/62/70 C) vs. isothermal mashing (20 and 60 C) (conditions: roller milling, 30% poorly modified malt, stands: 30 min (upward mashing), grist:liquor ratio 1:4; error bars: 95% of confidence level, n = 9). Fig. 4. β-glucan concentration in mash during mashing: variation of milling method (roller/hammer milling) (conditions: 100% poorly modified malt, stands: 30 min, grist:liquor ratio 1:4). extract by degradation. When this is not the case, hardly any extract is released due to lack of gelatinization and saccharification and low enzyme activity. The confidence intervals for extract were the smallest of the analytes investigated and demonstrated high reproducibility. Variation of milling procedure (roller/hammer milling; Table IV, no. 4/5) When applying roller milling the husks were conserved and an even distribution of fractions was provided. The grist originating from hammer milling primarily consisted of powder dust and fine fractions, husks and coarse grits did not occur (Table III). With hammer milling the β-glucan concentrations tended to be higher at the end of the first stand at 45 C compared to coarse grist obtained from roller milling (Fig. 4). As mashing proceeded and reached the 3rd stand at 70 C this relationship inversed with lower final values for the fine grist. Note that these observations are just trends, however, they are in agreement with those discussed above 7 and should be verified for significance. Thus, the use of hammer milling caused pre-modification of malt, that is mechanical decomposition and re- VOL. 111, NO. 3,

7 Fig. 5. FAN concentration in mash during mashing: variation of milling method (roller/hammer milling) (conditions: 100% poorly modified malt, stands: 30 min, grist:liquor ratio 1:4). Fig. 6. Extract concentration in mash during mashing: variation of grist:liquor ratio (1:4/1:5) and ratio 1:5 calculated to 1:4 (solid line), respectively (conditions: roller milling, 30% poorly modified malt, stands: 30 min; error bars: 95% of confidence level, n = 9). lease of malt components, to such an extent that the endoβ-glucanases were not able to degrade them all at once. This was indicated by occurrence of measurable concentrations even below the inactivation temperature of endoβ-glucanases, a hint for the substrate concentration not being the limiting factor in enzymatic degradation, but rather the enzyme concentration itself. In contrast, coarse grist mashes did not show any detectable concentration in this period. Subsequently, the absolute amount of β-glucan degraded should be larger with fine grist rather than coarse grist, and less β-glucan was available for release after gelatinization, when the β-glucanases became more and more inactive. Therefore, lower final β-glucan concentrations should be expected at the end of mashing with fine grist, which indeed tended to occur. Hammer milling caused higher FAN concentrations in mash after mashing-in which also seems to be a result of better mechanical pre-treatment (Fig. 5). However, towards the end of mashing, both levels aligned as mashing proceeded and resulted in a final FAN level of 30 mg/ JOURNAL OF THE INSTITUTE OF BREWING

8 Fig. 7. Temperature related β-glucan concentration in mash during mashing: variation of grist modification level (0/100% poorly modified malt) (conditions: roller milling, stands: 30 min, grist:liquor ratio 1:4). ml. These observations confirm earlier findings that finer grist causes a more intensive proteolysis with higher amino acid concentrations. 5,16 As another conclusion, it should be considered that intensive mechanical treatment may completely be compensated for by extension of mash stands. In our trials, the variation of milling did not lead to any difference in extract values throughout the entire mash process. Therefore, higher mechanical treatment by hammer milling did not provide a faster release nor higher final values if the amylolytic malt modification was sufficient. Here, the variation of milling procedure did not seem to affect enzymatic reaction sides. Again, this finding is in agreement with former findings of Einsiedler et al. 5, but is partly in disagreement with Schneider who observed higher extract concentrations due to finer milling with decreasing difference towards the end of mashing. 16 Variation of grist:liquor ratio (1:4/1:5; Table IV, no. 6/7) The β-glucan concentrations of the 1:4 and 1:5 mashes did not significantly differ at any stage of mashing, although their mash concentrations were different. This is in agreement with the observation of Schneider 16 for coarse grist. The thicker mash (1:4) had significantly higher FAN concentrations compared to the 1:5 mash throughout the entire process which could simply be an effect of dilution. Thus, proteolytic activity does not seem to be influenced by mash concentration. Similar to FAN, the extract had continuously higher values in 1:4 mashes compared to 1:5 which was due to dilution (Fig. 6). This can easily be demonstrated by converting 1:5 values into 1:4 level applying the following formula: extract(1:5 normalized) = extract(1:5)/4*5. By comparing the 1:4 values with the normalized 1:5 values (solid lines in Fig. 6) a significant difference could not be observed. The well known phenomena of higher activity of amylolytic enzymes at lower mash concentrations, 12 could not be confirmed by comparing the ratios of 1:4 to 1:5. Variation of time of mash stands (20/30/40 min; Table IV, no. 8 13) Trials were carried out varying the time of mash stands: 20, 30 and 40 min, that is, each stand of one mashing trial had a period of either 20, 30 or 40 min. According to the temperature-related diagram (Fig. 7) elongated stands caused an increase in β-glucan concentrations, which could particularly be observed for high loads of poorly modified malt. Here again, an increase occurred when 57 C was exceeded. The reason for this might be the low cytolytic modification and high β-glucan content of poorly modified malt (see Table I). In contrast, for well modified malt hardly any release even during the 40 min stands could be observed (Fig. 7). Therefore, keeping mash stands short, not only saves time and keeps final β-glucan concentrations low, but is an important measure to effectively suppress any excessive release when poorly modified malt qualities have to be processed in practical operations. In terms of FAN, 20 min stands were sufficient to result in final values of nearly 30 mg/100 ml for a 100% well modified malt type of mash (Fig. 8) which is enough for yeast nutrition. Longer stands caused slightly higher values of mg/100 ml. However, when mashings with 100% poorly modified malt were carried out, 30 min stands did not seem to be sufficient to obtain comparable final levels. Therefore, a poor proteolytic modification of malt may only partly be compensated by elongated mashing which is in agreement with the statements of Einsiedler 5 and Riis 15 discussed above. VOL. 111, NO. 3,

9 Fig. 8. FAN concentration in mash during mashing: variation of stand time (20/30/40 min) (conditions: roller milling, 0% poorly modified malt, grist:liquor ratio 1:4). Fig. 9. Extract concentration in mash during mashing: variation of stand time (20/30/40 min) (conditions: roller milling, 100% poorly modified malt, grist:liquor ratio 1:4). At a temperature of approximately 57 C, the respective extract concentration sharply increased independent of the duration of mash stand (Fig. 9). The gains were shifted by 10 min, respectively, due to the different stand times. Even with the shortest stand of 20 min a maximum extract yield of nearly 16% (w/w) was obtained. Thus, the variation of stand time does not have an influence on the extract release and an extension of mashing stands does not provide any advantage in terms of extract gain. This was also observed for the poorly modified malt since it had an amylolytic modification which was close to that of the well modified malt (Table I). Variation of grist modification level (0/30/50/70/100% of poorly modified malt; Table IV, no ) Increasing amounts of poorly modified malt resulted in increasing β-glucan concentrations of mashes (Fig. 10). The well modified malt had concentrations in final mashes of approximately 125 mg/l, while the poorly modified malt released approximately 950 mg/l. Commercially available malts have β-glucan concentrations of up to 400 mg/l (relative to an extract content of 16% (w/w) 1 which roughly corresponds to a poorly modified malt content of 324 JOURNAL OF THE INSTITUTE OF BREWING

10 Fig. 10. β-glucan concentration in mash during mashing: variation of grist modification level (0/30/50/ 70/100% poorly modified malt) (conditions: roller milling, stands: 30 min, grist:liquor ratio 1:4; error bars: 95% of confidence level, n = 9). Fig. 11. FAN concentration in mash during mashing: variation of grist modification level (0/30/70/ 100% poorly modified malt) (conditions: roller milling, stands: 30 min, grist:liquor ratio 1:4; error bars: 95% of confidence level, n = 9). approximately 30%. This strong increase in β-glucan concentration, depending on the grist modification level, clearly shows a correlation of malt quality and β-glucan concentration in the resulting wort. Thus, we observed a final concentration for poorly modified malt being 7.6 times that of well modified. Considering variations in malt as a natural raw material, this number corresponds well with the observation of Narziß and Litzenburger 13 who found a six-fold increase and of Einsiedler et al. 7. Lower β-glucanase activity in poorly modified malts has been reported earlier, 7,13 which might be assumed here as the reason for increasing β-glucan values. Beside enzyme activity, the modification itself and the physical degradation with higher temperatures might be responsible for the tremendous differences in release. When gradually increasing the poorly modified malt portion FAN concentrations decreased (Fig. 11). This decrease with increasing poorly modified malt portions (Fig. 11) is in agreement with Einsiedler s observation 5 of amino acid concentrations and is probably due to the VOL. 111, NO. 3,

11 Fig. 12. Temperature-related extract concentration in mash during mashing: variation of grist modification level (0/30/50/70/100% poorly modified malt); numbers: exemplary extract gains within the respective stands and heating steps (conditions: roller milling, stands: 30 min, grist:liquor ratio 1:4). lower proteolytic and cytolytic modification of poorly modified malt according to Table I. Detecting FAN concentrations in the range of 190 to 240 mg/l at the beginning of mashing parallels the findings of high initial amino acid concentrations according to Einsiedler et al. (200 to 250 mg/l). 5 Thus, malting seems to be more important than mashing for this characteristic. The extract concentrations did not show any differences, due to the similar amylolytic modification of both malts as discussed above. According to the temperature related extract diagram, the steepest increase was observed when exceeding 57 C (Fig. 12). The extract gain was highest when heating from 45 to 62 C and during the 62 C stand, corresponding to the temperature optima of β-amylase (60 to 65 C), while the α-amylase had its maximum at 70 to 75 C but might be sufficiently active already in the range of 45 to 62 C. On the other hand, the 45 and 70 C stands, as well as the heating step from 62 to 70 C, hardly contributed. A steep increase between 50 and 65 C was also observed by Narziß and Litzenburger 13. Accordingly, Einsiedler et al. 6 observed a fast reduction of starch content in a similar temperature range. CONCLUSIONS The β-glucan content in wort, being a key indicator for mashing, is determined by the malt quality expressed in the cytolytic key figures (Table I). The malt quality has substantial impact on cytolysis and therefore on β-glucan content in the resulting wort. A variation of milling procedure (mash filter/lauter tun grist) and of the grist:liquor ratio (1:4/1:5) is not of relevance in practical operation since any significant influence on the final β-glucan level could be detected in our trials. Mashing-in at low temperatures (45 C) causes a β-glucan degradation which, however, is not of relevance for the final β-glucan level in wort because during saccharification β-glucan is released in an amount which is again determined by the malt quality. The mash time is only important for poorly modified malts. That is, the longer mashing, the more β-glucan is released. On the other hand, this is not valid for well modified malts. An extension of the stands is of minor influence due to their low β-glucan potential. Therefore, short mashing is preferable, particularly for poorly modified malts, in order to minimize β-glucan input into wort. When short mashing is applied, lower FAN levels result from poorly modified malts since the cytolytic activity and physical release are determining, instead of the proteolytic enzyme activity during mashing. Similar to β- glucan, FAN release is predetermined by malt modification. Finer grist causes higher initial FAN values at mashing-in which, however, aligns towards the end of mashing even when short mashing (total mashing time: 85 min) is applied. A variation of the grist:liquor ratio (1:4/1:5) does not influence the FAN values significantly, while extending the β-glucan stand (approximately 50 C) causes a greater release of FAN, particularly from poorly modified malts. In case a high FAN yield is desired (adjunct brewing) one should consider an extend of the mash stand at a temperature below 50 C. Here, attention has to be paid to a good cytolytic modification of the malt in order to avoid an increased β-glucan release as discussed above. In contrast to FAN, an extension of the mashing time does not provide advantages in terms of extract yield. It is of fundamental importance to reach the gelatinization temperature which is well known from brewing with unmalted adjuncts (rice). The greatest gain in extract occurred around 57 C in this investigation. For an amylolytically well modified malt (Table I) finer milling (mash filter grist) and thinner mashing (grist:liquor ratio of 1:5) 326 JOURNAL OF THE INSTITUTE OF BREWING

12 result neither in more nor faster extract release. Therefore, it may be concluded that shortening of mash time is not necessarily related to a lower extract yield, however, wort quality should to be monitored (iodine test). The concentration of the key indicators for cytolysis (β-glucan), proteolysis (FAN), and amylolysis (extract) in wort are critically determined by malt modification. A variation of the investigated mashing parameters did not result in significant changes in terms of the key indicators for well modified malt. However, when poorly modified malts were used, the variation of mashing parameters had an impact on the key indicators in which cytolysis plays the dominating role. ACKNOWLEDGEMENTS This research project was gratefully supported by Arbeitsgemeinschaft industrieller Forschungsvereinigungen Otto von Guericke e.v. and Wissenschaftsförderung der Deutschen Brauwirtschaft e.v. REFERENCES 1. Arbeitsgemeinschaft zur Förderung des Qualitätsgerstenanbaus im Bundesgebiet e.v., Ed., Braugersten-Jahrbuch. Braugerstengemeinschaft: Eichenau, 2003, 41, pp Bamforth, C. W., Enzymolysis of β-glucan. Proceedings of the European Brewery Convention Congress, Copenhagen, IRL Press: London, 1991, pp Bamforth, C. W., Martin, H. L. and Wainwright, T., A role of carboxypeptidase in the solubilization of barley β-glucan. J. Inst. Brew., 1979, 85, Bamforth, C. W., Moore, J., Proudlove, M. O., Bartholome, B. and Wiliamson, G., The dissolution of β-glucans: new dimensions. Mschr. Brauwiss., 1997, 50(3/4), Einsiedler, F., Schwill-Miedaner, A. and Sommer, K., Experimentelle Untersuchungen und Modellierung komplexer biochemischer und technologischer Prozesse am Beispiel des Maischens. Teil 1: Proteolyse. Mschr. Brauwiss., 1997, 50(9/10), Einsiedler, F., Schwill-Miedaner, A., Sommer, K. and Hämäläinen, J., Experimentelle Untersuchungen und Modellierung komplexer biochemischer und technologischer Prozesse am Beispiel des Maischens. Teil 2: Amylolyse. Mschr. Brauwiss., 1997, 50(11/12), Einsiedler, F., Schwill-Miedaner, A., Sommer, K. and Hämäläinen, J., Experimentelle Untersuchungen und Modellierung komplexer biochemischer und technologischer Prozesse am Beispiel des Maischens. Teil 3: Cytolyse. Mschr. Brauwiss., 1998, 51(1/2), Erdal, K. and Gjertsen, P., β-glucans in malting and brewing. II. the fate of β-glucans during mashing. Proceedings of the European Brewery Convention Congress, Madrid, Elsevier: Amsterdam, 1967, pp Heyse, K.-U., Ed., Handbuch der Brauerei-Praxis. 3rd ed., Hans Carl: Nürnberg, 1995, p Miedaner, H., Brautechnische Analysenmethoden. 4th ed., Selbstverlag der MEBAK: Freising-Weihenstephan, 2002, 2, pp. 1 3, 42 45, 62 65, 74, Narziß, L., Abriß der Bierbrauerei. 6th ed., Ferdinand Enke: Stuttgart, 1995, pp. 25, Narziß, L., Die Technologie der Würzebereitung, 7th ed., Ferdinand Enke: Stuttgart, 1992, 2, p Narziß, L. and Litzenburger, K., Malzqualität, Maischeintensität und Gummistoffgehalt. Brauwissen., 1977, 30(9), Pfenninger, H., Brautechnische Analysenmethoden. 3rd ed., Selbstverlag der MEBAK: Freising-Weihenstephan, 1997, 1, pp , Riis, P., Proteolytic activity during mashing. Proceedings of the European Brewery Convention Congress, Dublin, CD-ROM, Fachverlag Hans Carl: Nürnberg, 2003, pp Schneider, J., Dynamische Mikrofiltration von Feinstschrotmaische mit oszillierenden Membranen, Dissertation, TU München, Freising-Weihenstephan, 2001, pp Schwill-Miedaner, A., Einsiedler, F. and Sommer, K., Barley endosperm cell walls: A review of cell wall polysaccharides and cell wall-degrading enzymes. Tech. Q. Master Brew. Assoc. Am., 1998, 14(4), Thompson, R. G. and LaBerge, D. E., Barley endosperm cell walls: A review of cell wall polysaccharides and cell walldegrading enzymes. Tech. Q. Master Brew. Assoc. Am., 1977, 14(4), (Manuscript accepted for publication October 2005) VOL. 111, NO. 3,