J. Inst. Brew., November-December, 1991, Vol. 97. pp COLD WATER EXTRACT OF MALTS: SOLUBLE CARBOHYDRATE AND MALTING BEHAVIOUR

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1 J. Inst. Brew., November-December, 1991, Vol. 97. pp COLD WATER EXTRACT OF MALTS: SOLUBLE CARBOHYDRATE AND MALTING BEHAVIOUR Malcolm Glennie Holmes N.S. W. Agriculture & Fisheries, Agricultural Research Institute, Wagga Wagga, N.S. W., Australia 2650 Received 2 February 1991 A rapid and simple method for the determination of the carbohydrate fraction of the Cold Water Extract of malts is described. The extraction is preceded by treatment of the malt with barium hydroxide, which is then removed by precipitation with zinc sulphate. This both deactivates enzymes and prevents solubilization of protein. The method was modified so that it was both faster and used smaller samples. A sustainable rate of 80 samples per day can be tested by one operator. This method was used to follow the pattern of modification through malted grain by analysis of pearler residues. Malting and non-malting cultivars differed in the localization of modification through the grain, with modification confined to the outer layers in non-malting cultivars. The method was also used to measure the proportion of soluble carbohydrate formed during malting with increasing modification. The proportion of extract solubilized during malting by malting cultivars was less than that solubilized by feed cultivars. Key Words: cold water extract, rapid tests. Introduction The components which make up Cold Water Extract (CWE) were examined as part of a programme to identify extra selection indices for the determination of malting potential in barley crossbreds. Cold Water Extract values occupy an ambiguous place in malt specifications. Dismissive statements are found in the literature such as... "The mis-named CWE determinations... give little information and relatively little attention is now paid by British brewers to CWE determinations and it is not a recommended method of the EBC or ASBCl}. In contrast, Preece14 remarked that'... it was a useful determination... On the assumption which is probably true that the dilute ammonium hydroxide solution used for extraction prevents enzyme action during the process and which is less certain does not itself have any extra solubilising effect, the cold water extract may be taken to be a measure of the total cold water solubles available at the time of experiment'. The method is still a recommended method of the Institute of Brewing10. The level of simple sugars formed during the malting process should be a useful index of malting perform ance, and also could offer information in experiments where patterns of modification through grain are under investigation, such as examination of pearler7 residues. Almost all 'CWE' determinations are misnamed, as mentioned above, for although the procedure was originally based on iced water17, the major methods of enzyme deactivation now in use appear to be dilute ammonia10 and mercuric3 salts. These methods extract a mixture of carbohydrate, protein and salts which is determined as total soluble solids by specific gravity or rcfractometry14. To determine the performed total soluble carbohydrates in malts, an extraction preceded by the enzyme inactivation method of Blakeney and Mutton2 was tested and found satis factory, with several advantages as described below. This was then further modified to provide a rapid, small-scale test for determination of the preformed total soluble carbo hydrate fraction in samples of malt from crossbreds of barley emanating from a breeding programme. The method was used to investigate the patterns of modification through grain by examination of pearler residues7 and the rate of solubilization of carbohydrate during malting of four barley cultivars. Materials and Methods Samples Micromalts were generated from barleys from the breeding programme at the Agricultural Research Institute, Wagga Wagga. A sample of commercial malt, made from Clipper barley, was provided by the Victorian makings of Barrett- Burston (Aust). Chemicals Barium hydroxide, Zinc sulphate and Ammonium hydroxide were AR grade chemicals from B.D.H. Chemicals. Barium hydroxide and Zinc sulphate solutions of 0.20 N were prepared in deionized water. MlCROMALTING Barleys are micromalted in a computer-controlled, automatic micromalter following the procedure described by Glennie Holmes et at*. The malts were shaken in their containers to remove the culms, the weights recorded, and the malts then stored in air-tight containers. Samples were ground on a Buhlcr Miag DLFU mill or Udy * Cyclone Grinder, fitted with I mm screen, as indicated. MlCROMASHING Determination of Hot Water Extracts Coarse-Concentrated Hot Water Extract (CCHWE) Samples were ground according to the Recommended Method of the Institute of Brewing (IoB # ) using a Buhler-Miag DLFU mill with the gap set at 1.00 mm. The extraction procedure IoB # was modified in that instead of 50.0 g malt made to a total volume of 515 ml, g was mashed and made to 100 g. Mashing was carried out on a Brewing Research Foundation [BRF] Mashing Bath, and times of extraction and conditions of stirring and temperature for the extraction were all as in IoB Method # for Infusion Mashing. The extracts were then centrifuged, [10 min/3,000 g] instead of filtered, (Glennie Holmes6) and determination of Total Soluble Solids was by the method of Essery5 using a Zeiss Immersion Refractometer. The refractometer was stated by the manufacturer to be accurate to the fifth decimal place and the temperature was stabilized to 2O±O.O2*C with a Haake F3 Digital Circulating Water Bath. Extract values were reported as percentage of total soluble solids. Modified lob methodfor Cold Water Extract The Institute of Brewing Method #2.5 was followed, with modifications to sample size and method of grinding. The sample was ground either with the Buhler-Miag DLFU or a Udy Cyclone Grinder fitted with a 1 mm screen. Sample size was 5.00 g, total sample volume ml [containing 6.00 ml 0.1 N Ammonium hydroxide]. The BRF Bath was set at 23 and the stirring motors were activated for one minute at 30

2 446 COLD WATER EXTRACTS OF MALT [J. Inst. Brew. minute intervals. Extraction time was 180 minutes, unless otherwise indicated. Centrifugation [3,000 g, lomin] and refractometry replaced filtration and determination of specific gravity respectively. Determination of the Carbohydrate Fraction of the CWE [1] On the BRF Mashing Bath: The samples (5.00 g) were placed in the mashing beakers with 80 ml of water at 23 C. Barium hydroxide solution was added [10.0 ml, 0.2 N], the sample stirred for 5 minutes, then zinc sulphate [10.0 ml, 0.2 N] added during the last minute of stirring. The procedure then followed the recommended method. The extract volume was made to a total of g before filtration and determination of Total Soluble Solids by refractometry. [2] In snaplop polythene centrifuge tubes (30 ml), samples (1.250 g) with 25 ml total extractant and deactivant solutions pro rata with those above, in (a) a Labmaster shaking water bath at 23 C or (b) shaken in a box shaker at ambient temperature, followed by centrifugation and rcfractometric determination. Results were expressed as Percentage of Total Soluble Solids. m o Diastatic power Diastatic power was determined by the method of Henry9. Speed of mashing extract Each sample was mashed 4 times under the conditions for grist/liquid ratio and temperature required for the determin ation of Fine Hot Water Extract Mashes. At 10 and 60 minutes after commencement, mashing was stopped in duplicate samples by the addition of barium hydroxide (10 ml, 0.2 N). The beakers were allowed to stand for 2 minutes. Coprecipitation was achieved by the addition of zinc sulphate (10 ml, 0.2 N), and standing for a further 2 minutes before removal from the bath, adjustment to weight and centri fugation. Total soluble solids were determined by refractometry and the results expressed as a percentage of the value obtained at 60 minutes Mill gap (mm) Error bars show standard deviation between triplicate results Fig. 1. Effect of the fineness of grinding of malt on the amount and accuracy of extraction of cold-water-soluble material by the IoB method. Results and Discussion Prior to the development of the Cold-Water-Soluble (CWS-) carbohydrate method, the effect of fineness of grinding of the samples and time of extraction on the IoB Method was examined. During the development of the CWS-carbohydrate procedure, the consequences of reduction in sample size, ph of the extractant solution and continuous shaking were also examined. Influence of Fineness of Grinding on IoB Method The recommended method (IoB 1977) specifies the use of a Buhler-Miag DLFU grinder which, even at its finest setting, produces a relatively coarse and gritty meal. As rapid and complete inactivation of enzymes and extraction of preformed sugars are both more easily obtained with finely ground material, the effect of fineness of grinding on the results of CWE determinations was measured. Samples of a micromalt, with a CWE of 23.1% by the IoB method, were ground on the cyclone grinder and on the DLFU grinder at 0.2, 0.5, 0.7 and 1.00 mm settings. [Williams16 showed that the mean particle size of meals ground through a 1 mm screen on a cyclone grinder was a: 75 u.) The results of analysing three replicates of each grinding by the modified IoB method is shown in Figure 1, which indicates that as particle size increased, both the actual extract and repeatability of the analyses decreased. The cyclone-ground sample gave a significantly higher result than the sample ground according to the IoB method, suggesting that the IoB method does not give complete extraction. The strongly curvilinear relationship of extract to particle size, which was not log/linear, but closer to that of a log/log relationship, indicated that the process was controlled by at least two rate-limiting steps, and that diffusion m O i O Clipper Minutes of extraction 180 cyclone-ground DLFU 0.2 mm DLFU 0.5 mm DLFU 0.7 mm cyclone-ground DLFU 0.2 mm DLFU 0.5 mm DLFU 0.7 mm Fig. 2. Effect on time of extraction and fineness of grind on the cold water extract values obtained by the IoB method on two malts, one made commercially from the malting variety Clipper, and the other a micromalt made from a feed variety, Galleon.

3 Vol. 97, 1991] COLD WATER EXTRACTS OF MALT 447 from the granule was limited by diffusion of the solutes through the extractant. Increasing the frequency and intensity of the mixing should therefore increase the rate of extraction. A commercial malt, (CWE 22.3% by the modified lob method), and a micromalt made from a feed barley, Galleon, (CWE 17.0%), were then tested. Samples of both malts were ground on the cyclone grinder and the DLFU grinder at 0.2, 0.S and 0.7 settings. Duplicate samples were extracted for 60, 120 and 180 minutes on the BRF bath by the modified IoB method. The results are shown in Figure 2. Both the cyclone-ground malts gave higher results after only one hour's extraction than those ground by the standard method did after three hours extraction. Fineness of grind was shown to be as influential as the time of extraction, confirming the results of Meddings and Potter12 who concluded that '... the overall rate of malt extraction could be spectacularly increased by reducing the particle size'. Samples for analysis were ground on the cyclone grinder thereafter unless specifically mentioned. 23-i Determination of the Carbohydrate Fraction of the CWE The use of a protein denaturant to deactivate enzymes, followed by a mechanism to remove the and allow extraction of preformed soluble material at neutral ph would offer a simple procedure for determination of the carbohydrate fraction of the CWE. Blakeney and Mutton2 described such a protein precipitant, based on barium hydroxide and zinc sulphate. Barium hydroxide denatures the soluble protein therefore enzymes should be completely deactivated zinc sulphate is then added, to produce dense Barium sulphate and gelatinous zinc hydroxide. Centrifugation or filtration removes both chemicals, leaving no residues to interfere with subsequent assays. They demonstrated, by use of C*14 radioactivelylabelled glucose, that the precipitation method did not remove any soluble sugars during centrifugation or filtration. Testing the clarified extract with Coomassie Brilliant Blue (Lewis et a/.") showed that co-precipitation had removed all soluble protein. Blakeney and Mutton2 used a slight excess of barium hydroxide to maintain a high ph which suited their method of sugar analysis (based on colorimetry using PAHBAH) but in this work equi-molecular concentrations were used. Blank determinations consistently gave zero readings (Scale zero, 14.50) on the refractometer. Unlike the IoB Method, in which the ammonium hydroxide gave a slightly turbid and yellow cast to the final solution, the barium hydroxide, after precipitation, leaves a water-clear extract ideal for refractometry. Although the method takes no account of dissolved salts14 (which would be measured as part of the CWS-carbohydrate), it is reasonable to expect the amount of dissolved salts to be relatively constant and thus their contribution may be ignored where changes with time or between cultivars is the object of the experiment. The method using barium/zinc salts was applied to the determination of Cold Water Extract to isolate the carbo hydrate fraction. A set of 20 barleys were selected at random from the routine crossbred assessment programme, micromalted with a three day modification period and tested by the modified IoB method and the method using co-precipitation. With a regression equation between the two methods of IoB = * co-precipitation, with a correlation coefficient of r= 0.99***, (Figure 3), the results were as expected, for the values obtained from the co-precipitation method were approximately 2.0 to 2.3% lower than those from the recom mended method. As co-precipitation has been shown not to remove sugars (vide supra), the lower result reflected the removal of protein from the extract. The mean soluble protein content of the extracts was shown to be 2.08% by the method of Lewis et al.u, which was lower than the 2.5% found by Windisch et alp for commercial malts, but which is explained by the shorter modification period used for the micromalting r CWE % by co-precipitation method Fio. 3. Comparison of the results of cold water extract on twenty 3-day micromults by the method of Ihe lob (1977) using ammonium hydroxide and co-precipitation. An alternative system suggested by Blakeney1, based on oxalic acid and calcium benzoate (which together yield the insoluble products calcium oxalate and benzoic acid) was also tried. This combination was found to be extremely effective in denaturing enzymes, particularly ot-amylase, but the blank determinations had high and variable refractometric readings and the combination was abandoned. This approach could be used for methods in which determination of sugars was by a method other than refractometry. Effect of the ph of the extractant To measure the effect which the ph of the extractant solution had on the method, CWS-carbohydrate determinations were carried out on eight micromalls, firstly as above and secondly by delaying the addition of the Zinc sulphate solution until after the three hours extraction. The higher results for the method using delayed precipitation (Figure 4) indicate that the long period of high alkalinity caused hydrolysis of polysaccharides to soluble sugars. The time of reaction was standardized at 5 minutes thereafter. Small scale test tube procedure The method was then reduced to a size comparable to the test-tube extract procedure of Glennie Holmes6: to suit small samples, such as those enanating from a micromaller. Samples (1.250 g) were weighed into centrifuge tubes (polythene, snaptop, 30 ml) made to a total volume of 25 ml (20 ml water plus 2.5 ml each of barium and zinc solutions) and shaken at half hour intervals for the three hour extraction period. A correlation coefficient of r=0.98*** between the test-tube and BRF Mashing Bath methods for the 20 samples indicated that reduction in sample weight had no effect on the ability of the method to rank samples in order of CWE values. Extraction under continuous shaking conditions To speed up the analysis, the use of continuous shaking for a shorter period was investigated. After the co-precipitation, an hour's shaking at room temperature in a laboratory box shaker before centrifugation gave a correlation r of 0.97*** for the 20 malts between the shaker/co-precipitation method and that using the BRF Mashing Bath. The shaker method was substantially faster than that method and ranked the malts in the same order.

4 448 COLD WATER EXTRACTS OF MALT [J. Inst. Brew. The sample of commercial malt was tested by the shaker method, with times of extraction from five to sixty minutes, and Figure 5 shows that extraction was complete within 20 minutes. From then on, 30 minutes was used as the time of extraction. When the samples of differently-ground material used to derive the results shown in Figure 1 were tested with the shaker method, a linear relationship was found (Figure 6) rather than the log/curvilinear relationship of the IoB extraction. Shaking removed the limiting diffusion process through the solution gradient, leaving only diffusion from the granule to control the 20 I g 19 Q. 6 U rate of dissolution. The extract achieved with cyclone-ground material with the shaker/co-precipitation method was 2.4% lower than that by the IoB method, comparable with the amount of prc-formed soluble protein found by Windisch el alp. However, the short time of extraction with the shaking method was insufficient for extraction of material from particles larger than those ground with the mill gap at 0.S mm. CWS-Carbohydrate Levels Through Malted Grain The co-precipitation/shaker method was then used to follow the patterns of modification developed in four barleys during micromalting as shown by CWS-carbohydrate values. The micromalts were prepared from the malting varieties Ark Royal & and the feed barleys &, by malting with a modification period of two and four days. The micromalts were pearled for varying periods of time (20,40 and 60 seconds), by the method of Glennie Holmes7. The pearls were ground on the cyclone grinder and CWS-carbohydrate values determined. The results (Figure 7) show that after two days the malting varieties had already developed a comparatively even level of CWS-carbohydrate throughout the grain whereas the nonmalting varieties exhibited a rapid reduction in the CWScarbohydrate towards the centre of each grain, although their overall CWS-carbohydrate levels were not substantially inferior to those of the malting types. Non-malting varieties were therefore characterized by having areas of modification that were confined to the outer layers of the grain. After 4 days, the differences between malting and feed varieties were still marked. Thus pearling accentuated the differences between the amounts of CWS-carbohydrate of malting and non-malting varieties at both two and four days of modification Co-precipitation before extraction CWE % Fig. 4. Comparison of the results of the cold water extract detenninations using co-precipitation before and afler the extraction period. The Proportion of Carbohydrate Solubilized During Malting and its Relationship to Malting Potential, a recently released Australian malting variety, has received a general commercial evaluation as a cultivar of good malting quality which is not consistent with its low diastatic power (MBIBTC 1990)13. -O 96' o co o ' 86' 84 ' t I 45 Extraction time (minutes) Fig. 5. Effect of time of shaking on extraction of soluble carbohydrate.

5 Vol. 97, 1991] COLD WATER EXTRACTS OF MALT TABLE 1. Barleys Used for Time-Of-Modification Experiment 95 - Cultivar Type & origin Thousand corn weight (g) Hardness P.R. Protein content % Total P- glucan % Malt 2-row U.K Feed 2-row W.A I 2 85H Feed 6-row N.S.W. Malt 2-row S.A Shaker/ coprecipitation Mill gap (mm) Fig. 6. Effect of fineness-of-grinding of malt on the efficiency of extraction with the IoB and shaker/co-precipitation methods % grain as pearls 20 FlO. 7. Cold water soluble (CWS) carbohydrate extract levels of the pearls made from four varieties of barley modified for two and four days. & are 2-row malting, & Galleon are 2-row feed varieties. To see if the proportion of carbohydrate solubilized during malting would reflect the reduced diastatic power [for the CCHWE certainly did not] determinations of diastatic power, CWS-carbohydrate and CCHWE were performed on malts made from four varieties of barley malted for one to six days. The barleys were samples of the varieties,, & selected from the 1988 Pure Seed Increase Trial at the A.R.I.. would be expected to have a low diastatic power not only intrinsically, but also due to the low protein content of the particular sample available (Table 1). When the CCHWE values of the malts were correlated with Diastatic Power, was anomalous. The other four cultivars has a relationship of DP to CCHWE of CCHWE = *DP (r=+0.92***, n = 24), indicating that, as expected, as DP increased, so did CCHWE. However, although also had a very close correlation between CCHWE and DP, and the slope was little different from that of the other four cultivars: CCHWE = *DP, (r= ***, it = 6), the intercept was significantly positively displaced from that of the other cultivars. Thus yielded CCHWE values significantly higher than the DP values would predict. The CWS-carbohydrate values of the malts were higher than those of other varieties (Figure 8), which was unexpected, considering the low diastatic power of the variety. The malting varieties both had higher levels of CWS-carbohydrate than the feed varieties in the early stages of malting, however, declined in Day 4 and then recovered, whereas did not. increased in CWS-carbohydrate from below the malting cultivars on Days 1 to 3 to equal by Day 6. is a six-row variety, a type known to develop high levels of diastatic power. The ratios of CWS-carbohydrate: CCHWE were then calculated (Figure 9). The percentage of the extract formed during malting in all cultivars declined between Days 1 and 2. The malting cultivars then showed an increase, whereas the increase for the feed varieties was delayed for a further day. and followed the same pattern, and the percentages of CWS-carbohydrate, particularly of, were consistently and significantly below those of the feed cultivars. Malting cultivars therefore degraded a smaller proportion of total extract during malting, and a greater proportion during mashing, than did the feed varieties. It was postulated that the malting quality of was a reflection of the speed at which extract was released in the mashing step rather than the amount of breakdown during malting, especially were it to be shown that the one-hour infusion of the IoB Mashing method was insufficient to extract the material solubilized by some varieties. Woodward & Oliver18 defined the speed of extract release as the time taken to achieve 99% of the extract achieved at 60 minutes, as deter mined by testing each sample at ten minute intervals through out the hour of mashing.

6 450 COLD WATER EXTRACTS OF MALT [J. Inst. Brew. ID 2 ( Day of modification Fig. 8. Changes in CWS-carbohydrale with lime of modification for four varieties: & 2-row malting, 2-row feed, and 6-row feed < Day of modification i 5 i 6 Fio. 9. Changes in CWS-carbohydrate/CCHWE with time of modification for four varieties: & are 2-row malting, 2-row feed, and 6-row feed. If the reaction kinetics of amylolysis were first order, the proportion of soluble sugars a brief time after commencement would be as accurate a determination of speed of extract as the multi-point method of Woodward & Oliver. Coulter & Potter4 demonstrated that the reaction kinetics of amylolysis for malt were adequately explained by a first order equation as long as the grist/liquid ratio was low and the sample finally ground. The speed of extract release, taken as the percentage of total carbohydrate extract achieved in 10 minutes, was then deter mined using conditions that satisfied the requirements above: the grist/liquid ratio was low (l:io) and the sample was finely ground (w75 u). The percentages of final extract achieved in 10 minutes are shown in Figure 10. For 60-minute results used in the calculations are not standard FHWE values, for cyclone-ground samples were used and the mashing was stopped with Barium hydroxide, thus dissolved protein was not measured. Malts of had substantially higher speeds of mashing extract, particularly in the malts given short periods of modification. Each variety followed an individual pattern, and with the exception of, the rate of increase with increasing modification was not closely related to extract potential, as the other malting variety,, had lower values than the feed varieties in this characteristic. In summary: had a higher CWS-carbohydrate level than other cultivars at the same time of modification. had a much higher speed of extract than the other cultivars at the same time of modification. However, had a CWS-carbohydrate level lower than that of the other cultivars

7 Vol. 97, 1991] COLD WATER EXTRACTS OF MALT o> 98 I- I 92 S 1"86 4) 96- goo i Day of modification FlC. 10. The speed of extract formation by malts of four cultivars malted for one to six days: Ark Royal & are 2-row malting, 2-row feed, and 6-row feed. at the same level of CCHWE. Therefore solubilized less of its total extract during malting than did the other varieties., which was shown to have a speed of extract below even the feed varieties, still behaved as a malting quality cuitivar, for the time to complete extraction was still shorter than the hour of infusion mashing used. The speed of mashing extract, therefore, did not offer a method of selection for malting potential. Conclusions The co-precipitation CWS-carbohydrate method enables the determination of preformed soluble carbohydrate in malts. Eighty determinations per day can be carried out by one operator using the small-scale co-precipitation shaking method as described. The method is rapid and accurate. It uses no polluting chemicals and gives a very clear extract on which determination of the refractive index is simple and un ambiguous. The rapid shaker, small-scale method is suitable for assessment of the carbohydrate fraction of the CWE in small samples, such as those obtained by pearling or generated from the early generation of the breeding programme. Results from CWS-carbohydrate determinations on pearler residues showed that feed varieties had modification confined to the outer layers of the grain whereas malting cultivars displayed more even modification through the grain. Studies on the changes in CWS-carbohydrate with time of modification suggest that malting cultivars degrade a smaller proportion of total extract to soluble forms during malting than feed varieties. Speed of extract was shown not to differen tiate between malting and non-malting cultivars. References 1. Blakeney, A. B. NSW Agriculture & Fisheries, Agricultural Research Institute, Yanco, 1989, Private Communication. 2. Blakeney, A. B. & Mutton, L. L. Journal ofthe Science offood and Agriculture, 1980,31, Blom, J. & Shwartz, B. European Brewery Convention, Proceedings of the 15th Congress, 1975, Nice, Coulter, P. R. & Potter, O. E. Journal of the Institute of Brewing, 1973,79, Essery, R. E. Journal of the Institute of Brewing, 1954,60, Glennie Holmes, M. R. Journal of the Institute of Brewing, 1990, 96, Glennie Holmes, M. R. Journal of the Institute of Brewing, 1990, 96, Glennie Holmes, M. R., Moon, R. & Cornish, P. B. Journal of the Institute of Brewing, 1990,96, Henry, R. J. Journal of the Institute of Brewing, 1984,90, Institute of Brewing, Recommended Methods ofanalysis Lewis, M. J., Krumland, S. C. & Muhleman, D. J. Journal of the American Society of Brewing Chemists, 1979,38, Meddings, P. J. & Potter, O. E. Journal of the Institute of Brewing, 1971,77, MBIBTC. Report of the Mailing & Brewing Industry Technical Committee to the Workshop on Research Needs in Barley Processing, 1990, CSIRO, North Ryde, Australia. 14. Preece, I. A. Journal of the Institute of Brewing, 1963, 69, Wainwright, T. & Buckee, G. K. Journal ofthe Institute of Brewing. 1977,83, Williams, P. C. Cereal Chemistry, 52, Windisch, W., Kolbach, P. & Schild, E. Wochenschrift Brauerei. 1931,48, Woodward, J. D. & Oliver, W. B. European Brewery Convention, Proceedings of the 22nd Conference Zurich, 299.