A review of malting and malt processing for whisky distillation

Transcription

1 Review article Received: 7 July 2015 Revised: 12 February 2016 Accepted: 15 March 2016 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI /jib.332 A review of malting and malt processing for whisky distillation George N. Bathgate* This paper encompasses a re-evaluation of published literature and data regarding wort attenuation in malt distilleries raising questions and discussing how the conventional wisdom has changed over time and what questions still need to be answered. Current knowledge is summarized in the following four points: (a) Under normal malting conditions, starch granules are partially degraded by a combination of α-amylase and α-glucosidase. This complex can open up the granule at specific sites on the surface and create characteristic pin-hole lesions, which may be widened by secondary hydrolysis by α- andβ-amylase, limit dextrinase and α-glucosidase (maltase). (b) All of these diastatic enzymes can survive mild kilning, probably by forming heat stable complexes on and within the starch granules and can continue a complete degradation of starch when mashed at ambient temperatures with glucose as the end product. (c) At normal mashing temperatures, starch granules gelatinize and dissolve with a concomitant rapid degradation to glucose, maltose, maltotriose and dextrins ranging from degree of polymerization (DP) 4 to > DP20. If there is immediate wort boiling after run-off, this is the final composition of starch derived carbohydrates according to the conventional paradigm. (d) All malt worts also contain a small amount of panose, isopanose as well as glucosyl maltodextrins, based on a core of 6 2 -α-glucosyl maltose (panose) or 6-α-maltosyl glucose (isopanose), which are remnants of the α-amylase/glucosidase degradation of granular starch. These dextrins are resistant to the action of debranching enzymes and their concentration may vary between 4 and 8% of the malt extract, depending on the degree of modification of the host starch granules. They may be created at the active sites of this enzyme complex when the granule is gelatinized. In a conventional mash of unboiled distilling wort, the spectrum ofwortdextrins produced from gelatinized starch is reduced to true limit dextrins of DP4 8 by continued α-amylolysis during early fermentation. These dextrins will contain side chains of either maltose or maltotriose residues surrounding the α-1,6-glucosidic linkage and can be debranched by limit dextrinase during late fermentation, leaving only the above glucosyl maltodextrins dextrins in the spent wash. Copyright 2016 The Keywords: distilling malt; fermentable extract; mashing; starch granules; α- and β-amylase; limit dextrinase (pullulanase) and α-glucosidase specificities; dextrins A review of malting and malt processing for whisky distillation The science and technology of brewing and of malt and grain distilling are remarkably similar. Indeed, it has been said that malt whisky is simply a distillation of unhopped beer and that grain whisky is based on an adjunct mash containing 90% of unmalted grain insteadofthe20%thatiscommoninmanystylesofbeer.however, there are now major differences in processing cereals in the distilling industry, which not only disprove this mantra, but also challenge some of the fundamentals underpinning brewing science. The most obvious example of this is the derivation of spirit yield. One of the most reliable statistics available to UK distillers is the historical returns made to Her Majesty s Customs and Excise declaring the actual volume of spirit produced and the degree of attenuation achieved. For Scotch malt distilleries, the declared wort fermentability has been between 86 and 87% for at least 150 years. If the above brewing mantra were valid, then the achieved fermentability would have been around 73%, as in a fully attenuated lager wort. Despite this obvious fact, there is no published scientific rationale that can explain why these attenuation limits are so different. However, is 87% the maximum attenuation that can be achieved from an all malt distilling wort? This was the question that scientists working in the Distillers Company research laboratories addressed over 40 years ago. They devised a simple test that involved mixing a finely ground sample of malt with water at ambient temperatures and fermenting the resulting mash directly with a distillers yeast. The alcohol from the fully fermented mash was then distilled off and the alcohol content calculated from the specific gravity of the distillate (1). They found that the attenuation limits were generally ~90%, depending on the malt quality. This method of assessing the maximum fermentability of malt extract, without prior gelatinization of starch, was based on the fact that, if given long enough, the enzymes of malt can completely hydrolyse granular starch. The method was never published as an analytical technique, but it was the basis for a patented distilling process that was successfully scaled up to full production levels (2). Although the spirit quality was markedly different from normal, the spirit yields were declared at 430 L of alcohol per tonne of malt mashed (L alc./t) at 5% malt moisture, some 5% higher than predicted from conventional mashing and equivalent to a wort * Correspondence to: Dr. G. Bathgate, Sunnyfield Consultancy, Albert Street, Nairn IV12 4HF, UK. g.n.bathgate@btinternet.com Sunnyfield Consultancy Albert Street, Nairn IV12 4HF, UK Abbreviations used: CWE, cold water extract; DP, diastatic power; F/C, fine/ coarse; FAN, free amino acid; FE, fermentable extract; FG, final gravity; F a, apparent fermentability; KI, Kolbach index; OG, original gravity; PRD, pullulanase resistant dextrins; PSY, predicted spirit yield; Fr, real fermentability; RG, residual gravity; RH, relative humidity; SNR, soluble nitrogen ratio; SWRI, Scotch Whisky Research Institute. 197 J. Inst. Brew. 2016; 122: Copyright 2016 The

2 198 fermentability of 91%. This patented process for low-temperature mashing and fermenting (usually termed all-grains-in fermentation) has also been used for unmalted cereal with great success. By combining the mashing and fermenting processes, the malt enzymes have up to 72 h to hydrolyse granular starch compared with ~3 h by conventional mashing of gelatinized starch. Therefore the same malt can have a maximum fermentability of 73% when used in brewing, up to 87% when mashed in a conventional malt distilling process, and 91% when mashed and fermented at ambient temperatures. These values are almost constants but, to the date of writing, there is no specific scientific explanation for their distinctive endpoints. Furthermore, they clearly demonstrate that malting and mashing to achieve maximum spirit yield are markedly different from the objectives of brewing and that a better understanding of the biological and biochemical transformations that take place during malting and the subsequent processing of both malted barley and raw cereals is now required. Therefore, the purpose of this review is to highlight the anomalies between current best practice in distilling and many of the basic assumptions derived from brewing science. Many of these anomalies can be partly explained by a re-examination of the existing research, much of which is focussed on brewing, and reinterpreting the results from a distilling viewpoint. This can reveal quite a different picture. Therefore the second objective of this article is to highlight where further research is required to answer some of the questions posed in the above examples. Since the purpose of malting barley for distilling is, by definition, to yield the maximum amount of fermentable extract, which will produce a spirit with the essential flavour and aroma characteristics of malt whisky, we will start by looking at how fermentable extract develops during the malting process and how we measure it. If the maltster has a batch of viable barley, of an approved variety with a protein content of ~10% and it has been screened over a 2.5 mm sieve to achieve a thousand corn weight of 40 g, what are the parameters that will define maximum fermentable extract? Normal practice would be to achieve an out-of-steep moisture of at least 45% and to germinate in a flow of fully humidified air (100% RH) at C for 5 days. This schedule would cater for the largest grain of the highest protein in a normal distribution. In temperate climates, such as Scotland, where the average day/night annual temperature is only 10 C, this is not too difficult but there is still a very fine balance in choosing when to go to kiln. A typical profile of extract development for the malting barley described above is depicted in Fig. 1. If malting were to continue from 5 to 7 days, to exaggerate underlying trends, we can see that there is quite a different pattern to maximizing fermentable extract (3 5) as compared with maximizing total extract (6). The conventional laboratory analyses of measuring total achievable extract by fine grinding (setting 2 on the laboratory mill) and the readily available extract by coarse grinding (setting 7) indicates that optimum conditions are achieved after 5.5 days of germination when the fine/coarse extract difference (F/C Diff.) (6) minimizes at %. In other words, a typical distillery specification of 82% coarse grind extract and a fermentability of 86.5% would be achieved after 5.5 days of germination. There would be no point in continuing to malt any longer since the F/C Diff. has reached its minimum and both extracts decline in parallel for the next 2 days. However, when we look at the pattern of wort fermentability over the same period, a different picture emerges. Wort fermentability peaks at 87% after 4.5 days and declines for the remainder of the malting period, therefore the maximum possible fermentable extract (FE), and G. Bathgate hence spirit yield, can be achieved half a day earlier when the F/C Diff. is 1.8% instead of 0.8%. Since most malt specifications are still set for maximum FE on a coarse grind, then a case can be made for specifying to maximum FE on the fine grind with a F/C Diff. of %. When laboratory analysis systems were standardized over 30 years ago, many Scotch malt whisky distilleries were still operating deep bed traditional mash-tuns, with up to four separate mashes per batch of malt and unsparged wort separations. It was therefore essential that malt had to be perfectly modified to avoid set mashes and so only the coarse extract could be achieved at a fermentability of around 86%. Now that nearly every distillery in Scotland has shallow bed lauter tuns, there is no reason to specify a F/C Diff. of 1 1.5%, when best practice technology could deliver perhaps 7 L more of spirit per tonne of malt with a 1.5 2% index. On such a specification, the maltster would be able to malt at a lower temperature of ~12 14 C if possible and benefit from a lower malting loss over the 5 days. It is therefore necessary to understand the small but significant changes in both wort concentration and composition that take place over the final 2 days of germination. In terms of mass balance, this is again determined by moisture, temperature and time of germination. More specifically we are talking about the finite time it takes for hydrolytic enzymes, synthesized initially in the upper (dorsal) cells of the aleurone layer and nearest (proximal) to the embryo, to progressively penetrate the endosperm to the bottom (ventral) cells most distant (distal) from the embryo. This is demonstrated in most text books (7) by the diagram in Fig. 2(a), which depicts a longitudinal section of a germinating grain of barley. The higher the moisture content (~45 48%) and the higher the temperature (~16 18 C), then the quicker will be the rate of modification, but at the expense of a much higher malting loss. This latter parameter is generally not discussed in a brewing context but it is vital in determining maximum spirit yield. It has long been known that FE is carbohydrate limiting or, in simpler terms, is dependent on obtaining the maximum concentration of fermentable sugars in the distillery wash (8). If a proportion of the barley starch is required to generate the synthesis of rootlets and acrospire in the growing grain, then there is less available for conversion to fermentable sugar. This is even more evident in the case of respiration loss (i.e. the amount of carbon dioxide and water respired from the grain in generating the energy required for germination). Every 6 units of carbon respired to the atmosphere is equivalent to a potential loss of 1 unit of glucose in the extract, therefore the argument for malting to the minimum acceptable degree of modification becomes even stronger. Furthermore, accelerated malting at higher moistures and temperatures for a shorter period (or with the use of gibberellic acid in a non-scotch malt) serves no purpose since this usually leads to the front half of the grain being overmodified and the distal half being partially undermodified, as shown in Fig. 3. The usual two-dimensional diagrams of longitudinal sections (Fig. 2) do not depict this, but scanning electron microscopy (9) is capable of depicting the small number of unmodified cells buried in the endosperm (Fig. 3a). Additional analyses such as friability (10) may detect these malts but not always, since only the cells in the deepest part of the endosperm may be affected. The average analysis, determined by F/C Diff., may be numerically similar to the model cooler, wetter, longer malt (Table 1) but the difference in spirit yield can only be attributed to overmalting and the loss of starch from the endosperm cells closest to the aleurone (Fig. 3a). The ideal malt on the other hand shows no such wileyonlinelibrary.com/journal/jib Copyright 2016 The J. Inst. Brew. 2016; 122:

3 Malting and malt processing for whisky distillation N.B. Maximum fine grind extract at day 5 and maximum coarse grind extract at day 5.5 with minimum C/F extract difference at day 6 N.B. Maximum fermentability at day 4.5 Figure 1. (a) Typical development of fine and coarse grind total extracts of distilling malt over an extended period of germination. NB, Maximum fine grind extract at day 5 and maximum coarse grind extract at day 5.5 with minimum C/F extract difference at day 6. (b) Development of distilling wort fermentability over the same period of germination as in (a). NB, Maximum fermentability at day 4.5. (c) Development of fermentable extract from fine and coarse grinds. (Applying equivalent fermentability values from (b) to extracts illustrated in (a).] (d) Development of predicted spirit yield (PSY = fermentable extract 6.06) from fine and coarse grind fermentable extracts shown in (c). NB, Maximum potential spirit yield on 5 day fine grind extract = 427 l.alc/t and maximum potential spirit yield on day coarse grind extract = 419 l.alc/t at minimum F/C grind extract difference (0.5%). Figure reproduced in colour in online version. starch loss (Fig. 3b) and has a higher predicted spirit yield (Table 1). The overall effect of overmodification and malting loss can also be demonstrated by the mass balance (11), shown in Fig. 4. While this is an oversimplification, it does illustrate the shift in the relative concentrations of the main constituents of malt extract. We have assumed that a 100 g sample of malting barley has a protein content of 10% and that, on a dry weight basis, it suffers a 10 g malting loss. Thus our 90 g of malt might have a total extract value of 82%, which would yield 74 g of wort solids. We have also assumed that the final value for protein solubilization is 45% (as measured by the Kolbach index), yielding 4.5 g of protein from the original 9 g of protein in malt (i.e. 90% of the original 10 g of barley protein). If we ignore the other minor constituents of the wort with a negligible contribution to gravity, we can assume that the remaining 69.5 g of wort solids consist of carbohydrates. Now if we were to carry out a chemical analysis of our original 90 g of malt, we would find that it contains 59 g of starch, 9 g of free sugars, 4.5 g of soluble protein, peptides and amino acids and 1.5 g of soluble glucans and pentosans, thus balancing out our 74 g of malt extract. On this basis, the maximum potential fermentability would be 68/74 or 91% of the potentially fermentable carbohydrate (starch + preformed sugars). This is a figure that matches the degree of attenuation achieved by the all-grains-in fermentation tests described above and therefore gives validity to the model. This simple model also illustrates another aspect of the measurement of distilling wort fermentability. We define real attenuation by the following formula: Real fermentability ðf r Þ ¼ OG RG=OG 100 where OG is the original gravity and RG is the residual gravity. 199 J. Inst. Brew. 2016; 122: Copyright 2016 The wileyonlinelibrary.com/journal/jib

4 G. Bathgate N.B. Maximum potential spirit yield on 5 day Fine Grind Extract = 427 l.alc/t and maximum potential spirit yield on day Coarse Grind Extract = 419 l.alc/t at minimum C/F Grind Extract Difference (0.5%) Figure 1. (Continued). 200 Figure 2. Longitudinal section of germinating barley grain. Then the final values are as dependant on the variable RG as much as on OG, especially if the wort gravity reaches a maximum value, while the RG continues to rise. In the malting scenario depicted in Table 1 and Figs 1 and 2, this is indeed what happens. The fine grind extract maximizes at day 5 as expected, when the last remaining unmodified parts of the endosperm are ruptured mechanically and become soluble. The fine grind extract then declines as an ever increasing amount of carbon is lost through grain respiration. Similarly the coarse extract maximizes at days 5 6 when optimum modification is reached and the F/C grind difference becomes constant at %. Both extracts then decline in parallel with an inverse correlation with malting loss. The only wileyonlinelibrary.com/journal/jib Copyright 2016 The J. Inst. Brew. 2016; 122:

5 Malting and malt processing for whisky distillation Figure 4. Mass balance of barley to malt extract. Figure 3. Scanning electron micrographs of distilling malts germinated with high temperature/low moisture and low temperature/high moisture regimes. (a) Scanning electron micrograph of distilling malt germinated for 5 days at 42-43% moisture and at C (cross-section of distal end). Note dissolution of starch granules adjacent to the aleurone layer on the left of picture and unmodified cells on the right. (b) Scanning electron micrograph of distilling malt germinated for 6 days at 45 46% moisture and at C. Note the intact starch granules adjacent to the aleurone layer on the left of the picture and evenly modified cells throughout the endosperm on the right. malt parameter that increases linearly throughout the latter part of the malting cycle is soluble protein. If the Kolbach index (soluble protein/total protein 100) were superimposed on Figs 1 and 2, it would show a linear increase from ~38% on day 4 to 45% on day 7. In the early stages of germination, some protein is lost to rootlet growth but as the culms wither in the latter stages, solubilized protein accumulates in the endosperm. Since most of this material is non-fermentable, it simply increases the residual gravity and now the overall picture of the development of fermentable extract emerges. During the first 4 days of germination, most of the available extract is carbohydrate with a high fermentability. However, during the critical last 24 h, an equilibrium is reached when the fermentable extract peaks before declining slowly as more carbon is respired to the atmosphere. What makes the equilibrium point even more difficult to detect is the soluble protein effect, which can inflate the residual gravity and final gravity to such an extent that fermentability values are depressed. This is even more apparent when distilling malt respires excessively (or sweats in old malting parlance) during the first phase of kilning. For example, the kilning cycle may be prolonged when the malt is subjected to heavy peating. Moist, acidic peat reek (smoke) slows the drying rate significantly and malt proteolysis is accelerated as the malt stews during the free drying stage. This can have a deleterious effect on the analysis of the finished malt so it is necessary, at this point, to review the fundamentals of distilling malt analysis. When a laboratory analysis is described as a measurement of spirit yield there may be a misconception that this infers an absolute measurement of the concentration of ethyl alcohol. Except where alcohol content and distillery wash OG are measured by a direct GLC/densitometer technique (12), all of the standard methods of analysis for extract, fermentability and spirit yield are anything but absolute. They merely infer values from the measurement of the gravity of various solutions, each containing a large number of constituents with different specific gravities. The best estimate of spirit yield is therefore obtained by distillation of a measured volume of fermented wash. When the volumes of both the distillate and the residual spent wash are restored to their original values using distilled water, the specific gravity of the alcohol solution and that of the residue can be measured. Statutory tables are then used to convert the specific gravity of the alcohol fraction into equivalent degrees of gravity lost during fermentation and, when this value is added to the gravity of the residual fraction, an estimate of the original gravity of the fermented wort and the wort fermentability can be calculated using the above formula. It is also necessary to emphasize the difference between measuring real attenuation and apparent attenuation at this point. In all of the following discussion on wort fermentability, we shall Table 1. Analytical data for malts depicted in Fig. 3 Germination temperature ( C) Moisture (%) day 2 Germination time (days) Coarse extraction (%) F/C Diff. (%) Friability (%) Homogeneity (%) PSY (L alc./t) Warm germination (Fig. 3a) Cool germination (Fig. 3b) F/C Diff., Fine/coarse extract difference; PSY, predicted spirit yield; L alc./t, L of alcohol per tonne of malt mashed. 201 J. Inst. Brew. 2016; 122: Copyright 2016 The wileyonlinelibrary.com/journal/jib

6 202 consider the concentration and constituents of residual gravity as much as fermentable extract, therefore it is essential to differentiate the two ways of measuring fermentable extract. The distillation method for measuring FE and spirit yield is accurate but it is a cumbersome and time-consuming form of analysis. The faster, but less accurate, method for measuring apparent fermentability has therefore become accepted (3) as the standard method in the UK. In this method the OG is taken from the standard method (6) for measuring coarse extract in the laboratory, and after fermentation of the extract, the final gravity (FG) is measured. In this case spirit indication tables cannot be used because they are specific only for alcohol water solutions, therefore a correction factor (once called the solution divisor), which is itself a variable, has to be used to convert apparent fermentability to an estimate of real fermentability as follows: Since Real Fermentability(F r )=OG RG/OG 100 and Apparent Fermentability(F a )=OG FG/OG 100 then Solution Divisor(SD) =F r /F a =OG RG/OG FG = to depending on wort composition. The Solution Divisor is a conversion factor which is not constant and changes with the relative amounts of alcohol and the composition of the residual gravity (3,4), but for a tightly specified malt of low soluble nitrogen the chosen value of does give a good correlation with true F r. Therefore, for brewing wort with a high residual dextrin concentration and low soluble protein (12), the factor may be Unfortunately, this does not allow for protein with a much higher specific gravity than carbohydrate, nor that the concentration of soluble protein may vary considerably in the fermented wort. While the standard method can give a fair estimate of fermentable extract for most distilling malts (4), it can be inaccurate for highly peated malts as described above, highly modified diastatic malts and malts that may have a high sulphur content. All of these types of malt have a much higher content of solubilized protein, which will therefore distort the conversion factor as shown by the following case study from actual commercial practice. The malt in question was highly peated,withslowsmouldering peat, to achieve the highest possible concentration of phenols. The malt kiln was direct fired with heavy fuel oil containing up to 3% sulphur. The peat also contained 1% sulphur. The concentration of sulphur dioxide on the finished malt was ppm. This resulted in ph values of around 4.8 in the mash leading to increased proteolysis, since this value is the ph optimum for carboxypeptidase. The malt already contained a high concentration of soluble protein owing to the stewing effect mentioned above, so it was not surprising that the Laboratory Fermentability was 85.9% and so deemed out of specification. These values were the result of using the FG method and a conversion factor of However, when this malt was mashed in the distillery, the declared attenuation (F r ) was 86.4%. It only takes simple arithmetic to show that a conversion factor of (the recommended brewing factor to allow for higher residual gravity) would have given the same degree of attenuation in the laboratory analysis. The reliability of the standard method for determining malt fermentability, and hence the predicted spirit yield (using the formula PSY = 6.06 FE, where the correlation coefficient of 6.06 is again empirically derived) is well documented (5), but it must be emphasized that this was for one specification of malt, within one group of distilleries, and that the PSY, being only a correlation with declared spirit yields, can be variable across the whole spectrum of distilling malt. G. Bathgate This is demonstrated in recently published data (14) (Fig. 5) and shows that the correlation between PSY and actual distillery yield, while still significant, is much more variable than might be expected (NB, the correlation coefficient of only 0.4). The other conclusion that can be drawn from the data in Fig. 5 is that the actual distillery yields, in the majority of the cases, were significantly higher than the PSY (by approximately 5 L alc./t and are therefore close to the values expected from the fine grind FE of a wellmodified malt with an F/C extract difference of ~1%. If there is doubt about the reliability of quoted figures for fermentable extract and PSY, then the malt soluble nitrogen ratio (or preferably the Kolbach index, KI) should be examined. The latter method is more indicative of variable RG since the results are more in line with the soluble protein values found in practice, because of thicker distillery mashes. There is also a direct method for estimating the potential RG in distillery wash and this can give a better indication of potential variations in fermentability. Measurement of the cold water extract (CWE) provides an estimate of the amount of free sugars, soluble protein, peptides and amino acids, soluble glucan and pentosan and other non-fermentable solutes in the malt (15). Assuming that the sugars and amino acids will be utilized by yeast and that their concentration is constant, then the other water soluble constituents will form the bulk of the RG. This simple analysis is still part of the method for determining amylase activity [diastatic power (DP) and dextrinizing units] in malt but has fallen out of favour as a malt parameter in its own right. This is unfortunate, since it has been shown that there is a strong inverse relationship between CWE and fermentability for the reasons given above (8). It is therefore vital that the quality of distilling malt is depicted by all of the specified parameters and that PSY is taken only as an estimate of the minimum yield that can be achieved in practice, given the above caveats. Nevertheless, the standard method for estimating spirit yield in UK malt is probably more reliable at present (4) than at any other time over the last 30 years for the following reasons. Firstly, there are now very few directly fired kilns still in operation in which heavy fuel oil is burned. The specification for heavy fuel oil allows for a sulphur content of up to 3% and at this level, as in the example described above, the concentration of sulphur dioxide in the malt can be ppm with a concomitant drop in wort ph. Similarly, the need to burn rock sulphur in gas fired kilns to combat the formation of nitrosamines (16,17) is no longer a necessity since nearly all maltsters have converted to some form of indirect heating using heat exchangers (18). Therefore the soluble nitrogen effect, brought about by enhanced proteolysis in the mash, is no longer a concern except in highly peated and enzymic malts. Secondly, in the UK there are a series of new malting varieties that have intrinsically lower soluble nitrogen ratio (SNR) values. Shortly after the standard method for measuring fermentable extract became established, the variety Triumph was the dominant malting variety grown in the UK. This cultivar, and several other subsequent crosses, all displayed a higher level of proteolysis during malting, with SNR values between 42 and 45%. The standard method had been calibrated against unsulphured malt made from Golden Promise with SNRs in the range 36 38%, thus the conversion factor of made it extremely difficult to achieve specified fermentability with Triumph barley, especially at higher total nitrogen concentrations. After the demise of Triumph, new breeding lines resulted in varieties at the other end of the spectrum. The first of these was Chariot with typical SNRs in the range of 35 36% and all subsequent cultivars have had similar values giving high wileyonlinelibrary.com/journal/jib Copyright 2016 The J. Inst. Brew. 2016; 122:

7 Malting and malt processing for whisky distillation Figure 5. Correlation of malt PSY with an actual distillery yield over 18 months. [Reproduced from Bringhurst (13) with permission.] Correlation coefficient (r) = 0.4. apparent fermentability figures. Such traits are obviously generic in British malting barley but the trend may be in the opposite direction in other countries. In general, attempting to achieve maximum modification from poorer varieties with higher nitrogen levels may be counter-productive because of the higher malting loss and the soluble nitrogen effect may also distort estimates of PSY. Therefore, distillers who wish to specify fermentable extract and PSY in their specifications should check their method of analysis to see if the solution divisor requires re-calibration for the type of malt required. This can be accomplished by using the distillation method for F r and then deriving a specific solution divisor for the quick method (F a ). It was necessary to describe in detail the development of fermentable extract, and how it is measured in absolute terms, to provide the context and framework for the following discussion on the original questions posed above, viz. Why does the same malt have an apparent fermentability of 73% when used for brewing, always attenuates to 87% in normal distilling practice but never yields its maximum potential of 92% and what happens in a malt mash at temperatures below the starch gelatinization temperature? The conclusion that can be drawn from the above observations and models of malting is that real fermentability values for unboiled distilling malt wort are dependent on (a) the total amount of fermentable carbohydrate that can be extracted and (b) the variable amount of residual gravity, especially if its soluble protein content is high. However, these variations in fermentability and fermentable extract are minor compared with the difference in the above three values of wort fermentability. We therefore have to look at the three critical differences in mashing listed below and what effect they would have on the enzymolysis of malt starch to derive an answer to our questions: (1) for wort boiled immediately after run-off; (2) for wort from a conventional distilling infusion mash at say, 65 C; (3) for wort from an all-grains-in mash/fermentation at ambient temperatures. For many years the enzymic degradation of starch in a conventional malt mash has been attributed to the action of only α- and β -amylase and the other two known diastatic enzymes, limit dextrinase and α-glucosidase (maltase), were thought to have little or no effect on the overall fermentability of the derived wort (19,20). An all malt brewing wort was reported to contain as much as 25% of the original starch in the form of dextrins ranging from a degree of polymerisation of 4 to over 21 (21,22). These definitive studies by Enevoldsen and Schmidt (21,22) on the structure of brewing wort dextrins were carried out using a refined Sephadex dextran gel chromatographic system. When worts from simulated distilling mashes (8) were chromatographed, at the equivalent concentrations and on the same apparatus, a completely different pattern of dextrinization emerged. Because there was little evidence of higher dextrins in the distilling wort, it was assumed that they had been debranched by limit dextrinase, thus increasing the relative concentration of maltotriose and maltotetraose. That shift in the dextrin profile is demonstrated in Fig. 6(a) by superimposing a chromatogram of distilling wort onto the dextrin profile of a lager beer at the same column loading. The wort from this highly enzymatic malt (DP = 170) was unboiled and had been incubated for 36 h in the presence of a strong bactericide to prevent interference from the growth of natural malt microorganisms. However, re-examination of the chromatographic data of this distilling wort, and others produced by Bathgate et al. (8), reveals a more complex pattern of starch hydrolysis. The laboratory method (23) used was one that replicated a traditional Scotch whisky distillery mash in that three separate malt mashes were made at progressively increasing temperatures (65, 77 and 88 C). Each mash was centrifuged after a 1 h stand and the wort cooled to ambient temperature before being added to 203 J. Inst. Brew. 2016; 122: Copyright 2016 The wileyonlinelibrary.com/journal/jib

8 204 the previous centrifuged wort. [This method is still used as a laboratory standard (4) for producing distilling worts for analytical purposes.] Some of the distilling wort was inactivated by heating to boiling point after final collection (i.e. 3 h after the first mash) and was then compared with active wort incubated at 30 C in the presence of sodium azide for a further 36h to simulate the conditions for a standard laboratory fermentability analysis (3). The results of these HPLC profiles have now been quantified by integration of the original graphs (Fig. 6a c and Table 2). On reexamination of all of the HPLC analyses of these worts, and of the fermented wash derived from them, it was noted that the relative amounts of excluded unfermentable polysaccharide were constant. This provided a useful internal marker to quantify the relative amounts of fermentable sugars and the residual dextrins. Had this been done at the time of publication a different conclusion would have been drawn. Firstly, there was only a very small increase in fermentable sugars from the time final worts were collected and 36 h later (Table 2 and Fig. 6c). The only significant change was the reduction in higher dextrin to true limit dextrins with a degree of polymerisation of 4 7 glucose units (Table 2 and Fig. 6a and b). Secondly, comparison of the profiles between wort, which had been incubated for 36 h, and the fermented wash derived from the same wort clearly shows that limit dextrinase was only active in the fermenting wash (Table 2 and Fig. 6b). The third oversight was that the fractions deemed to be maltotriose were, in fact, mixtures of true maltotriose and unfermentable trisaccharides which remained present in the wash despite being fermented with DCL M yeast, a known maltotriose fermenter (12). A similar result had been reported in a brewing context a few years previously (24). The residual trisaccharides in the brewing wort, which had been fermented with a maltotriose fermenting strain of yeast, were identified as panose and isopanose, that is, 6 2 -α-glucosyl maltose and 6-α-maltosyl-glucose (25). These misconceptions about dextrin formation and the action of limit dextrinase are only now evident because of the rigorous investigations carried out more recently by research teams at the Scotch Whisky Research Institute (SWRI). They have examined the role of distilling malt saccharifying enzymes in much more detail (26,27) and have confirmed the importance of limit dextrinase in producing a more highly fermentable wort in distilling practice. Prior to this research, it was assumed that, as in brewing malt, limit dextrinase had little effect on the composition of wort (22) during mashing. Using a high-performance anion exchange chromatographic system, Bringhurst et al. (27) at the SWRI not only identified dextrins with a degree of polymerisation of 4 7 glucose units in a distilling malt wort but also separated the linear from branched isomers in this range. The purified fractions with 6 and 7 glucose units were treated with pullulanase in the same manner that Enevoldsen and Bathgate (28) had used to elucidate the structure of the equivalent brewing wort dextrins. These digests, together with further characterization by sophisticated mass spectrometry, clearly showed that the branched dextrins with a degree of polymerisation of 6 and 7 in a distilling wort were nearly all isomers of either maltose or maltotriose attached by α-1,6-glucosidic linkages to either maltotriose or maltotetraose. However, there were very small amounts of residual 6 and 7 unit dextrins, which were not degraded by pullulanase and which may be of some significance. The postulated structures for the hydrolysed dextrins therefore complied with a proposal by Vinogradov and Bock (29) that all α-1,6 branched dextrins of 10 glucose units or less have maltose or maltotriose as the side chain, thus making them amenable to hydrolysis by pullulanase or limit dextrinase. G. Bathgate Bringhurst et al. (27) inferred from this theory that the fractions containing 8 and 9 glucose units that they had identified must, therefore, have an analogous structure. The SWRI team also demonstrated (26) that the majority of dextrins were formed during mashing and that, during the early stages of fermentation of an unboiled wort, there was continued accumulation of true limit dextrins by further α-amylolysis of higher dextrins. What was most revealing in this latter work was the action of limit dextrinase in hydrolysing most of the branched dextrins, but not until the fermentation was well under way. By monitoring the concentrations of 6 and 7 unit branched dextrins during the course of fermentation, they clearly demonstrated that the relative amounts of each of these fractions more than doubled during the first h of fermentation, when α-amylase was at its highest activity and then declined to around 40% of their original concentrations over the next 20 h, when limit dextrinase rose to its maximum activity. It is interesting to note that the concentration of the residual dextrins then remained constant until the end of a 60 h fermentation. The exact mechanism whereby limit dextrinase remains active during the first 36 h of fermentation is not entirely clear, but it would appear that there is continuous release of free limit dextrinase from the bound form as described below. The implication is that all of the components of this fraction that can be debranched are hydrolysed during the first half of fermentation when all true limit dextrins have been formed by post-mashing α-amylolysis. It is now well established that limit dextrinase is present in malt, mostly as an inactive form linked to an inhibitor (30,31). However, the theory that limit dextrinase must thereby be inactive during mashing has been disproved by the revelation that its proteinaceous inhibitor can be uncoupled by a cysteine proteinase (32) in malt and that some free limit dextrinase activity can be present in wort (33). What the definitive research at SWRI has proven is that limit dextrinase is not heat inactivated during mashing but can pass into the wort to contribute significantly to the increase in fermentability during the middle stage of fermentation. So whether free or in a bound form, what is now proved both by the SWRI work and the revision of the earlier work by Bathgate et al. (8) is that limit dextrinase only becomes fully active in a fermenting wort. The principal enzyme activity in unboiled wort, even when incubated 36 h after wort separation, is that of α-amylase (34) since there was no appreciable increase in total fermentable sugars (Table 2 and Fig. 6b). The inference is that β-amylase may only be active during mashing, or in the early runnings of cooled wort to the fermenter, because there was no overall increase in maltose (Table 2). However, there may have been some turnover in maltose and maltotriose by α-glucosidase (maltase) since there was a small but measurable increase in the glucose concentration (Fig. 6c and Table 2). The initiation of debranching activity during fermentation is related to the drop in ph from around 5.0 in wort to about 4.5 in an actively fermenting wash (35).While α-amylase is most active at ph values between , the debranching enzyme in fermenting wash has an optimum ph of The conclusion is that, while free limit dextrinase may be present in wort, it is the bound form, uncoupled from its inhibitor, which becomes active in the fermentation of an unboiled wort, when yeast activity reduces the ph to the latter values (36). These results can also be substantiated by now returning to the model of malt extract (37) depicted in Fig. 4. It was postulated that 59 g of the total extract of 74 g consisted of starch. If we make another simplification and estimate that the amylopectin content of wileyonlinelibrary.com/journal/jib Copyright 2016 The J. Inst. Brew. 2016; 122:

9 Malting and malt processing for whisky distillation Data from Enevoldsen and Schmidt 60 and Bathgate, Martinez-Frias and Stark 8at Data from Bathgate, Martinez-Frias and Stark 8 Figure 6. (a) Comparison of lager beer [data from Enevoldsen and Schmidt (21)] and distilling wort [data from Bathgate et al. (8)] dextrinsbyhplc.(b) Glucose, maltoseand maltotriose/trisaccharide profiles in distilling wort and fermented wash [data from Bathgate et al. (8)]. (c) Changes in dextrin profiles in distilling wort and fermented wash [data from Bathgate et al. (8)]. Figure reproduced in colour in online version. malt starch is 80%, with an average chain length of 25 glucose units (38,39), then this would result in a molecule with four α-1,6- glucosidic linked branch points for every 100 units in α-1,4-linked chains. If we now assume that the absolute limit of α-amylolysis around a branch point is two glucose units (29,40), that is, 6 3 -α-maltosy-maltopentaose, as postulated by Vinogradov and Bock (29) then we can envisage the simplest structure for amylopectin as being one with an equal number of A:B chains (38,39) 205 J. Inst. Brew. 2016; 122: Copyright 2016 The wileyonlinelibrary.com/journal/jib

10 G. Bathgate Data from Bathgate, Martinez-Frias and Stark 8 Figure 6. (Continued). Table 2. Chromatographic data from HPLC of distilling wort and fermented wash dextrins and equivalent concentrations (arbitrary units) of fermentable sugars (Fig. 6a c) Glucose Maltose DP3 DP4 DP5 DP6 DP7 DP8 DP > 8 Total Wort (3 h) Wort (36 h) Wash Data from Bathgate et al. (8). 206 and six glucose units around each of the four branch points as shown in Fig. 7. In other words 7 4 glucose units in every 100 in the amylopectin molecule would form limit dextrins. If we also assume that all of the amylose can be hydrolysed by amylases to fermentable sugar, then 20% of the original 59 g of malt starch would yield 11.8 g of maltose and maltotriose. That would leave 80% of the starch as amylopectin of which 28% would be dextrin, assuming that there was no further hydrolysis by limit dextrinase and glucosidase. The outer chains of amylopectin could therefore yield 72% of fermentable sugars. Thus 47.2 g of amylopectin would yield 34 g of fermentable carbohydrate (72% of 47.2 g). This theoretical composition of fermentable extract is summarized in Table 3 for two scenarios: one in which all of the amylopectin branch points are intact (i.e. no de-branching) and a second in which all of the branch points are hydrolysed by limit dextrinase. Since the model dextrin used was the smallest postulated, then the calculated fermentability of 74% represents the highest value expected for a brewing (boiled) wort, fermented with a maltotriose fermenting yeast, and in which there has been no debranching activity. The range of fermentabilities reported when DCL M yeast was recommended for a standard brewing wort fermentability method (12) was 73.4 ± 0.9%, so matching the above theoretical calculation. Similarly, the theoretical value for fully debranched amylopectin (92.1%) matches the results reported (90 91%) for ambient temperature malt mashing/fermentations using the DCL all-grains-in test for maximum attenuation. This further corroborates the above reviews in that: There is no limit dextrinase activity during normal mashing and wort run-off when the only active enzymes are α- and β-amylase. There is considerable α-amylase activity during mashing, wort run-off and early fermentation, creating a turnover from higher molecular weight dextrin to true limit dextrin. There is considerable debranching by limit dextrinase, but only when fermentation is established and the wort ph drops to There is possibly a small contribution from α-glucosidase in increasing the relative amount of glucose in the collected wort. We now have a clearer picture of what happens during normal malt distilling practice, but this still does not explain the residual dextrin left in fully fermented wash. This only amounts to about 5% of the total carbohydrate in wort (Table 2) and is of the same order of magnitude as reported by Bringhurst et al. (27). However, the latter authors did not include non-linear trisaccharides, which appear to be present in their chromatographic analyses of wort. The analyses carried out on Enevoldsen s apparatus (8), on the other hand, demonstrated that significant amounts of unfermented trisaccharide were present in the fully fermented wash wileyonlinelibrary.com/journal/jib Copyright 2016 The J. Inst. Brew. 2016; 122:

11 Malting and malt processing for whisky distillation Figure 7. Structure of outer chains of amylopectin and corresponding smallest α-amylase limit dextrin [as proposed by Vinogradov and Bock (29)]. Table 3. Theoretical fermentable extracts based on model malt (Figs 4 and 7) Component Total extract ( g) Fermentable extract (no debranching) Fermentable extract (total debranching) Starch: amylose amylopectin Free sugars Soluble protein Glucans etc. 1.5 Total Fermentability 74.3% 92.1% of this high enzymatic malt. (Fig. 6b and c, Table 2) Assuming that linear maltotriose was completely fermented, those residual trisaccharides might be panose and isopanose and represent about 30% of the original wort trisaccharides. Similarly, the DP4 7 residual dextrins, which are apparently resistant to limit dextrinase or pullulanase, might be glucosyl-maltotriose, -maltotetraose, - maltopentaose and -maltohexaose. The total concentration of this residual fraction is very small relative to the original concentration of maltose (Table 2) but it is still significant with respect to the final attenuation and requires further investigation. The puzzle that now presents itself is that none of these pullulanase resistant dextrins (PRD) should exist according to the above model of Vinogradov and Bock (29). Indeed,if panose and isopanose are present, then glucosyl-maltose and maltosyl-glucose must be the smallest dextrins containing an α-1,6 glucosidic linkage. The essential test would be to isolate the PRD present in the wash and treat them first with pullulanase and, if they are resistant, then with amyloglucosidase (41). If all of the remaining dextrins are hydrolysed to glucose then the above formulations are correct. This would be relatively easy to carry out, since the substrate can be sourced, quite literally by the ton, from any malt distillery s spent wash. On the assumption that PRD are residual dextrins with a degree of polymerisation of 3-7 then they should have the structures shown in Fig. 8. Given that α- and β-amylase cannot hydrolyse maltose or maltotriose on either side of the glucosyl-α-1,6-linkage then the predominant isomers will have a degree of polymerisation of 4 6 as illustrated. However, the revised data from Bathgate et al. (8) in Fig. 6(c) would suggest that the trisaccharides are the most prevalent residues, therefore the core dextrin for PRD is either panose or isopanose and not maltosyl-maltotetraose as depicted in Fig. 7. The existence of PRD in a fully fermented malt wort may also explain why attenuation maximizes at ~87% in a normal mash but never reaches the theoretical maximum. Since they are not present in an ambient-temperature all-grains-in mash and fermentation, which does go to completion at 91% fermentability, we can conclude that saccharification of starch granules follows a completely different pathway from gelatinized starch. Therefore, 207 J. Inst. Brew. 2016; 122: Copyright 2016 The wileyonlinelibrary.com/journal/jib