Letter to the Editor: Update on Knowledge Regarding Starch Structure and Degradation by Malt Enzymes (DP/DU and Limit Dextrinase)

Transcription

1 Letter to the Editor: Update on Knowledge Regarding Starch Structure and Degradation by Malt Enzymes (DP/DU and Limit Dextrinase) George N. Bathgate and Tom A. Bringhurst J. Inst. Brew. 117(1), 33 38, 2011 Dear Editor, We are currently developing a Diploma in Distilling (cereal, molasses and grape based). However, the teaching material covering the syllabus for each of these options tends to be out of date or nonexistent. For example, the following is a short summary of current information on starch structure and its enzymic breakdown during the mashing of malted barley and the fermentation of its unboiled wort, only some of which appears in available text books. We would therefore like to make this available for the next diet of exams and also to encourage prospective candidates to use this Journal as a means of keeping their knowledge of brewing and distilling science up to date. George N. Bathgate for IBD Board of Examiners and Tom A. Bringhurst Scotch Whisky Research Institute INTRODUCTION Currently, starch and its structure and properties continue to be a major area of both academic and applied research, and our knowledge in this area is continually expanding. While some fundamental aspects of this subject are now reasonably well understood, at least in principle, our knowledge of the details continues to evolve as researchers focus more closely on the fine structure and functions of starch in cereals and other materials. Much of this is underpinned by the application of more modern analytical techniques and rapid developments in the understanding of the genetic factors underlying the quality traits required by barley end users. It is important to emphasise that although starch has been studied for many years, we still do not know everything, and our evolving knowledge of the fine structure and functions of barley (and other cereal) starch remains a work in progress. Tester et al. 24 present a review of fairly recent knowledge of starch biosynthesis and structure which highlights the dynamic nature of this field of research. This review summarises the biosynthesis of starch from its starting point in sucrose from photosynthesis in the initial stages of α-glucan deposition, to the synthesis Publication no. G The Institute of Brewing & Distilling of starch granules, but also includes relatively detailed descriptions of amylose and amylopectin, which relate to cereal and other starches. MacGregor and Fincher 21 continue to provide the standard reference for the carbohydrate components of barley, concisely summarising information on starch structure and enzymes which continues to be of fundamental relevance to brewers, maltsters and distillers. More recently Bamforth 1 and Evans et al. 18,19 have reported studies which have provided more recent data. While much of this work is focussed on brewing, it is still of great relevance to distillers. STARCH STRUCTURE Malted barley contains a bimodal distribution of starch granules. These consist of large lenticular A granules (diameter ~15 25 microns) and small spherical B granules (diameter < 10 microns). The small granules account for approximately 80 90% of the total number of starch granules, but only about 10 15% of the total weight of starch 23. Starch granules are composed of 2 main polysaccharides (α-glucans), amylose and amylopectin. It is important to note that cereal starches also contain integral (internal) lipids (lysophospholipids (LPL) and free fatty acids), which are largely associated with the amylose fraction, and which can be present at up to 2% of the total starch weight in high amylose barley 24. Starch granules are also contaminated with surface lipids such as triglycerides, glycolipids, phospholipids and free amino acids deriving from the amyloplast membrane and non-starch sources 24. Starch lipids and proteins can occur on the surface of the starch granules as well as being embedded within the matrix of the granules and can have the potential to moderate the functionality of the starch 24. In lipid containing starch granules, part of the amylose fraction is present as an amylase inclusion complex, where fatty acid chains occupy a hydrophobic core within the single amylose helix 24. This can range from <15 to >55% of the amylose fraction in cereal starches. Oat starches are particularly rich in lipids and complexed amylose. Amylose and amylopectin have different structures and properties. Amylose is essentially linear and has a molecular weight of and is composed of approximately 500 5,000 α-(l,4) linked glucose units and contains a small number of α-(l,6) branch points (approximately 9 20 per molecule) 1. VOL. 117, NO. 1,

2 Amylopectin is a much larger molecule, with a more complex, highly branched structure and is comprised of three different types of α-(l,4) glucosyl chains, linked via α-(l,6) branches to form a molecule of molecular weight The C chain terminates with the sole reducing group, and contains approximately glucose residues 1. The B chains branch from the C chains, (and other B chains) and also contain approximately glucose residues. The A chains, which contain approximately 15 glucose residues, are terminal linear branches from the B chains 1. At our current stage of knowledge, barley starch is typically composed of 25 30% amylose and 70 75% amylopectin 1. These values are not far removed from the standard textbook values that have been used in the malting, brewing and distilling industries, where it has been accepted that the starch of malting quality barley is composed (on average) of 20% amylose and 80% amylopectin, and suggests that amylopectin contains α-(l,6) branch points, on average for every (approximate) 25 glucose units 21. Both amylose and exterior (A and B) chains of amylopectin can entwine to form double helices, which promote the formation of more crystalline structures 24. However not all the amylopectin forms double helices, and the proportion can vary for different starch sources (no figures are given for barley, but for wheat, the proportion is between 32% and 46%). The degree of crystallinity is important, since it influences the degree of packing of the helices within the structure of the molecule, which can influence the structure of the starch granules and their ordering within the cereal. Cereals are generally characterised by a relatively compact crystalline structure, compared with high amylose starches associated with tuber, legume, root, fruit and stem starches, which have more amorphous, open structures. Barley is associated with about 20 24% crystallinity. The crystalline regions of amylopectin impart structure to the molecule (crystalline lamellae), giving rise to regular three dimensional patterns, which can associate together to form ordered regions, and ultimately to form starch granules. Branch points constitute amorphous regions within the structure 24. STARCH DEGRADING ENZYMES AND MALT FERMENTABILITY The actions of the main starch degrading enzymes in barley, α-amylase, β-amylase and limit dextrinase (and their various iso-forms) together with α-glucosidase, have been well established for many years 21. More recent work by Bamforth 1 confirms that α-amylase, β-amylase and limit dextrinase are of primary importance in the hydrolysis of starch, but suggests, in the absence of other evidence to the contrary, that the contribution of α-glucosidase is minimal in comparison with the other enzymes. Evans et al. 15 present a useful summary of the functions and actions of the starch degrading enzymes and references some information 20 regarding the typical fermentable sugar composition of barley malt wort (maltose (65.4%), maltotriose (17.6%), hexoses (glucose/fructose) (11.2%) and sucrose (5.1%)) deriving from the combined action of the diastatic power (DP) enzymes. The enzyme α-amylase randomly attacks amylose and amylopectin chains to produce a mixture of maltose, maltotriose and a range of dextrins. The enzyme β-amylase removes maltose units from the non-reducing ends of the branches. Both of these enzymes are very active during mashing. The combined action of these enzymes can efficiently degrade starch amylose and amylopectin, but predominantly operate up to 2 units from the branch points, so there will be a large proportion of residual limit dextrins, containing at least 4 8 glucose units 8,26. However, the smallest dextrins found in all-malt wort are the trisaccharides panose (6 2 α-glucosyl-maltose) and iso-panose (6α-maltosyl-glucose) 3. Similarly, it has been demonstrated 12,13 that 6α-glucosyl-maltose and 6α-glucosyl-maltotriose are also present in wort. This is proof that it is possible for the combined action of all the amylolytic enzymes in malt to hydrolyse amylopectin down to within one glucose unit of the branch point so that they are out with the specificity of limit dextrinase and pullulanase. However the concentration of these resistant dextrins is relatively small 6. Since on average there are approximately two branch points for every 50 glucose units, this means that in the absence of de-branching enzymes such as limit dextrinase and α-glucosidase (maltase), approximately 12% of the original molecule would remain as a limit dextrin. Although these two enzymes are heat sensitive during mashing, and would have limited action during mashing, they can break down at least half of the residual limit dextrins during subsequent secondary conversion in a distilling wort, to give further linear dextrins (3, 4, 5 glucose units) which can then be hydrolysed to fermentable sugars by α- and β-amylase 5,6. Such secondary conversion can yield an additional 15% in fermentability 5. This figure was deduced by comparing the fermentabilities of inactivated (brewing) and active (distilling) worts made from the same Institute of Brewing check malt extracted by the standard methods of the Institute and using the same distillers yeast 4,10. LIMIT DEXTRINASE While the actions and activities of α- and β-amylase have been well established and well documented in the literature for some time, our understanding of limit dextrinase has progressed significantly during the last decade, as researchers have focussed more closely on this enzyme. Bryce et al. 9 provide a concise summary review of recent knowledge regarding this enzyme with respect to malting and distilling. Our current understanding of limit dextrinase is that it is largely present in malt in an inactive form, where it is bound to a protein inhibitor, which protects the enzyme against the relative high temperatures encountered during mashing. The active enzyme is only partially released during mashing, but is denatured at mashing temperatures. Thus the debranching activity is relatively small during mashing. However, recent work 22,25 has shown that the active, free enzyme can be released during the early stages of fermentation, as the ph falls from a wort ph of 5.5 to ph , where it becomes available to degrade α-(l,6) branched dextrins in the fermenting wort, and is thus an 34 JOURNAL OF THE INSTITUTE OF BREWING

3 important factor in the secondary conversion of dextrins to fermentable sugars during fermentation. Since, for whisky production, the wort is not boiled, the efficient degradation of starch to fermentable sugars is a result of the combined action of α- and β-amylase, limit dextrinase and yeast enzymes during mashing/extraction and fermentation. HYDROLYSIS OF DEXTRINS BY ENZYMES Enevoldson and Bathgate 12, Enevoldson and Schmidt 13 and Bringhurst et al. 8 have produced papers summarising the products of the hydrolysis of starch and higher dextrins into fermentable sugars, short chain branched and linear dextrins and oligosaccharides, which contribute greatly to our fundamental understanding of these processes in relation to mashing and fermentation in both brewing and distilling. These give some indication of the structures and properties of both linear and branched dextrins which can help us to appreciate their importance in determining wort fermentability, since some of them are easily degraded to fermentable substrates, while others can only be partially, or slowly degraded, or are limit dextrins and hence cannot be fully hydrolysed by the standard set of starch degrading enzymes (α-amylase, β-amylase and limit dextrinase 8,12,13 ). Vriesekoop et al. 26 further elucidated the actions and activity of the main starch degrading enzymes α- amylase, β- amylase and limit dextrinase, and describe in some detail the turnover of dextrins during mashing and fermentation of brewing (boiled) and distilling (unboiled) type worts. They confirmed the previous suggestion 8 that there is a dynamic equilibrium as larger dextrins are hydrolysed and then further degraded and debranched to produce smaller dextrins. However, they did not fully address the diversity of dextrin structures (linear, and branched dextrins (singly and possibly double branched)), which are also highly important wort components that will have an impact on fermentability 8,12,13. These will affect the subsequent hydrolysis by β-amylase and limit dextrinase in the continuing breakdown of higher dextrins during fermentation, and might help to account for the residual levels of maltotriose that were observed during the fermentation of the distillers worts. However, this also could be due to any of a number of factors affecting fermentation efficiency, including the yeast, fermentation conditions or the presence of bacterial infection, as well as a result of the random turnover of higher dextrins by the enzymes. It is also possible that maltotriose is fully fermented and what has been observed could be panose and iso-panose (i.e., glucosyl- 1,6-maltose and/or maltosyl-1,6-glucose) 3,6. In mashing, the wide range of dextrin structures including every possible isomer, from 3 8 glucose units is produced by the combined and simultaneous action of α- and β-amylase (and possibly α-glucosidase) on gelatinised starch granules. Vriesekoop et al. 26 suggest that there is a dynamic equilibrium, which results in a low utilization of maltohexaose and maltoheptaose and that this could be due to the combined activity of limit dextrinase and α- amylase, although further work is necessary to confirm this. There was also a preferred accumulation of maltopentaose and maltohexaose during mashing, which might relate to steric hindrance in close proximity to an α-1-6- branch point (i.e., within 3 glucose residues). This is supported by Enevoldsen and Schmidt 13, who observed that 7- to 8-glucose unit dextrins may contain two α-1,6- linkages which makes them inaccessible to α-amylase and limit dextrinase. Vrieskoop et al. 26 suggest that there is a dynamic equilibrium as larger dextrins are hydrolysed and then debranched, and it is clear that there is an underlying concentration of dextrins that cannot be attacked by any of the active malt enzymes or utilized by yeast during fermentation 8. As a result, there is a residue of unfermentable dextrins that still requires further study 26. This might include 4- to 7-glucose unit dextrins containing glucosyl- 1,6-maltotriose 12,13, (-maltotetraose, -maltopentaose and -maltohexaose) which cannot be hydrolysed by limit dextrinase or pullulanase. PREDICTION OF MALT FERMENTABILITY Bathgate et al. 6 provide a framework for our understanding of the fundamental parameters controlling the fermentability and fermentable extract of distilling malt. This provides the basis of our understanding of the biochemistry underlying the standard fermentability method 7,10,11, which is still one of the principal barley malt quality parameters, recognised by both distillers and sales maltsters for commercial trading purposes. In recent years there have been some attempts to find ways of predicting the fermentability of barley malt from first principles, based on our detailed knowledge of the structure of barley starch, and the known activities of the enzymes that are developed and/or released during malting. Two models are described, one based on the hypothetical structure of typical barley starch fractions 5, to predict the fermentable extract of distillers barley malt. The other is based on the relative activities of the different malt enzymes 15,which is more focussed on brewing. PREDICTION OF FERMENTABLE EXTRACT FROM STARCH STRUCTURE (BATHGATE MODEL) The combined action of the starch degrading DP/DU and limit dextrinase enzymes can efficiently degrade starch amylose and amylopectin. Normally α and β amylase can only operate up to 2 units from the branch points, so there will be a large proportion of residual limit dextrins, containing 4 8 glucose units. As shown above, there will also be some 3- to 5-glucose unit dextrins that are resistant to further enzymic hydrolysis. In terms of malt distilling, the inference is that there will always be some residual dextrins that cannot be fully broken down to fermentable sugars. The exceptions to this might be the trisaccharide fraction that can be fermented by some strains of distilling yeast 8. However, in the simplistic model VOL. 117, NO. 1,

4 shown below, it is assumed that normal limit dextrin contains 7 glucose units, i.e., 3 maltose units surrounding the glucose unit containing the α-1,6-linkage. The amylopectin content of barley starch is usually in the range of 70 to 80%, but for the sake of simplicity in the following model it is assumed to be at the higher end of this range. It is also assumed that the average chain length of amylopectin is 25 glucose units, so that there are on average 2 branch points for every 50 glucose units. This means that in the absence of de-branching enzymes, such as limit dextrinase and α-glucosidase (maltase), approximately 12% of the original molecule would remain as a limit dextrin. Although these two enzymes are heat sensitive during mashing, and would have limited action during mashing, they can break down at least half of the residual limit dextrins to give further linear dextrins (3, 4, 5 glucose units), which can be hydrolysed to fermentable sugars by α- and β-amylase, and can give an additional 15% in fermentability as demonstrated above. On the basis of our current knowledge and assumptions, it is possible to make an estimate from first principles of the soluble extract, fermentability and fermentable extract of a typical distilling malt sample, which can show the effects of the debranching enzymes on the fermentable extract. This can be done by deriving the soluble extract from the various components of the malt, in terms of starch, pre-hydrolysed sugars, soluble protein and other soluble material, and using our knowledge of starch structure and composition to estimate the relative contributions of amylose and amylopectin degradation, taking into account the actions of α- and β-amylase and the debranching enzymes (limit dextrinase, α-glucosidase (maltase). It is then possible to calculate the fermentability and fermentable extract for a given proportion of limit dextrins and to give approximations for the secondary conversion of dextrins to fermentable sugars. This is illustrated by the example shown below. EXAMPLE Soluble extract Typical barley at 65% starch and 10% protein (TN = 1.60%), is malted to a Kohlbach Index of 45% to give 4.5 g soluble protein/100 g. Since the malting loss is normally in the region of 10%, this is equates to the malt containing 59% starch. Thus 100 g malt would contain 59 g starch and 4.5 g soluble protein, together with prehydrolysed sugars (estimated at 9 g) and other soluble material (e.g., glucans and minerals at approximately 1.5 g). Table I shows how these data can be combined to give an estimate of soluble extract. Table I. Individual components of soluble extract. Relative amount deriving from Component 100 g barley (g) Malt starch (g) 59 Prehydrolysed sugars (g) 9 Soluble protein (g) 4.5 Other solutes/glucans/minerals (g) 1.5 Total soluble extract (g) 74 Since on average, 100 g barley gives 90 g malt for a malting loss of 10%, the percentage soluble extract (SE) of malt can be calculated as follows: SE (%) = 74/ = 82% Fermentable extract If barley malt starch is composed of 20% amylose and 80% amylopectin, 11.8 g of fermentable extract will derive from the amylose fraction of 59 g starch, assuming that all of the amylose is degraded to fermentable sugars. Amylopectin accounts for the remainder (47.2 g). Assuming that the ratio of A to B chains is 1:1, there are 2 branch point units per 50 glucose units (4 per 100), amylopectin will be degraded to 28% limit dextrin (i.e., 4 7 glucose units around each α-1-6 linkage), and the rest (72%) to fermentable sugars. Thus 47.2 g amylopectin will yield 34 g fermentable sugars (72 % of 47.2 g). Table II summarises the contributions of the components of the fermentable extract (FE) based on the assumption that there is no significant post-conversion action of limit dextrinase and α-glucosidase in degrading limit dextrins. Based on this value for the fermentable extract, and the total extract (74 g) a value of 74.3% can be estimated for the apparent fermentability (i.e., 55/74 100), assuming no significant post-conversion activity of limit dextrinase and α-glucosidase. Apparent fermentability (%) = 55/ = 74.3% Since the percentage fermentable extract is the product of the soluble extract and the apparent fermentability: Fermentable extract (%) = ( )/100 = 60.93% This figure would be considered to be equivalent to the fermentability of a boiled (brewing) wort. An idealised figure for the fermentable extract can also be calculated for the total fermentable extract, assuming all of the branch points in amylopectin are hydrolysed and the dextrins are completely converted to fermentable sugars (Table III). Table II. Individual components of Fermentable Extract (FE) (no LD/αglucosidase activity). Relative amount deriving Component from 100 g barley (g) Preformed sugars in malt (g) 9 FE from amylose hydrolysis (g) 11.8 FE from dextrinised amylopectin (g) 34 Assimilable amino nitrogen and other nutrients (g) 0.2 Total fermentable extract (g) 55 Table III. Individual components of Fermentable Extract (FE) (assuming complete conversion of dextrins). Relative amount deriving Components from 100 g barley (g) Preformed sugars in malt (g) 9 FE from amylose hydrolysis (g) 11.8 FE from complete amylopectin hydrolysis 47.2 (g) Assimilable amino nitrogen and other 0.2 nutrients (g) Total fermentable extract (g) JOURNAL OF THE INSTITUTE OF BREWING

5 Similarly, based on a total extract of 74 g, a value of 92.2% can be estimated for the apparent fermentability (68.2/74 100). Apparent fermentability (%) = 68.2/ = 92.2% This would equate to a fermentable extract of 75.6% for unboiled wort. Fermentable extract (%) = ( )/100 = 75.6% In this case, the difference in fermentability resulting from the complete degradation of amylopectin was 17.9% ( ). This equates to a difference in fermentable extract of 14.7%. The above value of 74.3% fermentability equates almost exactly to that found for the laboratory extracted wort from a standard lager brewing malt 4 and which had been inactivated immediately after wort filtration. On the other hand, the maximum theoretical fermentability is some 5% higher than the comparable fermentability of unboiled wort from the same malt, indicating that about 4% (5% of 82%) of the original extract remains unfermentable. This is also similar, in order of magnitude, to the amount of unfermented dextrin found in distilling worts 6, thus the model provides empirical values which are comparable to distilling practice. However, it is important to emphasise that the above case study is based on an idealised situation, since in the real world, the starch structure and composition of barley malt grains will vary considerably, even within individual barley grains, and within the complex structures of the starch granules themselves. Nevertheless, the exercise shows that it is possible to demonstrate how our current understanding of the micro structure and composition of malted barley starch, can influence the properties of the grain, and how we can use this information to provide meaningful data regarding the macro quality and properties of a given sample of barley malt. PREDICTION OF APPARENT ATTENUATION LIMIT (AAL) FROM MALT ENZYME ACTIVITY (EVANS MODEL) Evans et al. 15 examined the combined effects of α-amylase (Megazyme Ceralpha method), β-amylase (Megazyme Betamyl method) and limit dextrinase (Megazyme Limit DextriZyme method) to develop a predictive model for fermentability based on the apparent attenuation limit (AAL) of small scale mashes based on EBC method (EBC reference) This data was combined into the following algorithm: Apparent Attenuation Limit (AAL) = A B C D E 0.001DE Where: A = α-amylase activity (U/g); B = total limit dextrinase activity (U/kg); C = Kohlbach Index (KI) (%); D = total β-amylase activity (U/g); E = β-amylase thermostability (%). The R 2 value was While this equation was derived in the context of brewing applications, it is still relevant to distillers, since it offers a theoretical baseline against which to assess the relative effects of different enzyme activities. More recent work by Evans et al. 16, has refined this model to include the effects of other parameters such as limit dextrinase thermostability, Kohlbach Index (KI), free amino nitrogen (FAN) and wort β-glucan in multilinear regression (MLR) and partial least squares (PLS) multivariate models. It should be emphasised that the aim of this model was to identify predictive factors that barley breeders could use to support breeding programmes directed at maltsters and brewers, rather than to determine a universal model to predict malt fermentability. It should be emphasised that both these models represent to some degree idealised situations, but are important in that they can provide empirically derived baseline models with which to define the theoretical limits, which can be used to predict the quality of malted barley. CONCLUSIONS Our knowledge of starch composition and structure is very much a work in progress, and there has been much progress in recent years in elucidating the structural and biochemical complexity of this substrate, and it is important to be aware that while it is possible to develop macro models relating the structural features to the actions of different starch degrading enzymes to provide fermentable sugars, these by their very nature provide a somewhat idealised and simplified picture. However, these models are important in providing a theoretical baseline to allow us to understand more fully the basis for comparisons between different barley malt samples, and to provide meaningful information that will be useful in helping to predict their properties REFERENCES 1. Bamforth, C. W., Barley and malt starch in brewing: A general review. Tech. Q. Master Brew. Assoc. Am., 2003, 40, Bamforth, C. W., Current perspectives on the role of enzymes in brewing. J. 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