STRUCTURAL ANALYSIS OF WORT DEXTRINS BY MEANS OF β-AMYLASE AND THE DEBRANCHING ENZYME,PULLULANASE

Dextrins from a sweet wort, prepared by infusion mashing, were fractionated by paper chromatography according to their molecular weights and further into linear and branched dextrins. Each fraction was treated with β-amylase and with the debranching enzyme, pullulanase. The products of this enzymic hydrolysis were identified by paper chromatography. From the pattern of oligosaccharides so obtained, structures have been postulated for the dextrins with a degree of polymerization of 4 to 8 glucose units. The linear dextrins, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and malto-octaose, were shown to be present in wort. In addition, at least eleven branched dextrins were found: 6²-α-maltosyl-maltose, 6-α-glucosyl-maltotriose, two branched pentasaccharides, two branched hexasaccharides, two branched heptasaccharides and three branched octasaccharides.     

I. CONDITIONS OF MASHING AND CARBOHYDRATE COMPOSITION OF THE WORT

Experimental mashings of ungerminated barley and 5–10% of malt with addition of the debranching enzyme pullulanase have been carried out. Worts with high attenuation are obtained in good yield. Of the fermentable sugars, there is less glucose, and more maltose and maltotriose than normally observed. The dextrin pattern is different from, but not necessarily inferior to, that traditionally seen. The worts resulting from the action of pullulanase are deprived of the dextrins with 8–14 glucose units, whereas the amounts of dextrins with 4–6 glucose units are close to those normally observed. The pullulanase preparations used are accompanied by proteolytic activity. It is suggested that debranching enzymes such as pullulanase offer an alternative choice of carbohydrases to be used in brewing from unmalted cereals.     

DEXTRINS IN BREWING

The dextrins in beer exhibit a characteristic wavy distribution with respect to their molecular weight, so that these dextrins seem to fall into distinct groups. These groups of dextrins (I, II, III and IV corresponding to DP 5–10, DP 11–16, DP 17–21 and DP 22–27) have been isolated by gel-chromatography on Bio-Gel P-2. Debranching of the megalosaccharides in Groups II, III and IV by means of pullulanase shows that they contain two, three and four α-1,6 linkages respectively. The dextrins in Group I are either linear or singly-branched. The distribution patterns of the constituent, linear maltosaccharides have been determined and some likely structures for the multiply-branched dextrins are suggested. The results indicate that the majority of the α-1,6 linkages in amylopectin survive the brewing process; 25% of the α-1,6 linkages show up as singly-branched oligosaccharides and at least 35% as multiply-branched megalosaccharides of DP 11–30. With respect to the fine structure of amylopectin, the implication is that although the average interior chain length is about 6 glucose units, at least 35% of the α-1,6 linkages occur in densely branched clusters (pairs, triplets, quadruplets, etc.), the interior chain length of which is about 3 glucose units. This in turn offers an explanation of the wavy distribution of the dextrins in wort and beer.     

Pilot Scale Production of a Lager Beer from a Grist Containing 50% Unmalted Sorghum

Pilot scale (1000 L) brews were carried out with a grist comprising of unmalted sorghum (50% of total wet weight of grain) (South African variety) and malted barley (50% of total wet weight of grain) grist using a mashing program with rests at 50°C, 95°C and 60°C. Mashes were supplemented with a high heat stable bacterial α‐amylase, a bacterial neutral protease and a fungal α‐amylase. A control brew containing 100% malted barley was also carried out. Saccharification difficulties were encountered during mashing, and extraction of the grist was lower for the sorghum mashes. The sorghum mashes showed comparable lautering behaviour to that of the control mash. At mashing off the sorghum worts were starch positive. Apparent degree of fermentation of the sorghum gyles were less than the control gyles. Green beer filtration proved unproblematic. The sorghum beers compared quite closely with the control beer with regard to colour, pH and colloidal stability. Foam stability deficiencies were apparent with the sorghum beer. However, the fermentability of the sorghum worts were lower. Hence the sorghum beers were lower in total alcohol. Sensory analysis indicated that no significant differences existed between the sorghum beer and both the control beer and a commercial malted barley beer with regard to aroma, mouth‐feel, after‐taste and clarity. However, the sorghum beer was found to be significantly different to both of the other beers with regard to colour, initial taste and foam stability.     

ROLE OF ALPHA-GLUCOSIDASE IN THE FERMENTABLE SUGAR COMPOSITION OF SORGHUM MALT MASHES*

The cause of the high glucose to maltose ratio in sorghum malt worts was studied. Mashing temperature and pH strongly affected both the amount of glucose and the proportion of glucose relative to total fermentable sugars. The relative proportion of glucose was higher when mashing was performed. at pH 4.0, close to the pH optimum for sorghum alpha-glucosidase, than at the natural pH of the mash (pH 6.0–5.5). Mashing according to the EBC procedure using an enzymic malt extract with pre-cooked malt insoluble solids producing a wort containing maltose and glucose in an approximately 4:1 ratio, whereas mashing with a malt extract without pre-cooking the malt insoluble solids resulted in a wort with approximately equal amounts of maltose and glucose. Both treatments gave the same quantity of total fermentable sugars and amount of wort extract. Sorghum alpha-glucosidase was confirmed to be highly insoluble in water. All or virtually all activity was associated with the insoluble solids. Hence, it appears that the high amount of glucose formed when sorghum malt is mashed conventionally is due to alpha-glucosidase activity. Pre-cooking the malt insoluble solids inactivates the alpha-glucosidase, preventing the hydrolysis of maltose to glucose.     

外加酶法酿制低糖啤酒糖化工艺的研究

以普鲁兰酶Ｐｒｏｍｏｚｙｍｅ１２０Ｌ为重点，综合分析了外加酶糖化过程中影响麦汁总还原糖量和糖组成的各种因素，如各酶制剂的用量、糖化温度、料水比、物料比及各因素之间的相互作用等，确定了一套最优的糖化工艺方案。所得麦芽汁浸出率高、色度浅、粘度低、还原糖含量高；经过高效液相色谱分析（ＨＰＬＣ）表明，其糖组成合理；经过十天的发酵，发酵度达８２．２％，酒精分为６．４５５％（Ｗ／Ｗ）。     

EXTRUDED SORGHUM AS A BREWING RAW MATERIAL

Small scale mashes (50 g total grist) with grists containing up to 50% by weight of extruded whole sorghum produced worts of high extract yield and low viscosity. Increasing the proportion of extruded sorghum in the grist resulted in decreasing wort filtration volume, total nitrogen and free amino nitrogen content. The wort filtration behaviour of mashes containing sorghum extruded at 175°C was superior to that of mashes containing sorghum extruded at 165°C or 185°C. The results from such small scale mashing experiments suggested that extruded sorghum compared favourably to extruded barley and extruded wheat as a brewing adjunct. Worts and beers were produced on a pilot brewery scale (100 1) from grists comprising 70% malt + 30% extruded sorghum and 100% malt under isothermal infusion mashing conditions. Mashes containing sorghum extruded at 175°C showed comparable wort filtration behaviour to that of the all malt control mash whereas mashes containing sorghum extruded at 165°C or 185°C showed poor wort filtration behaviour. Worts produced from grists containing extruded sorghum fermented more quickly than the control wort and attained lower values of final gravity. The resulting beers were filtered without difficulty. Beers produced from grists containing extruded sorghum contained lower levels of total nitrogen and free amino nitrogen compared to the control beer consistent with extruded sorghum contributing little or no nitrogenous material to the wort and beer. Beers brewed from grists containing extruded sorghum were of sound flavour and showed reasonable foam stability behaviour.     

EFFECT OF CALCIUM IONS IN SORGHUM BEER MASHING

One of the problems in sorghum beer brewing is that of sugar production. This is because sorghum malts are low in diastatic activity, the grist contains a high proportion of adjunct and in some brewing processes conversion is carried out at pH 4. Since the positive effects of calcium ions on alpha-amylase activity are well described, the use of additional calcium in sorghum mashing was investigated. Mashing at pH 4.5 in the presence of 0.1% calcium acetate (227 ppm Ca) resulted in almost complete conservation of diastatic activity during conversion and higher reducing sugar production compared with tap water (31 ppm Ca). At a mash pH of 4, a calcium ion concentration of approximately 200 ppm gave maximum reducing sugar production and wort yield and increased extract. Under these conditions, some alpha-amylase activity was detected at the end of the conversion, whereas without calcium no alpha-amylase activity was detected. It, therefore, appears that improved conversion took place due to the conservation of alpha-amylase activity by calcium ions. The inclusion of additional calcium ions in sorghum beer mashes also enabled the same amount of sugar production compared with straight tap water but using a substantially lower proportion of malt in the grist.     

Current Developments in Malting and Brewing Trials with Sorghum in Nigeria: A Review

Initially, large‐scale lager beer brewing with sorghum malts proved highly intractable due to a number of biochemical problems including: high malting losses estimated at 10–30% as against 8–10% for barley; high gelatinisation temperatures which limited starch solubilisation/ hydrolysis by the amylolytic enzymes during mashing; low extract yield/low diastatic power (DP) due to inadequate hydrolytic enzyme activities especially β‐amylase; low free α‐amino nitrogen (FAN) due to inadequate proteolysis limiting yeast growth during fermentation; high wort viscosities/beer filtration problems due to low endo‐β‐1,3; 1–4‐glucanase activities on the endosperm cell walls causing the release of some β‐glucans. Strident research efforts using improved Nigerian sorghum malt varieties (SK5912, KSV8 and ICSV400) have reported some encouraging results. The knowledge of the biochemical integrity of the endo‐β‐glucanases of the sorghum malt is helping to elucidate their mode of activity in the depolymerisation of the β‐glucans. This is bound to ensure process efficiency in sorghum beer brewing, reduce beer production costs and ultimately, produce a Pilsner‐type of lager beer with 100% sorghum malt.     

Increased protein–thiol solubilization in sweet wort by addition of proteases during mashing

The flavour stability of beer is of major concern for breweries and is closely linked to oxidative processes in the beer. In this study, a new approach to boost the antioxidative capacity of beer was investigated. Protease treatment during mashing was performed in order to solubilize or extract more thiol‐containing protein‐derived compounds into the wort. Five different proteases were tested, and they were all capable of solubilizing increased amounts of thiols in wort during mashing but with different efficiencies. The wort was characterized by measuring total nitrogen by Kjeldahl, protein concentration by the Bradford method and protein composition by SDS–PAGE, in combination with matrix‐assisted laser desorption ionization–mass spectrometry. The results indicated that the proteases increased thiol concentrations by solubilizing thiol‐containing peptide fractions and not full‐length proteins. Copyright © 2014 The Institute of Brewing & Distilling     

Starch Hydrolysis During Sorghum Beer Brewing

Samples were taken at different stages of sorghum beer brewing and the starch isolated. The physico-chemical properties of the starch were determined to establish what changes occurred at the molecular level of the starch during brewing. Complete hydrolysis of the starch to fermentable sugars is never the aim of mashing during sorghum beer brewing. A certain amount of starch in the beer is required to give it its characteristic viscosity and body. This residual starch was found to be much reduced in amylose content as well as in molecular size compared with the starch in the raw materials. Debranching with isoamylase followed by gel filtration chromatography indicated that the amylopectin side chains were reduced in length by approximately half.     

Free α-amino nitrogen production in sorghum beer mashing

Free α-amino nitrogen (FAN) is an essential nutrient for yeast growth during fermentation. Under normal conditions of sorghum beer mashing, 60°C at pH 4.0, production of FAN by proteolysis accounts for approximately 30% of wort FAN, the remaining 70% being preformed in the malt and adjunct. The quality of the FAN in sorghum beer worts is good as it does not contain a high percentage of proline. Optimum conditions for FAN production during mashing are 51°C and pH 4.6. Wort FAN was increased proportionally by raising the ratio of sorghum malt to adjunct and conversely decreased by raising the ratio of adjunct to malt. FAN was also increased by the addition to the mash of a microbial proteolytic enzyme. Wort FAN is directly proportional to malt FAN.     

The Identification and Behaviour of Branched Dextrins in the Production of Scotch Whisky

Application of high performance anion chromatography (HPAEC) to distillers worts was able to resolve a series of individual fractions (A, B, C, D, E and F) which eluted between α(1–4) linear dextrins (DP4-7) and which were ubiquitous in worts. Their positions in the chromatogram and their behaviour suggested that they might be α(1–6) branched dextrins. Two of these fractions (C and D) were purified and shown to be α(1–6) branched dextrins by treatment with an α(1–6) debranching enzyme (pullulanase). Further characterisation by HPAEC and Matrix Assisted Desorption Ionisation/Time of Flight (MALDI-TOF) mass spectrometry revealed that the unknown fractions, C and D, were α(1–6) branched dextrins consisting of 6 and 7 glucose units respectively. Possible structures were suggested for these two fractions and it was shown that they were comprised of maltosyl and maltotriosyl branches attached by α(1–6) links to maltotriose, maltotetraose and maltopentaose. The behaviour of these dextrins was studied in the context of the Scotch whisky process under both laboratory and production conditions and they were found to be important substrates for the debranching enzyme, limit dextrinase, during fermentation. The study of these dextrins provided a useful tool for monitoring the effects of enzymes (α-, β-amylase and limit dextrinase) in the Scotch malt whisky production process.     

THE CONTRIBUTION OF DEXTRINS OF BEER SENSORY PROPERTIES PART II. AFTERTASTE

We investigated the possibility that dextrins could contribute to the aftertaste of beer by being broken down to sweet sugars by salivary α-amylase after beer Ingestion. The volume, pH and temperature of beer and saliva present in the oral cavity after swallowing were measured on several subjects and found adequate for the hydrolysis of beer dextrins by salivary amylase. The in vitro hydrolysis of a 6% dextrin solution and of commercial beers by pancreatic α-amylase yielded significant amounts of glucose and maltose in addition to maltotriose. Similarly, the hydrolysis of commercial beers by human saliva produced between 2.7 and 7.4 g/litre of sweet-tasting maltose and glucose, depending on the duration of the hydrolysis (30 sec to 2 min) and the amount of saliva added to 2.5 ml of beer (0.5 or 2 ml). This much sugar, because it is produced over a period of time, may not cause a detectable sweet taste but it might partially mask the bitter aftertaste of beer.     

Brewing Beer with Sorghum

Research reports on extracts, proteins, total nitrogen and free amino nitrogen content of sorghum malt and worts obtained from mashes indicate that sorghum is potentially an alternative substrate for conventional beer brewing in the tropics. Remarkable variations in biochemical characteristics among different sorghum cultivars affect their optimal malting conditions. Factors such as temperature and time of steeping and germinating of grains with their intrinsic enzymic activities, and kilning temperature determine the quality of malt. Further works on mashing, viscosity and fermentability of worts as well as the character of the resulting beers, such as alcoholic content, colour, taste and specific gravity tend to confirm the status of sorghum as a credible substitute for barley in beer brewing. This review reports on progress made in the use of sorghum for brewing beer.     

THE DEGRADATION OF β-GLUCAN DURING MALTING AND MASHING: THE ROLE OF β-GLUCANASE

Significant β-glucanolysis takes place during mashing and is catalysed by a β-glucanase which is specific to mixed-linkage β-glucans. The enzyme develops during the germination of barley, but is rapidly and extensively destroyed in kilning. Partially-purified preparations of β-glucanase are protected from denaturation by heat if their solutions are adjusted to pH 4 or if bovine serum albumin is added. However the most effective stabiliser of the enzyme is reduced glutathione. Oligosaccharides containing three and four glucosyl units are produced by the action of β-glucanase and they are further converted during malting and mashing by a different enzyme(s) to disaccharides and glucose.     

Simultaneous Determination of Mono‐, Di‐, and Trihydroxyoctadecenoic Acids in Beer and Wort

Monohydroxy, dihydroxy‐, and trihydroxyoctadecenoic acids in beer and wort were simultaneously determined using gas chromatography after a solid extraction method. These three acids were detected at ppm levels in the wort. The monohydroxyoctadecenoic acids were not detected after wort boiling, but the dihydroxy‐ and trihydroxyoctadecenoic acids were transferred through wort boiling, fermentation and lagering into the finished beer. During the mashing using a laboratory mash bath, they gradually increased to about twice the levels those at mashing‐in. The amounts of dihydroxyoctadecenoic acid and trihydroxyoctadecenoic acid in commercial beer samples varied from 0.6 to 1.6 ppm and 6 to 15 ppm, respectively.     

THE CONTRIBUTION OF DEXTRINS TO BEER SENSORY PROPERTIES PART I. MOUTHFEEL

The effect of dextrins on the mouthfeel of beer was assessed using instrumental and sensory techniques. By measuring the viscosity of beer with a rheogoniometer, it was shown that beer is a newtonian fluid, i.e., shear stress increased linearly with shear rate. A capillary viscometer and a rheogoniometer gave viscosity measurements that did not differ significantly for six beers ranging in viscosity from 1.1 to 2.6 centipoise (cP). In carbonated beer, the amount of dextrins needed to produce an increase in viscosity detectable by judges was 52 g/litre, which raised the viscosity by 0.31 cP as measured by capillary viscometry. Since beer usually contains between 10 and 50 g/litre of dextrins, it is concluded that dextrins are not the sole determinant of beer viscosity.     

The Evolution of Dextrins During the Mashing and Fermentation of All‐malt Whisky Production

The production of malt whisky involves the mashing of barley malt, followed by the fermentation of the resulting wort without further treatment. While this process has many parallels to the production of an all‐malt beer, one of the main differentiating steps during substrate preparation is the inclusion of a boiling step for the wort in the production of beer. Other than the destructive action of the boiling process on microorganisms, the boiling also destroys all malt enzyme activity. Since a typical whisky wash is not boiled it carries through a certain proportion of microbial activity associated with the malt, but more importantly it retains some enzyme activity that has been activated during the malting and mashing processes. The changes in sugars and dextrins during both mashing and fermentation of the resulting wash were investigated. Evidence of the continuous amylolytic activity during an unboiled, all‐malt wash fermentation is shown; while no ongoing amylolytic activity could be deduced during the fermentation of a boiled all‐malt wort. Furthermore, the data suggests that the amylolytic activity during mashing and fermentation are different with regards to α‐amylase action linked to its multiple‐attack action pattern as a function of substrate conformation, temperature, and effectiveness of potential hydrolytic events.     

Molekulare Struktur und physikalischchemische Eigenschaften von löslichen Stärken und Dextrinen

Molecular Structure and Physico-chemical Properties of Soluble Starches and Dextrins. Physico-chemically modificated starches, so called soluble starches show about ten times increases of reductivity and decrease of molecular weight as compared with native starch. Roasted starch without chemicals undergoes the most significant changes. Smaller and smaller changes are present successively in lintnerizated starches with hydrochloric, nitric and phosphoric acids. Soluble starches slightly α-amylase hydrolyzed in dependence on the reaction time change into high-molecular or medium-molecular dextrins with increasing during reaction degree of reductivity from 0,4 to 4 DE and decreasing molecular weight from 40000 to 6000. During α-amylase hydrolysis of soluble starches to dextrins average number of end-groups decreases in molecules from 11 to 4 and the number of branchings from 10 to 3.