Characterisation and Assessment of the Role of Barley Malt Endoproteases During Malting and Mashing1

Barley malt endoproteases (EC.3.4.21) develop as multiple isoforms mainly during grain germination and pass through kilning almost intact. Thermostability, under simulated mashing conditions, varied from low to high depending on the substrate used in the assay. This suggests that individual enzymes respond differently to heat exposure and to protein substrates. The optimal pH with haemoglobin was pH 3.5, with hordein pH 4 and with glutelin pH 5. The optimal temperature with hordein was 40°C, with glutelin 50°C and with haemoglobin 60°C. These differences suggest that it is not possible to comprehensively characterise all malt endoproteases under one set of assay conditions. In brewing, most of the barley protein degradation (> 70 %) occurs during malting. But some proteinases remain active during mashing and contribute to wort soluble proteins and free amino nitrogen. Their contribution to all malt EBC mash total free amino nitrogen was 25 % in Schooner (Australian) and 30 % in Morex (USA). The importance of proteolytic activity during mashing and the possibility that the levels may not be adequate, at high solid adjunct ratios, are acknowledged.     

Effect of malting time,mashing temperature and added commercial enzymes on extract recovery from a Nigerian malted yellow sorghum variety

Preliminary microbiological studies carried out on sorghum grains showed that the major microorganisms found were mainly bacteria and that aflatoxin‐producing fungi were not found. The effect of added commercial enzyme preparations and different infusion mashing temperatures on extract yield, from sorghum malted at 30 °C, was studied. The infusion mashing method (65 °C) developed for mashing well‐modified barley malt produces poor extract yields with sorghum malt. The extract yield from the sorghum malt in this study was very low with infusion mashing at 65 °C, without the addition of commercial enzyme preparations. A higher extract yield was obtained from the sorghum malt, without the commercial enzyme addition, when using infusion mashing at 85 °C. Both infusion mashing temperatures (65 and 85 °C) showed an improved extract yield over the control malt when commercial enzyme preparations were used during mashing of the sorghum malt. The added enzyme preparations resulted in a higher extract yield from the germinated sorghum when infusion mashing was performed at 65 °C over mashing at 85 °C. The use of individual commercial enzymes (α‐amylase, β‐glucanase, protease, xylanase, saccharifying enzyme and combinations of some hydrolytic enzyme) increased extract yields, when complemented with the enzymes that had developed in the sorghum malt. Copyright © 2016 The Institute of Brewing & Distilling     

Optimisation of the Mashing Procedure for 100% Malted Proso Millet (Panicum miliaceum L.) as a Raw Material for Gluten‐free Beverages and Beers

Proso millet is a gluten‐free cereal and is therefore considered a suitable raw material for the manufacturing of foods and beverages for people suffering from celiac disease. The objective of this study was to develop an optimal mashing procedure for 100% proso millet malt with a specific emphasis on high amylolytic activity. Therefore, the influence of temperature and pH on the amylolytic enzyme activity during mashing was investigated. Size exclusion chromatography was used to extract different amylolytic enzyme fractions from proso millet malt. These enzymes were added into a pH‐adjusted, cold water extract of proso millet malt and an isothermal mashing procedure was applied. The temperatures and pH optima for amylolytic enzyme activities were determined. The α‐amylase enzyme showed highest activity at a temperature of 60°C and at pH 5.0, whereas the β‐amylase activity was optimum at 40°C and pH 5.3. The limit dextrinase enzyme reached maximum activity at 50°C and pH 5.3. In the subsequent mashing regimen, the mash was separated and 40% was held for 10 min at 68°C to achieve gelatinisation. The next step in the mashing procedure was the mixture of the part mashes. The combined mash was then subjected to an infusion mashing regimen, taking the temperature optima of the various amylolytic enzymes into account. It was possible to obtain full saccharification of the wort with this mashing regimen. The analytical data obtained with the optimised proso millet mash were comparable to barley wort, which served as a control.     

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 simultaneous saccharification and fermentation of malt dust and use in the acidification of mash

In brewing, the mash or wort is frequently acidified by the addition of lactic acid or the bioacidification of the mash. The present study provides an alternative approach for mash or wort acidification by the simultaneous saccharification and fermentation (SSF) of malt dust. In this method, fermentable carbohydrates released by the enzymatic breakdown of the cellulosic portion of the malt dust are converted to lactic acid by lactic acid bacteria. The effect of temperature, ranging between 45 and 51°C, solid loading of malt dust at 2, 5 and 10% (w/v) on a dry basis, and enzyme loading at 0.65, 2.6 and 6.5 filter paper units (FPU) per gram malt dust on SSF and change in pH in mash acidification were examined. The final pH and lactic acid concentration and final glucose concentration of the SSF media were significantly affected by the temperature of the process (p < 0.05). The highest lactic acid titre (9.7 g/L) and the lowest pH (3.12) were obtained by SSF of 10% (w/v) malt dust at 45°C with 6.5 FPU/g. The pH of the mashing solution [containing 20% (w/v) ground malt] decreased to around 5.4 and 5.2 after adding 1.9 and 2.9% of SSF media with pH 3.39. © 2019 The Institute of Brewing & Distilling     

Studies on the mashing conditions of teff (Eragrostis tef) malt as a raw material for lactic acid‐fermented gluten‐free beverage

Formation of extracts and fermentable sugars during mashing can be limited by incomplete starch gelatinisation. The aim of this research was to develop mashing programme for 100% teff malt as a potential raw material for gluten‐free lactic acid‐fermented beverage. Isothermal mashing at temperatures ranging between 60 and 84 °C was conducted, and the highest extract (85%) was observed for the wort samples produced at temperatures higher than 76 °C. Sixty‐minute rest at 71 °C resulted in higher fermentable sugars than other tested conversion rest temperatures. Inclusion of lower mashing‐in temperature in the mashing programme also substantially improved the concentrations of free amino nitrogen (128 mg L^?1) and fermentable sugar (58 g L^?1) in the final wort. Therefore, 30‐min rest at 40 °C followed by 60‐min rest at 71 °C and 10‐min rest at 78 °C was found to be a suitable mashing programme for teff malt.     

Optimisation of a Mashing Program for 100% Malted Buckwheat

The objective of this study was to develop a temperature programmed mashing profile for 100% buckwheat malt. Both standard brewing methods and a rheological tool (Rapid Visco Analyser) were used to characterise worts and mashes. An optimal grist: liquor ratio of 1:4 was observed. At this ratio, buckwheat malt showed a gelatinisation temperature of 67°C and barley malt 62°C. A one hour stand at 65°C exhibited higher FAN levels, fermentable extracts and lower viscosity values than stands at 67°C or 69°C, and was therefore used in further mashing trials. An extra mashing step of 30 min, at any of the tested temperatures, increased extract values a minimum of 4%, decreased viscosities a minimum of 0.20 mPas, and increased fermentable extracts 12%. Best results were obtained when a mashing‐in temperature was used in the range of 35°C to 45°C. These mashing‐in temperatures were used to design an optimal mashing procedure: 15 min at 35°C; 15 min at 45°C; 40 min at 65°C; 30 min at 72°C; 10 min at 78°C. This program showed higher extract values and fermentable extract values (72.7% and 49.9%) than obtained by congress mashing (65.3% and 40.0%), thus successfully optimising the mashing program.     

α-GLUCOSIDASE ACTIVITY OF SORGHUM AND BARLEY MALTS

Sorghum malt α-glucosidase activity was highest at pH 3.75 while that of barley malt was highest at pH 4.6. At pH 5.4 employed in mashing sorghum malt α-glucosidase was more active than the corresponding enzyme of barley malt. α-Glucosidase was partly extracted in water but was readily extracted when L-cysteine was included in the extraction buffer, pH 8. Sorghum malt made at 30°C had higher α-glucosidase activities than the corresponding malts made at 20°C and 25°C. Nevertheless, the sorghum malts made at 20°C and 25°C produced worts which contained more glucose than worts of malt made at 30°C. Although barley malts contained more α-glucosidase activity than sorghum malts, the worts of barley had the lowest levels of glucose. The limitation to maltose production in sorghum worts, produced at 65°C, is due to inadequate gelatinization of starch and not to limitation to β-amylase and α-amylase activities. Gelatinization of the starch granules of sorghum malt in the decantation mashing procedure resulted in the production of sorghum worts which contained high levels of maltose, especially when sorghum malt was produced at 30°C. Although the β-amylase and α-amylase levels of barley malt was significantly higher than those of sorghum malted optimally at 30°C, sorghum worts contained higher levels of glucose and equivalent levels of maltose to those of barley malt. It would appear that the individual activities of α-glucosidase, α-amylase and β-amylase of sorghum malts or barley malts do not correlate with the sugar profile of the corresponding worts. In consequence, specifications for enzymes such as α-amylase and β-amylase in malt is best set at a range of values rather than as single values.     

Increasing fermentable sugar yields by high‐pressure treatment during beer mashing

High‐pressure treatment, which is an effective means of enhancing enzymatic reactions, was implemented during beer mashing to increase the production yield of fermentable sugar (FS). The malt solution was heated (62, 67, and 72°C) under pressure [0.1 (1 atm), 2, 50 and 100 MPa], and the FS was measured. The amount of FS reached an equilibrium level, which was the highest at 67°C and at 2 MPa. The pressures were 2, 50, 100, and 0.1 MPa in decreasing order of FS amount at 67°C. The temperatures were 67, 72 and 62°C in decreasing order of FS amount at 2 MPa. With a mechanistic approach, only the effect of pressure on gelatinization was analysed. The gelatinization degrees were also higher at pressures higher than 0.1 MPa. This observation highlights the positive effect (increasing the FS yield) of high‐pressure treatment on beer mashing. Copyright © 2015 The Institute of Brewing & Distilling     

Formulation and shelf-life of a bottled pawpaw juice beverage

A simple procedure was developed for production of bottled pawpaw beverage juice by peeling and macerating peeled tissue in 25% water, straining through a 0.8-mm sieve, adjustment of juice pH with citric acid and flavour adjustment with sucrose. Fresh juice was optimized for acceptability at pH 3.9 and 10% (w/w) sucrose. Heating for 6 min at 72.2°C was required to achieve commercial pasteurization. Samples of juice were prepared with no preservative, and containing sodium benzoate (125mg/100ml), sodium metabisulphite (50mg/100ml) and sodium metabisulphite/sodium benzoate combination (25mg and 60mg/100ml) for trials in which acceptability, pH, specific gravity, brix, total acidity, vitamin C and biomass concentrations were measured over 90 weeks storage at 10°C and 30.2°C. Sodium benzoate alone extended the shelf-life at 30°C up to 80 weeks but the other preservatives were not effective after 20 weeks. The control juice was already deteriorating by 10 weeks at 30°C. At 10°C all preserved samples were stable up to 80 weeks, although the control deteriorated rapidly after 20 weeks.     

HEAT SENSITIVITY,OPTIMUM pH AND CHANGES IN ACTIVITY OF SORGHUM PEROXIDASE DURING MALTING AND MASHING

Peroxidase activity was demonstrated in dry and germinating sorghum seeds. The specific activity increased about 14-fold during malting for a 96-hour period. On the average about 41% of peroxidase activity was located in the endosperm, and the remaining 56% in the acrospire and rootlet of sorghum malt. The crude enzyme extract retained 77%, 17.5% and 7.6% of activity after heating at 60°, 70° and 80°C, respectively. More than 50% of the peroxidase activity in the finished malt survived mashing at 65°C. Optimum activity was recorded at pH 5.5 which falls within the observed pH range of sorghum worts. The level of residual peroxidase activity in the wort differed with sorghum species.     

CHANGES IN ACTIVITY OF SORGHUM LIPASE DURING MALTING AND MASHING

Lipase activity was monitored during malting and mashing of sorghum grains. All three sorghum varieties contained detectable lipase activity in the ungerminated form. Lipase activity changed only slightly during steeping for 24 hours but increased several fold in the course of germination. Between 24% and 60% of the lipase activity of the green malt was retained after kilning at 48°C but no activity was detected in the wort after mashing at 65°C. About 68% of the lipase activity of 72 hours old malt was detected in the plumule, while 29% and 3% were detected in the endosperm and radicle, respectively. Optimum activity was observed at pH 7.0.     

Overcoming technical barriers to brewing with green (non-kilned) malt: a feasibility study

Brewing using enzyme rich ‘green’ (germinated, but not kilned) malt has the potential to unlock considerable energy savings in the malting and brewing chain. This paper examines the major quality issues associated with green malt, by monitoring lipoxygenase (LOX) activity and S-methyl methionine (SMM) levels through a micromalting cycle both with and without rootlets after 48 h of germination. The data suggest that rootlets are a major concern when brewing with green malt and that their influence on wort and beer quality needs to be further investigated. Lipoxygenase activity and nonenal potential were measured following treatment under varying conditions of pH, temperature and pre-treatment. Results indicated that lipoxygenase activity can be controlled to a substantial degree by manipulating these limiting factors, while preserving diastatic enzyme activity. Green malt worts were then prepared from (i) whole green malt immediately post-germination; (ii) heat treated green malt (65°C x 1 h); (iii) re-steeped green malt and (iv) endosperm-rich extracts of green malt after the husk and rootlets had been removed; using laboratory mashing with a ‘LOX-hostile’ mash schedule. Data were compared with mashing of kilned pale malt made from the same green malt, as a reference point. Based on the present data, re-steeping of green malt in combination with a LOX hostile mashing environment (63°C, pH 5.2) could help to control LOX activity and the trans-2-nonenal potential of green malt. The resultant brewing process would need to be optimised to deal with the elevated SMM levels in green malt worts. © 2020 The Authors. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

Optimisation of pectin acid extraction from passion fruit peel (Passiflora edulis flavicarpa) using response surface methodology

Pectin was extracted from passion fruit peel using three different acids (citric, hydrochloric or nitric) at different temperatures (40–90 °C), pH (1.2–2.6) and extraction times (10–90 min), with and without skins using a 2⁴ factorial design. Temperature, pH and extraction time had highly significant effects on the pectin yield. A central composite design with face centring was used to optimise the extraction process conditions for citric acid without skins. Pectin yields varied from 10% to 70%. The optimal conditions for maximisation of pectin yield were the use of citric acid at 80 °C and pH 1 with an extraction time of 10 min considering model extrapolation.     

Effect of extraction conditions on the yield and purity of apple pomace pectin precipitated but not washed by alcohol

ABSTRACT:  A study of the influence of extraction conditions (pH: 1.5 to 2; temperature: 80 to 90 °C; extraction time: 1 to 3 h), on the yield and purity of apple pomace pectin without elimination of impurities by alcohol washing was carried out. The alcohol precipitate yields varied from 2.9% to 8.9% depending on the pH. At pH 1.5, these yields were higher than those obtained at pH 2 contrary to the galacturonic acid purity (%w/w). Compounds other than pectins were solubilized from the cell walls of apple pomace at pH 1.5, and they were precipitated with alcohol. The apple pectins obtained from the different extraction procedures were highly methylated (54.5% to 79.5%), especially when the conditions (temperature, pH) were drastic. Similar conclusions can be drawn for the neutral sugar content that decreased at pH 1.5 (arabinose, xylose, and galactose) or at the highest temperatures and extraction times (arabinose and galactose). The phenomenon of demethylation and pectin degradation of neutral sugars chains can be observed at acid pH, and for long extraction times. The presence of high quantities of mannose or fructose, glucose, and xylose in the alcohol precipitate showed that pectin precipitation with ethanol was not specific.     

RATE-LIMITING ENZYMES IN THE LIBERATION OF AMINO ACIDS IN MASHING

When 29 malts representing a wide variation in free amino nitrogen of wort were mashed for 3 h at 45°C, the increases in TCA-soluble amino nitrogen varied from 47 to 116 μmoles per gramme of malt. The carboxypeptidase activities of the malts showed only small positive correlations with the increase of amino nitrogen during mashing. Inactivation of the different malt carboxypeptidases by 70 to 90% with diisopropylfluorophosphate decreased the liberation of amino nitrogen by only 5 to 25%. Moreover, addition of purified malt carboxypeptidase I to the mashing suspensions had no effect on the liberation of amino nitrogen. It is therefore evident that the carboxypeptidases do not have a major rate-limiting role in the liberation of amino acids in mashing, although it has earlier been shown that they are essential enzymes in this process. The proteinase activities of the malts, determined using two methods, correlated strongly with the liberation of amino nitrogen in mashing. Addition of crude malt proteinase to the mash increased, and addition of purified barley proteinase inhibitor decreased the measured proteinase activity and the liberation of amino acids roughly to the same extent. These results indicate that the most important rate-limiting enzyme activity in the liberation of amino acids in mashing is the proteinase activity of the malt.     

Quality Assessment of a Sorghum Variety Malted Commercially under Tropical Conditions and Controlled Tropical Temperatures in the Laboratory

A sorghum variety was supplied as commercial malt and as an unmalted cereal by a maltster. The commercial sample had been malted in a tropical country. Sub‐samples of the unmalted cereal were malted in the laboratory under controlled germination temperatures of 28°C and 30°C. Laboratory and commercially malted sorghum were studied for their brewing qualities. The α‐amylase development in sorghum malt was enhanced when germination was carried out at the higher temperature of 30°C rather than at 28°C. Hot water extract (HWE) was more variable. With infusion mashing, results showed a significant difference for germination time (3–6 days), but no significant difference relating to germination temperature. With the decantation mashing method the reverse was observed. The low numerical values of HWE obtained from sorghum malt in the infusion mashing process confirmed that this process is not suitable to produce optimal extract development from malted sorghum. The 28°C germinated sorghum released more FAN products into the worts than the 30°C malt, using both the infusion and decantation methods. With regard to the parameters tested, commercially malted sorghum gave lower analytical values than laboratory malted sorghum. It was also observed that variations in malting temperatures and mashing processes can cause unexpected variations in the analyses of sorghum malt. These findings suggest that careful process control is required during the malting and mashing of sorghum.     

Effect of unmalted oats (Avena sativa L.) on the quality of high-gravity mashes and worts without or with exogenous enzyme addition

Barley malt is the preferred brewing material these days because of its high extract content and high enzyme activities. However, when substituting malted barley with oats to create a unique beer flavor and aroma, endogenous malt enzymes become the limiting factor. Therefore, the objectives of this study were to evaluate the effect of 10–40 % unmalted oats on the quality of high-gravity mashes/worts and to investigate the limitations of endogenous malt enzymes as well as the benefits of the application of industrial enzymes. The enzyme mix Ondea^® Pro was found to be particularly suitable for mashing with unmalted oats and was therefore used in the present rheological tests and laboratory-scale mashing trials. In order to gain detailed information about the biochemical processes occurring during mashing, the quality of mashes was comprehensively analyzed after each mash rest using standard methods described by Mitteleuropäische Brautechnische Analysenkommission and Lab-on-a-Chip capillary electrophoresis. Mashing with up to 40 % oats resulted in increased mash consistencies, color/pH (20 °C) values, β-glucan concentrations, wort viscosities 12.0 %, and filtration times as well as decreased FAN and extract contents. The application of Ondea^® Pro enormously increased the color of worts despite lower pH values but considerably improved the quality and processability of 30 or 40 % oat-containing mashes/worts. However, the substitution of up to 20 % barley malt with unmalted oats can easily be realized without the addition of exogenous enzymes.     

REVERSIBLE GELATION OF WHEY PROTEINS: MELTING, THERMODYNAMICS AND VISCOELASTIC BEHAVIOR

Whey protein isolates formed reversible gels following heating at 90°C for 15 min under certain conditions i.e., pH 6.5 to 8.5 with protein concentration of 9.0–10.5%. The melting temperatures of the gels formed at pH 8.0 ranged from 24.5°C to 57.8°C. The maximum enthalpy of formation (ΔH_f) was –858 call mole of crosslinks. A maximum storage modulus (G') of 240 dynes/cm² was obtained following holding for 7 h at 8°C.     

乳酸菌种混合发酵研制大米饮料的工艺研究

以大米,麦芽为主要原料,经粉碎、糊化、糖化、乳酸菌种发酵等一系列操作以后,加入调味剂和辅助剂,即可制备发酵米乳饮料。实验时确定了发酵米乳的最佳制作工艺：大米与水以1：10的比例蒸煮糊化,糖化时麦芽汁的加入量为原料的30%,糖化酶加入量为发酵醪液的1%;按照3：2的比例分别接种保加利亚乳杆菌和嗜热链球菌到大米培养基中培养制备母发酵液,前发酵时母发酵液的接入量为1%~3%,时间为72~80 h,温度为37℃,后发酵时间为15~30 d,温度为4~5℃。经此一系列操作后可制得口感细腻、米香、麦芽香浓郁,营养健康的功能性发酵米乳饮料。