Exploration of high‐gravity fermentation to improve lactic acid bacteria performance and consumer's acceptance of malt wort‐fermented beverages

Lactic acid bacteria (LAB) fermentation performance is essential for aroma metabolites formation and product flavour quality. Hence, this study appraises high‐gravity malt wort fermentation (HGF) by three LAB strains to improve the fermentation performance and consumer's acceptance of lactic acid‐fermented malt‐based beverages (LAFMB). HGF at 20% (w/w) provided higher amino acid content and buffering capacity that allowed greater cell development, viable cell count and sugar utilisation. Moreover, the pH change was lesser although marked lactic acid accumulation. It is noteworthy that HGF significantly incremented the content of higher alcohols (+0 – 161%), 2‐phenylethanol (+11–147%), acetaldehyde (+27–44%) and β‐damascenone (+25 – 66%) comparing to low‐gravity malt wort at 12%. Thus, HG‐fermented beverages were significantly preferred with greater hedonic scores (4.6 ± 2.1). Our results indicate that HGF is a valuable strategy for improving LAB fermentation performance in malt wort, which in turn increases key aroma compound content resulting in enhanced acceptance of LAFMB.     

Preparation of a Low Carbohydrate Beer by Mashing at High Temperature with Glucoamylase

A malt grist, supplemented with glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3), was mashed isothermally at 70°C to produce wort with a real fermentability of over 87% and beer with a residual carbohydrate content of less than 0.75% w/v at an original gravity of 42°S. The effects of varying mash pH, calcium content, glucoamylase addition rate and mash conversion time were investigated. The process was effective even with undermodified malt. Pilot scale brewing trials shozved no adverse effect on beer foam quality when the glucoamylase preparation had a specified range of protease side-activity. The process has the advantage of ensuring glucoamylase inactivation during wort boiling while being shorter than the alternative of enzyme treatment of wort prior to boiling.     

Influence of malted barley and exogenous enzymes on the glucose/maltose balance of worts with sorghum or barley as an adjunct

The sugar profile of wort from laboratory malted barley, malted sorghum, unmalted barley and unmalted sorghum grains mashed with commercial enzyme preparations were studied. Similar levels of glucose to maltose (1:7) were observed in wort of malted barley and malted sorghum. Mashing barley or sorghum grains with commercial enzymes changed the glucose to maltose ratio in both worts, with a greater change in wort from sorghum grains. Although hydrolysis with commercial enzymes released more glucose from maltose in sorghum wort, the same treatment retained more maltose in barley wort. Adding malted barley to sorghum grains mashed with commercial enzymes restored the glucose to maltose ratio in sorghum mash. Fermentation of wort produced from all barley malt (ABM) mash and commercial enzyme/barley malt/sorghum adjunct (CEBMSA) mash of similar wort gravity was also studied. ABM and CEBMSA worts exhibited similar glucose to maltose ratios and similar amino acid spectra. However, ABM released more individual amino acids and five times more proline than wort from commercial enzyme/barley malt/sorghum adjunct. ABM produced 27% more glucose and 7% more maltose than CEBMSA. After fermentation, ABM mash produced 9.45% ABV whilst commercial enzyme/barley malt/sorghum adjunct mash produced 9.06% ABV. Restoration of the glucose/maltose ratio in the CEBMSA mash produced wort with a sugar balance required for high gravity brewing. © 2020 The Institute of Brewing & Distilling     

Application of Biological Acidification to Improve the Quality and Processability of Wort Produced from 50% Raw Barley

In this study four strains of lactic acid bacteria (LAB) were chosen to bioacidify a mash containing 50% barley and 50% malt. The strains were isolated from malted and unmalted barley and assayed for extracellular enzymatic activities (proteases, amylases, β‐glucanases). The biologically acidified mash was compared to a chemically acidified mash, 100% malt mash un‐acidified and 50% malt and 50% barley mash unacidified. Characteristics such as pH, extract, colour, viscosity, total soluble nitrogen (TSN), free amino nitrogen (FAN), apparent fermentability, β‐glucan and lautering performance of the resultant worts were determined. A model lautering system replicating one used in a brewery was designed and built in University College Cork (UCC) to measure the lautering performance of the bioacidified mashes. The new system was compared to the filtration method used in EBC method 4.5.1. Overall the addition of LAB to bioacidify a mash of 50% barley and 50% malt resulted in faster filtration times, which correlated with decreased β‐glucan levels. Proteolytic LAB had a positive influence on the quality of wort and resulted in increased FAN levels. Lighter colour worts were observed along with increased extract levels.     

生物酸化技术在啤酒生产中的应用

生物酸化技术应用于啤酒生产可降低糖化醪pH值，实现对麦芽、糖化醪和麦芽汁的酸化。生物酸化可增加酶活。生物酸化技术在啤酒生产中的应用不仅能降低生产成本，而且还能提高啤酒质量；但对乳酸茵和工艺有严格的要求。（孙悟）     

Sour beer production: impact of pitching sequence of yeast and lactic acid bacteria

Lactic acid bacteria have long been used in brewing to acidify mash or wort and in the production of traditional sour beer styles (Lambic, Gueuze) using spontaneous fermentation. This approach is time consuming (some sour beers are matured for three years to obtain the appropriate flavour), so many brewers choose a faster approach using mixed cultures (yeast and bacteria). In this study, the influence of the pitching sequence of bacteria and yeast on the fermentation process was evaluated (by rate of fermentation and acidification, attenuation degree and ethanol concentration). The trials were performed using three procedures: (i) the addition of bacteria followed by the yeast; (ii) the addition of the yeast followed by the bacteria and (iii) the simultaneous addition of yeast and bacteria. In each trial the following time intervals were applied: 24, 48 and 72 h. Weight loss of samples and drop in pH were measured daily, and ethanol content, real extract and lactic acid concentration measured at the end of the process. It was shown that the order of addition of yeast and bacteria determines the success of the lactic acid fermentation. The appropriate yield of lactic acid (ca. 6 g/L) and the drop in pH (<3.6) can be achieved by inoculating the bacteria prior to the inoculation of yeast. When yeast was pitched before the bacteria, the lactic acid content was ca. 2 g/L and pH was 3.9–4.2. © 2019 The Institute of Brewing & Distilling     

The Influence of Lactic Acid Bacteria on the Quality of Malt

In this study three strains of lactic acid bacteria were applied during the malting process to evaluate the impact on malt and wort quality. The trials were performed in a micromalting plant simulating an industrial malting programme. The samples were compared to chemically acidified as well as non‐acidified malt. Bacterial cultures were chosen with reference to their enzymatic (proteolytic/amylolytic) activity, or their good acidifying properties. The effects of lactic acid bacteria on wort characteristics were investigated and compared to wort produced from 100% unacidified malt. A chemical food grade lactic acid was also used to acidify the barley for comparison purposes. Characteristics such as pH, extract, colour, viscosity, total soluble nitrogen, free amino nitrogen, apparent fermentability, β‐glucan and lautering performance of the resultant worts were determined. Results showed improved levels of β‐glucanase in the malt although reduced malt friability was observed where LAB was employed. An improved lautering performance, lower wort viscosity and elevated TSN levels were also reported where LAB exhibiting protease activity were applied.     

SMALL SCALE MASHING EXPERIMENTS WITH GRISTS CONTAINING HIGH PROPORTIONS OF RAW SORGHUM

Small scale mashes (50 g total grist) with grists containing high proportions of raw sorghum (50%–80% malt replacement) showed high values of extract recovery and produced worts of lower total nitrogen, free amino nitrogen, viscosity and colour but higher values of pH compared to worts produced from all malt mashes. Increasing the proportion of raw sorghum in the grist relative to malt resulted in a decline in extract recovery, wort total nitrogen, free amino nitrogen and an increase in wort pH. Addition of industrial enzyme preparations to mashes containing raw sorghum resulted in higher values of extract recovery (enzyme preparations containing α amylase and β glucanase), higher values of wort total nitrogen and free amino nitrogen (enzyme preparations containing a neutral proteinase) and decreased wort viscosity (enzyme preparations containing β glucanase or cellulases) compared to worts produced from untreated mashes. Worts and beers were produced on a pilot brewery scale from 50% malt and 50% polished (whole) sorghum (single decoction mashing regime) and 20% malt and 80% raw sorghum supplemented with an industrial enzyme preparation (double mashing regime). Mashes comprising 50% malt and 50% polished sorghum showed comparable wort filtration behaviour (lautering) to that of control mashes (70% malt and 30% maize grists) whereas wort produced from 20% malt and 80% raw sorghum filtered slowly. Worts produced from grists containing sorghum were of high fermentability and showed lower levels of total nitrogen and free amino nitrogen compared to control worts. Analysis of worts produced from small scale mashes containing raw sorghum and a pilot brewery scale mash comprising 20% malt and 80% raw sorghum demonstrated that the levels of total nitrogen and free amino nitrogen were higher than expected from the reduction in the malt content of the mash, consistent with the release of nitrogenous components (polypeptides, peptides and amino acids) derived from sorghum into the wort. Beers produced from 50% malt and 50% polished sorghum and 20% malt and 80% raw sorghum were filtered without difficulty and were of sound flavour. Beers produced from 50% malt and 50% polished sorghum contained lower levels of isobutanol, 2-methylbutanol, dimethylsulphide and higher levels of n propanol and diacetyl compared to control beers.     

Validation and application of osmolyte concentration as an indicator to evaluate fermentability of wort and malt

The objective was to develop a new simple and quick approach to predict fermentability, based on osmolyte concentration (OC). Eight malts were assayed for diastatic power, starch‐degrading enzymes [α ‐amylase, β ‐amylase and limit dextrinase (LD)] and malt OC (MOC). All malts were mashed to determine wort OC (WOC), real degree of fermentation (RDF) and sugar contents in a small‐scale mashing protocol. The results showed that MOC was correlated with malt α ‐amylase, LD, the resultant WOC, RDF and fermentable sugar (r  = 0.813, 0.762, 0.795, 0.867, 0.744, respectively), suggesting that MOC was discriminating in predicting levels of malt amylolytic enzymes, wort sugar and RDF without the mashing and fermentation process. Moreover, WOC showed stronger correlations with malt α ‐amylase, LD, RDF and fermentable sugars (r  = 0.796, 0.841, 0.884, 0.982, respectively), suggesting that WOC can be used to quickly predict wort sugar contents and RDF without a fermentation step. Furthermore, the effects of mashing temperature and duration on WOC, RDF and sugar contents are discussed. Adjusting mash temperature to 65°C or extending the mash duration dramatically increased RDF and WOC, whereas malt extract was relatively stable. Similarly, WOC showed significant correlations with RDF and fermentable sugars (r  = 0.912 and 0.942, respectively), suggesting that WOC provides a simple and reliable tool to assist brewers to optimize mash parameters towards the production of ideal wort fermentability. In conclusion, the ability of OC to predict malt fermentability and sugar content allows brewers to keep better control of fermentability in the face of variation of malt quality, and to quickly adjust mashing conditions for the consistency of wort fermentability. Copyright © 2017 The Institute of Brewing & Distilling     

Physicochemical characterization of special persimmon fruit beers using bohemian pilsner malt as a base

This study involved the production of special fruit ale beers with different concentrations (100:0%, 75:25%, 50:50% and 25:75% v /v) of barley malt and persimmon juice from the ‘Rojo Brillante’ variety. Fermentation took place under beer quality control parameters and the influence of persimmon juice on beer quality was investigated. Colour, turbidity, pH, titratable acidity, total soluble solids, sugars, organic acids, total phenolic compounds, antioxidant capacity and ethanol formation were determined during the fermentation process. These fruit beers, whose alcoholic contents were within the standards (3.6–5.63% v /v ethanol), were characterized by a normal acid pH (3.97–4.13) with citric and lactic acids the most abundant organic acids, a clear golden colour without turbidity [2.05–2.83 European Brewery Convention units], intermediate total phenolic compound values (283.0–327.1 mg GAE/L) and antioxidant activities between 1.65 and 5.78 mm TE/L. The persimmon beverage which contained 75% fruit juice was the most valued and preferred by the panelists followed by the 50:50% wort–persimmon beer. Copyright © 2017 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.     

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.     

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.     

DETERMINATION OF FATTY ACID HYDROPEROXIDES PRODUCED DURING THE PRODUCTION OF WORT

Linoleic and linolenic acid hydroperoxides in malt, mash, or wort were determined with high sensitivity and high selectivity by the chemiluminescence-high performance liquid chromatography (CL-HPLC) method using isoluminol-microperoxidase solution as a luminescing reagent. The determination limit of this method for both hydroperoxides was 0.1 μM in mash or wort. During the mashing in a laboratory mash bath, the hydroperoxides started to increase just after mashing-in, reached a maximum at 65°C, and then decreased. Though the hydroperoxides were detected in mash just before the lautering in a pilot scale brewing, they disappeared during the lautering and could not be detected during the subsequent stages of wort production. Therefore, it was thought that the mashing process is the most important of the lipid oxidation reactions during wort production. It is also expected that the CL-HPLC method can give useful information on lipid oxidation mechanisms during wort production.     

Effect of wheat malt on the concentration of metal ions in wort and brewhouse by‐products

The ionic composition of brewer's wort depends on the raw materials and processing employed. The macroelement content is usually sufficient for yeast, but some of the microelements (mainly zinc) often need to be supplemented to the wort. Wheat malt is used as an adjunct in the production of beer, replacing up to 60% of barley malt. In this study, the effect of replacing barley malt with wheat malt on the concentration of magnesium, manganese, iron and zinc ions in brewer's wort was investigated. The ionic content of both the raw materials and by‐products were analysed. Similar amounts of magnesium ions were found in wheat malt and barley malt, whereas, manganese, zinc and iron were more abundant in the wheat malt. Nevertheless, wheat malt did not cause a significant change in ion concentration in the first wort (except for magnesium; its content decreased). The ionic content in the spent grains increased owing to wheat malt addition; the concentration of ions in the wort decreased after wort boiling (Mn²⁺ and Fe) or remained unaffected (Mg²⁺ and Zn²⁺). It was concluded that the ionic composition of the wort depends mainly on the removal rate of ions from the wort during mash filtration and hot trub separation, rather than on the actual amount of ions in the raw materials. Copyright © 2015 The Institute of Brewing & Distilling     

LIPIDS IN WORT

Material balances have been constructed for lipids during wort preparation in commercial breweries. 4–5% of malt lipids were released into the wort using the mash filter, 1·0% with the lauter tun but only 0–3% with the mash tun. Up to 30% of malt lipids may be oxidised during mashing. Hop products make a contribution to the lipids in the copper though these lipids are not readily extracted. Efficient removal of spent hops and hot break will ensure a low level of wort lipids in the fermentation vessel.     

A New Approach to Limit Dextrinase and its Role in Mashing*

Limit dextrinase (EC 3.1.2.41) is a debranching enzyme catalyzing the hydrolysis of α-1,6-glucosidic linkages in starch. The role of this debranching enzyme in beer brewing has been questioned due to its assumed heat lability. In the present work the effectiveness of limit dextrinase was studied under conditions mimicking brewery practice rather than in a buffer solution. It was demonstrated that typical conversion temperatures of 63–65 °C and a mash pH of 5.4–5.5 favour the action of malt limit dextrinase. The temperature optimum for the limit dextrinase of a malt extract was 60–62.5 °C, as opposed to 50 °C for purified limit dextrinase. Lowering the mash pH from 5.8 to 5.4 increased wort fermentability due to increased limit dextrinase activity. Wort fermentability was more strongly correlated to the free limit dextrinase activity of malt than to the α- and β-amylase activities.     

Survival of human derived Lactobacillus plantarum in fermented cereal extracts during refrigerated storage

The aim of this study was to investigate the survival of a potentially probiotic Lactobacillus plantarum strain in barley, wheat and barley malt extracts. The extracts were produced from three flour/water suspensions, i.e., 5%, 20%, 30% w/w. After inoculation, the cultures were incubated for 24 h at 37 °C, and were subsequently stored at 4 °C for up to seventy days. The lactic acid and reducing sugar concentrations at the beginning of storage were significantly different between the fermented media, ranging from 0.5 g/L to 17 g/L and from 0.8 g/L to 6.5 g/L respectively, while the pH ranged between 2.9 and 3.4. It was observed that the cells survived much better in the malt extracts compared to barley and wheat extracts during refrigerated storage. Based on the results from a study using model media and supplemented cereal extracts it was derived that this was most likely due to their higher sugar concentration and the presence of protective unidentified compounds, albeit the fact that the malt extracts contained higher amounts of lactic acid.     

Effect of varying starch properties and mashing conditions on wort sugar profiles

The aim of this research was to investigate the relationship between starch composition in barley and its malted counterpart alongside malt enzyme activity and determine how these factors contribute to the fermentable sugar profile of wort. Two Australian malting barley varieties, Commander and Gairdner, were sourced from eight growing locations alongside a commercial sample of each. For barley and malt, total starch and gelatinisation temperature were taken, and for malt, α‐ and β‐amylase activities were measured. Samples were mashed using two mashing profiles (infusion and Congress) and the subsequent wort sugar composition and other quality measures (colour, original gravity, soluble nitrogen) were tested. Variety had no significant (<0.05) effect on any barley, malt, enzyme or wort characteristics. However, growing location impacted gelatinisation temperature, colour, malt protein content and original gravity. The gelatinisation temperature in malt samples was higher, by ~0.8°C, than in the equivalent barley sample. Several malt samples, even with protein contents <12.0%, had gelatinisation temperature >65°C. The fermentable sugars measured in the malt prior to mashing showed a higher proportion of maltose than glucose or maltotriose. In addition, there were significant differences in the amount of sugar produced by each mashing method with the high temperature infusion producing a higher amount of sugar and proportionally more maltose. There is scope for further research on the effect of genetics and growing environment on gelatinisation temperature, mash performance and fermentable sugar development. Routinely measuring gelatinisation temperature and providing this information on malt specification sheets could help brewers optimise performance. © 2019 The Institute of Brewing & Distilling