Acid Washing and Serial Repitching a Brewing Ale Strain of Saccharomyces cerevisiae in High Gravity Wort and the Role of Wort Oxygenation Conditions†

Acid washing pitching yeast is an effective method for removing bacterial contamination, but if the yeast is washed incorrectly decreased fermentation performance and beer quality problems may result. Various factors can affect the acid resistance of yeast strains during brewery fermentations. Yeast from shaking flask experiments was more resistant to the combination of high gravity and acid washing conditions than yeast cropped from static fermentations. Yeast harvested from static high gravity wort (20° Plato; 1.083 OG) fermentations was more adversely affected by acid washing than yeast from standard gravity (12° Plato; 1.048 OG) wort. Wort oxygenation resulted in enhanced yeast fermentation performance and healthier yeast crops when yeast was serially repitched into 20° Plato wort. Yeast cropped from fermentations with air saturated high gravity wort responded poorly when acid washed. These results suggest that the structure of the plasma membrane particularly the sterol and fatty acid composition, may have an important role in tolerating high gravity wort and acid washing conditions.     

The Yeast Vacuole ‐A Scanning Electron Microscopy Study During High Gravity Wort Fermentations

The yeast vacuole has been shown to exhibit morphological responses to environmental conditions when exposed to worts of different gravity during fermentation. Marked effects of high gravity wort (20° Plato) on yeast morphology compared to more conventional wort gravity (12° Plato) were observed. High gravity worts caused vacuolar enlargement compared to conventional gravity wort. These results suggested that yeast cells experienced severe alterations with the vacuolar tonoplast when exposed to high osmotic pressure and elevated levels of ethanol.     

Brewing with Rice Malt — A Gluten‐free Alternative

The preparation of beer‐like beverages with rice malt as the only raw material is reported. Several tests were performed on a laboratory scale and in a 25 L‐capacity pilot plant. Both the decoction and the infusion procedure were tested; malt and water were mixed in a ratio 1:3.5 for both methods and the mash was brewed without adding exogenous enzymes. The obtained worts were fermented using bottom fermenting yeasts, while “beers” were re‐fermented utilizing top fermenting yeasts and adding either sterile wort or sugar. A maximum ethanol of 4.5% vol. was obtained after the primary fermentation from an initial wort with an original gravity of 11.8°Plato. All parameters of the beer were found to be acceptable using a standard beer analysis. Owing to a suitable hop addition, an aroma very similar to that of a normal beer was obtained.     

The Loss of Hydrophobic Polypeptides during Fermentation and Conditioning of High Gravity and Low Gravity Brewed Beer

The object of this study was to investigate the loss of hydrophobic polypeptides, which are important for foam quality and stability in finished beer. Loss of hydrophobic polypeptide due to fermenter foaming occurs during transfer of fermented wort since a gradient of hydrophobic polypeptides towards the surface is created during fermentation. Due to higher polyphenol levels in high gravity (20°Plato) wort, more hydrophobic polypeptides are lost due to cold break (cold trub) precipitation compared to low gravity (12°Plato) wort. Another important factor affecting the loss of hydrophobic polypeptides could be proteinase A activity during fermentation, especially in high gravity fermentation where the yeast is exposed the higher stress. During high gravity fermentation, where osmotic pressures are higher, ethanol levels become greater, and nitrogen‐carbohydrate ratios are lower, more proteinase A is released by the yeast. This release of proteinase A into fermenting wort could have implications for the foam stability of the finished product.     

Comparing the Impact of Environmental Factors During Very High Gravity Brewing Fermentations

The impact of the initial dissolved oxygen, fermentation temperature, wort concentration and yeast pitching rate on the major fermentation process responses were evaluated by full factorial design and statistical analysis by JMP 5.01 (SAS software) software. Fermentation trials were carried out in 2L‐EBC tall tubes using an industrial lager brewing yeast strain. The yeast viability, ethanol production, apparent extract and real degree of fermentation were monitored. The results obtained demonstrate that very high gravity worts at 22°P can be fermented in the same period of time as a 15°P wort, by raising the temperature to 18°C, the oxygen level to about 22 ppm, and increasing the pitching rate to 22 × 10⁶ cell/mL. When diluting to obtain an 11.5°P beer extract, the volumetric brewing capacity increased 91% for the 22°P wort fermentation and 30% using the 15°P wort. After dilution, the fermentation of the 22°P wort resulted in a beer with higher esters levels, primarily the compound ethyl acetate.     

Yeast Proteolytic Activity During High and Low Gravity Wort Fermentations and its Effect on Head Retention

The aim was to discover the effect of high gravity brewing on yeast protease activity during fermentation, on the loss of hydrophobic polypeptides from wort during fermentation, and on the foam stability of stored beer. The hydrophobic polypeptide content of low (10° Plato) gravity worts showed a steady decline throughout fermentation, but for the 20° Plato wort there was a rapid decline over the first 8 days of fermentation, followed by little change over the remaining period. The decrease in hydrophobic polypeptides was greater in the high gravity fermentation. Proteinase A increased during fermentations with the highest levels being present at the end of fermentations. High gravity fermentations exhibited levels of yeast protease that from the 3rd to 11th day of fermentation were at least twice the values of the low gravity fermentations. The high gravity brewed beer contained significantly higher levels of proteinase A activity than the low gravity brewed beer. The inclusion of FERMCAP™, an antifoam, in high gravity wort did not affect either the hydrophobic polypeptide levels or foam stability of the resultant beer. This suggests that proteinase A, rather than fermenter foaming, must be the major contributor to the lack of foam stability of high gravity brewed beer. Head retention measurements conducted on the high and low gravity brewed bottled beers, over a five month period, demonstrated a steady decline in foam stability for both beers. The declines in head retention did not occur in high and low gravity beers that had been pasteurised.     

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.     

Corn grist adjunct – application and influence on the brewing process and beer quality

In the brewing industry, barley malt is often partially replaced with adjuncts (unmalted barley, wheat, rice, sorghum and corn in different forms). It is crucial, however, to preserve constant quality in the beer to meet the expectations of consumers. In this work, how the addition of corn grist (10 and 20%) influences the quality of wort and beer was examined. The following parameters were analysed: wort colour, dimethyl sulphide (DMS) and protein content, non‐fermentable extract, extract drop during fermentation, alcohol content and the attenuation level of the beer, together with filtration performance. The samples (all‐malt, and adjunct at 10 and 20% corn grist) were industrial worts and the beers produced in a commercial brewery (3000 hL fermentation tanks). The application of 10 and 20% corn grist had an effect on the wort colour, making it slightly lighter (11.1 and 10.5°EBC, respectively) than the reference barley malt wort (12.2°EBC). The free amino nitrogen level, DMS and non‐fermentable extract were significantly lower in the worts produced with the adjunct; the alcohol content and attenuation levels were higher in the beers produced with adjunct. The use of corn grist, at the level of up to 20% of total load, appears to affect some of the technological aspects of wort and beer production, but it does not significantly influence the final product characteristics. Copyright © 2014 The Institute of Brewing & Distilling     

The Selection of a Dried Yeast Strain for Use in the Apparent Attenuation Limit Malt Analysis (AAL) Procedure

This investigation identifies that Mauribrew Lager 497 strain of dried yeast can be used as a standard strain for the determination of malt apparent attenuation limit (AAL). It provides ferment‐ability results for malt quality evaluation laboratories that are comparable to fresh brewery yeast. It was found that the optimal pitching rate in Congress wort (EBC Analytica, 1998, method 4.5.1), was 1 g per 200 mL, pitched at 25°C and fermented for 24 h at 20°C with agitation to complete attenuation. Preliminary trials also indicated that the Mauribrew Lager 497 dry yeast may be useful to brewers for determining the wort batch attenuation characteristics by the limit gravity test. In this case a pitching temperature of 35°C was found to be optimal with all other conditions as above. For the purpose of malt quality evaluation and brewery quality control the advantages of using a standard dry yeast strain include ease and convenience of use, consistency of quality, and uniformity between laboratories when they are located in separate geographic regions.     

Variation in maltose in sweet wort from barley malt and rice adjuncts with differences in amylose structure

Starch from malt and solid adjuncts provides the majority of fermentable sugars for fermentation. However, there is no current data on the variation in starch structure (particularly long chained amylose) and its impact on the final wort composition of fermentable sugars, specifically maltose. This is the first study to report variation in amylose structure from barley malt and rice used as an adjunct and how this impacts the production of maltose. We compared four commercial malts with two rice adjuncts mashes, in solid and liquid additions, with an all‐malt mash used as a control. All combinations of malt and rice adjuncts were tested under two grist‐to‐liquor (G:L) ratios (1:3 and 1:4) in a 65°C ramped mash. After mashing, the wort original gravity and maltose concentration were measured. The commercial malts had different malt quality but very similar gelatinisation temperatures (~65°C). The malts varied in starch and amylose contents but had only minor variations in average amylose chain lengths. The two rice adjuncts also had similar average amylose chains lengths, but quite different amylose contents, and hence different gelatinisation temperatures. The results showed that liquid adjunct mashes had higher original gravity and maltose concentration for both G:L ratios. However, there was no consistent result in original gravity or maltose between G:L ratio or adjunct type, suggesting interactions between each malt and rice adjunct. Knowing amylose chain length could improve understanding of the potential maltose levels of the sweet wort prior to fermentation. © 2018 The Institute of Brewing & Distilling     

PITCHING RATE IN HIGH GRAVITY BREWING

The optimal pitching rate in high gravity worts (12–16°P) was about 0.3 g/l wet weight (2.3 × 10⁶ counted cells/ml) and per one percent of original wort gravity. In very high gravity worts (20–23°P) the corresponding figure was 0.4 g/l (2.9 × 10⁶ cells/ml). Higher amounts of yeast did not improve the fermentation rate. With increased original wort gravity, flocculation of the yeast weakened and the amount of cropped yeast decreased. The viability of the crop yeast was good. In the conditions used, excessive production of acetate esters occurred only with pitching rates lower than the recommended rate. As the original wort gravity increased, more fermentable extract was metabolized to ethanol rather than utilized for yeast growth. The highest ethanol yield obtained was 10.9% (v/v).     

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.     

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.     

Vitamins in brewing: presence and influence of thiamine and riboflavin on wort fermentation

Thiamine and riboflavin vitamers are present in a wide range of foods including beer. These vitamers play critical roles in a variety of enzymatic complexes and can promote and maintain metabolism. Currently, the presence and role of these vitamers in the malting and brewing industry have not been widely explored. This research investigated the effects of various fermentation conditions that may lead to the variations in the vitamin content in beer observed by previous researchers. The present research found that during fermentation, the thiamine content of wort is quickly utilized within the first 6 h of a standard fermentation and the uptake of this vitamin is not affected by increases in wort gravity. While no significant changes were observed in extracellular phosphorylated vitamers of thiamine, both free thiamine and thiamine diphosphate accumulated intracellularly during the wort fermentation. Meanwhile extracellular riboflavin vitamers were only poorly utilized during beer fermentations, however flavin mononucleotide rapidly accumulated intracellularly and more so under aerobic conditions. When yeast was exposed to an all‐malt high‐gravity wort, the thiamine or riboflavin utilization was not affected. However, thiamine utilization was reduced in adjunct‐driven high‐gravity worts. Notwithstanding the lowered thiamine uptake under high‐gravity conditions; there were some minor improvements in fermentation performance and yeast viability. The addition of thiamine to an all‐malt wort did appear to enhance yeast viability, both under normal and high‐gravity conditions. Copyright © 2016 The Institute of Brewing & Distilling     

Impact of Dark Specialty Malts on Extract Composition and Wort Fermentation

Dark specialty malts are important ingredients for the production of several beer styles. These malts not only impart colour, flavour and antioxidative activity to wort and beer, they also affect the course of wort fermentations and the production of flavour‐active yeast metabolites. The application of considerable levels of dark malt was found to lower the attenuation, mainly as a result of lower levels of fermentable sugars and amino acids in dark wort samples. In fact, from the darkest caramel malts and from roasted malts, practically no fermentable material can be hydrolysed by pilsner malt enzymes during mashing. Compared to wort brewed with 50% pilsner malt and 50% dark caramel malt or roasted malt, wort brewed with 100% pilsner malt contained nearly twice as much fermentable sugars and amino acids. Reduced levels of yeast nutrients also lowered the fermentation rate, ranging from 1.7°P/day for the reference pilsner wort of 9 EBC to 1.1°P/day for the darkest wort (890 EBC units), brewed with 50% roasted malt. This additionally indicates that lower attenuation values for dark wort are partially due to the inhibitory effects of Maillard compounds on yeast metabolism. The application of dark caramel or roasted malts further led to elevated levels of the vicinal diketones diacetyl and 2,3‐pentanedione. Only large levels of roasted malt gave rise to two significant diacetyl peaks during fermentation. The level of ethyl acetate in beer was inversely related to colour, whereas the level of isoamyl acetate appeared to be affected by the use of roasted malt. With large levels of this malt type, negligible isoamyl acetate was generated during fermentation.     

WORT-BOILING IN RELATION TO BEER QUALITY

Comparisons of beers from worts mashed with pale ale malts (nitrogen 1·3%) at 150°F. (65·5°C.) and boiled for 1/2, 1 and 2 hr. show that increase in boiling time leads to slightly increased hop-utilization, decreased head-retention, improved non-biological stability and slight differences in flavour. Findings were very similar when the mashing temperature was either 145°F. (63°C.) or 155°F. (68°C.) except that, in the latter case, no difference in flavour was detectable between beers from long and short boiled worts. Stirring can replace vigorous boiling to secure normal hop-utilization and shelf-life but simmered worts give beers of characteristic flavour probably as a result of enhanced contents of hop oil. Use of a lightly kilned malt of high nitrogen content (1·8%) produced a characteristically different flavour from that obtained with pale-ale malt and reduced the utilization of hop substances by provoking a greater loss of this material during fermentation.     

Influence of temperature on continuous high gravity brewing with yeasts immobilized on spent grains

Flavor compounds’ formation and fermentative parameters of continuous high gravity brewing with yeasts immobilized on spent grains were evaluated at three different temperatures (7, 10 and 15 °C). The assays were performed in a bubble column reactor at constant dilution rate (0.05 h⁻¹) and total gas flow rate (240 ml/min of CO₂ and 10 ml/min of air), with high-gravity all-malt wort (15°Plato). The results revealed that as the fermentation temperature was increased from 7 to 15 °C, the apparent and real degrees of fermentation, rate of extract consumption, ethanol volumetric productivity and consumption of free amino nitrogen (FAN) increased. In addition, beer produced at 15 °C presented a higher alcohols to esters ratio (2.2–2.4:1) similar to the optimum values described in the literature. It was thus concluded that primary high-gravity (15°Plato) all-malt wort fermentation by continuous process with yeasts immobilized on spent grains, can be carried out with a good performance at 15 °C.     

Brewing with 100% Oat Malt

Oats are a cereal with beneficial nutritional properties and also unrealized brewing potential. Furthermore, oats can be tolerated by the majority of people who suffer from celiac disease. Malting of oats produced a malt, which was found suitable for brewing a 100% oat malt beer. The mashing regime, designed by using mathematical modelling, was successfully transferred to a pilot scale plant. The improved lautering performance of oat malt was due to its higher husk content, which also led to a lower extract content in oat wort when compared to barley wort. The protein profile of oat wort, as measured by using Lab‐on‐a‐Chip analysis, revealed that there was no significant difference in the protein profile between oat and barley wort. The fermentation of oat and barley worts followed the same trend; differences could only be seen in the higher pH and lower alcohol content of the oat beer. The flavour analysis of oat beer revealed some special characteristics such as a strong berry flavour and a lower amount of staling compounds when forced aged. This study revealed that it was possible to brew a 100% oat malt beer and that the produced beer was comparable to a barley malt beer.     

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     

High gravity primary continuous beer fermentation using flocculent yeast biomass

The current work assessed a new immobilized cell reactor system throughout a long‐term (54 days) continuous primary fermentation of lager‐type wort of high specific gravity. The experiment was performed in a 4 L airlift bioreactor and immobilization of biomass was attained solely by flocculation. Despite the constant liquid agitation and washout of biomass, up to 53 g dry wt/L of yeast remained immobilized in the system. Two types of beer were produced without interrupting the reactor, based on two types of wort: a Pilsener type with high specific gravity of 15.6 ± 0.3°P; and a dark lager wort with specific gravity of 14.4 ± 0.03°P. Even during the inlet of high gravity wort, the desired attenuation was achieved without the need for either recirculation or an auxiliary second stage bioreactor. The specific saccharide consumption rate was kept around 7.9 ± 0.4 g/L/h and ethanol productivity oscillated at 3.36 ± 0.2 g/L/h for nearly a month. During this period the volumetric productivity of the current bioreactor reached 1.6 L beer/L/day. The green beers produced from the Pilsener and dark lager worts met the standards of regular finished primary beer fermentation. The productivity of diacetyl through the entire experiment could be correlated to the free amino nitrogen consumption rate. Copyright © 2014 The Institute of Brewing & Distilling