NITRATE REDUCTION AND ATNC FORMATION BY BREWERY WILD YEASTS

Control of NAD(P)H-dependent nitrate reductase (NR) and nitrite reductase (NiR) synthesis and activity in Hansenula anomala, Rhodotorula glutinis, Candida versatilis and Brettanomyces anomalus was investigated. Activities of both enzymes were high in all four yeasts when cultured in a medium containing nitrate as the sole source of cell nitrogen, but ammonia and amino-nitrogen were shown to rapidly repress nitrate assimilation and reduction. Little or no NR or NiR activity was detected in wort or beer-grown cultures. Only B. anomalus was found to excrete nitrite when grown in wort, but not at a concentration which could be chemically reduced to allow formation of a detectable concentration of N-nitrosamines. Cask beer (containing 16 mgl^?1 nitrate) contaminated with nitrate reducing wild yeasts, pre-grown on nitrate, contained < 10 μgl^?1 Apparent Total N-Nitrosocompounds (ATNC) following 10 weeks storage. It was concluded that contamination of wort, fermentation and finished beer by nitrate-reducing wild yeast is unlikely to result in formation of detectable ATNC .     

The effect of hopping regime,cultivar and β‐glucosidase activity on monoterpene alcohol concentrations in wort and beer

Previous studies show that the complexity of hop aroma in beer can be partly attributed to the hydrolysis of glycosidically bound monoterpene alcohols extracted from hops during the brewing process to release volatile aglycones. However, fundamental studies that examine the extraction of glycosides during brewing and their subsequent hydrolysis by yeast have not been performed. Furthermore, extraction of other hop‐derived compounds into beer shows a strong dependency on the hop cultivar being used and the point at which it is added. This study focused on the extent of glycoside extraction owing to hopping regime and cultivar, and their hydrolysis by yeast β‐glucosidase activity. Glycoside concentrations of wort made with three different hopping regimes and three cultivars were measured by the difference in volatile aglycone concentrations between samples treated with purified β‐glucosidase and untreated samples. Aglycone concentrations were measured by solid‐phase microextraction gas chromatography–mass spectrometry. Additionally, β‐glucosidase activities for 80 different yeast strains and their effect on aglycone concentration in wort were determined. Results showed that yeast have a wide range of abilities to hydrolyse glycosides with a maximum hydrolysis occurring after 3 days of fermentation regardless of yeast activity. Although it was shown that yeast are capable of glycoside hydrolysis, glycoside concentrations in wort are low and make small contributions to hop aroma. These results help explain the extent to which different brewing yeasts and hopping regimes contribute to hoppy beer aroma through the hydrolysis of non‐volatile hop‐derived compounds. Copyright © 2017 The Institute of Brewing & Distilling     

The temperature dependent functionality of Brettanomyces bruxellensis strains in wort fermentations

In recent years, Brettanomyces bruxellensis has found increasing application in brewery fermentations. Indeed, B. bruxellensis contributes to the flavour profile of many Belgian beers, typically during secondary or spontaneous fermentation. In North America, the yeast is used in primary fermentation to produce beers with ‘Brett’ characteristics with ‘fruity’ and/or ‘funky’ sensory profiles associated with the production of volatile esters and phenols. However, little is understood about the factors that influence flavour metabolite production or fermentation rate in this yeast. Here, the impact of temperature is reported on fermentation efficiency, flavour metabolite production and carbon utilisation of one commonly used and eight poorly characterised B. bruxellensis strains during wort fermentation. A high degree of strain and temperature‐dependent variability was found in fermentation efficiency and metabolite production amongst B. bruxellensis strains. Further, fermentation efficiency and carbon utilisation were temperature dependent, while ester production increased at higher temperature and phenol production was strain and temperature independent. These results indicate significant strain and temperature dependent variation, suggesting the potential application of strain variability as a tool to achieve product diversity in B. bruxellensis primary fermentations. © 2019 The Institute of Brewing & Distilling     

HOP SUBSTANCES AND YEAST BEHAVIOUR

Top fermentation yeast strains vary in their ability to form a yeast head in the absence of hop substances. However, the addition of isohumulone or humulone to unhopped wort stimulates yeast-head formation. A quantitative method for the estimation of hop substances bound to the yeast cell is described and is used to demonstrate that in top fermentation, most of the adsorbed bitter substances are present in the head. It is shown that most of the hop substances in the yeast head are transported there by bubbles of carbon dioxide; in bottom fermentation, considerable quantities are present in the surface foam. In comparison with hopped wort the fermentation of unhopped wort with Saccharomyces cerevisiae (N.C.Y.C. 240) is characterized by an increased amount of yeast in suspension during fermentation, although there is no increase in the rate of attenuation.     

Possibilities to improve the antioxidative capacity of beer by optimized hopping regimes

Different hopping regimes were evaluated to investigate the effect on the oxidative stability of wort and beer. Compared with a single hop dosage at the beginning of wort boil, it was possible to increase the concentration of α‐acids in pitching wort and beer by applying incremental hop dosage, dry hopping or the use of a pre‐isomerized hop product in combination with an α‐acid extract, which concomitantly resulted in lower iron concentrations and an enhanced flavour stability as indicated by standard wort and beer analyses, atomic absorption spectroscopy, electron spin resonance spectroscopy and sensory analysis of fresh and force‐aged beers. The functional principle of hop dosage variations is explained by saving of α‐acids throughout the wort production process, which yields an increased formation and precipitation of pro‐oxidative acting transition metal ions (e.g. Fe) in α‐acid‐complexes during the whirlpool rest and fermentation. Consequently, fewer reactive oxygen species are generated. Additional laboratory trials simulating wort cooling and beer storage in buffered model solutions proved that un‐isomerized α‐acids are strong iron chelators and confirmed the functional principle of the applied hopping regimes. Negative effects of higher α‐acid contents on fermentation performance and depletion of the zinc concentration, which is an essential nutrient for yeast, could be excluded. Copyright © 2014 The Institute of Brewing & Distilling     

Evaluation of different yeast strains on the quality of beer produced from malted proso millet (Panicum miliaceum L.)

In order to investigate the influence of temperature on pH, alcohol production, carbohydrate profile and aroma compounds, a variety of top-fermenting yeast strains were used to ferment wort obtained from proso millet malt. Champagne (Saccharomyces bayanus), altbier, kölsch and ale (Saccharomyces cerevisiae), as well as Brettanomyces bruxellensis were used in two isothermal (12 and 18 °C) fermentation trials. During the fermentation, changes in pH, extract conversion and the monosaccharide profile were monitored. After 2 weeks of storage, the levels of higher alcohols and esters were also measured in the finished beers. In many cases, a strong resemblance in the behaviour of the selected strains to Saccharomyces was observed during the fermentation. All other influencing fermenting parameters like pitching rate, yeast generation, fermenting vessels geometry, amount of assimilable nitrogen, yeast nutrients, oxygen and CO₂ content and the sugar content were the same for all trials regardless of the yeast applied. However, at higher temperatures Brettanomyces showed more rapid rates of sugar conversion. Concerning the development of aroma components, no uniform picture was evident in these trials, neither with respect to temperature dependence nor to what extent the incurred values are comparable to those already known for beers brewed using barley malt wort. The values for aliphatic alcohols obtained during experimentation, ranged from 23 to 267 μg/L, and the sum of aromatic alcohols, from 2,437 to 11,844 μg/L. Values for ester content were recorded between 188 and 2,543 μg/L. Overall, wort produced using proso millet malt was found to be a substrate that lends itself to alcoholic fermentation, exhibiting a sufficient decrease in pH over the course of fermentation, as well as reasonable alcohol yield and a broad range of aroma components.     

Effect of the respiro-fermentative balance during yeast propagation on fermentation and wort attenuation

Breweries use different yeast strains to create beers with different flavours and aromas. Yeast propagation must produce yeast that performs consistently from the first fermentation to harvesting and re-pitching in subsequent fermentations. Breweries propagate yeast in wort leading to low efficiency fermentative growth in Crabtree-positive yeast. There is limited knowledge on the impact on beer production when fermenting with yeast propagated in sugar limited and nutrient supplemented wort. It was hypothesised that propagating yeast in this way would have a positive impact on subsequent fermentation performance. Saccharomyces cerevisiae was propagated at the laboratory scale in standard wort with a high carbon to nitrogen (C:N) ratio (850) or in modified wort supplemented with yeast extract to achieve a low C:N ratio (100) and at varying sugar concentrations. Propagation in low C:N wort with 2°P sugar yielded a 27% decrease in fermentation efficiency and a 46% increase in cell production compared to 2°P high C:N wort. This suggests nitrogen is critical to the respiro-fermentative balance during growth. Yeast propagated in standard wort resulted in slower fermentations and significant under-attenuation compared to yeast grown in the modified wort with low sugar and high nitrogen. The results of this study suggest the nitrogen and sugar content drive the respiro-fermentative balance during yeast propagation. The metabolism of yeast during propagation induces significant downstream impacts on the subsequent fermentation performance and wort attenuation. © 2020 The Institute of Brewing & Distilling     

INFLUENCE OF NITRATE AND BACTERIAL CONTAMINATION ON THE FORMATION OF APPARENT TOTAL N-NITROSO COMPOUNDS (ATNC) DURING FERMENTATION

Formation of Apparent Total N-Nitroso Compounds (ATNC) was monitored throughout fermentations of all-malt ale worts supplemented with nitrate (0–100 mg Litre^?1 (0–100 ppm)). The pitching yeasts were obtained from commercial breweries and contained different levels of the contaminant bacterium Obesumbacterium proteus (0–2.1% by number). Levels of ATNC present at the end of fermentation were dependent on both initial wort nitrate levels and the initial level of bacterial contamination of the pitching yeast. Only relatively low nitrate levels were required to produce ATNC levels greater than the Brewers' Society recommended limit of 20 μg Litre^?1 (ppb), provided that the bacteria were present. Indeed, the use of whole hops alone would contribute sufficient nitrate to the wort to produce excessive amounts of ATNC, provided O.proteus was present. The only feasible solution to ATNC production during fermentation is to remove the contaminating bacteria from both the pitching yeast and brewing plant. Effective removal of O.proteus can be achieved by acid washing the pitching yeast under carefully controlled conditions, prior to fermentation.     

Overexpression of vacuolar H+‐ATPase‐related genes in bottom‐fermenting yeast enhances ethanol tolerance and fermentation rates during high‐gravity fermentation

Vacuolar H⁺‐ATPase (V‐ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom‐fermenting yeast strains, in which the V‐ATPase‐related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high‐sugar medium simulating high‐gravity fermentation. Among the strains examined, the DBF2‐overexpressing bottom‐fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high‐gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V‐ATPase is a stress target in high‐gravity fermentation and suggests that enhancing the V‐ATPase activity increases the ethanol tolerance of bottom‐fermenting yeast, thereby improving the fermentation rate and cell viability under high‐gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling     

Review: Pure non‐Saccharomyces starter cultures for beer fermentation with a focus on secondary metabolites and practical applications

Recently there has been increased interest in using non‐Saccharomyces yeasts to ferment beer. The worldwide growth of craft beer and microbreweries has revitalised the use of different yeast strains with a pronounced impact on aroma and flavour. Using non‐conventional yeast gives brewers a unique selling point to differentiate themselves. Belgian brewers have been very successful in using wild yeasts and mixed fermentations that often contain non‐Saccharomyces yeasts. Historically, ancient beers and beers produced before the domestication of commonly used Saccharomyces strains most likely included non‐Saccharomyces species. Given the renewed interest in using non‐Saccharomyces yeasts to brew traditional beers and their potential application to produce low‐alcohol or alcohol‐free beer, the fermentation and flavour characteristics of different species of non‐Saccharomyces pure culture yeast were screened for brewing potential (Brettanomyces anomalus and bruxellensis, Candida tropicalis and shehatae, Saccharomycodes ludwigii, Torulaspora delbrueckii, Pichia kluyveri, Zygosaccharomyces rouxii). Alcohol‐free beer is already industrially produced using S. ludwigii, a maltose‐negative species, which is a good example of the introduction of non‐Saccharomyces yeast to breweries. Overall, non‐Saccharomyces yeasts represent a large resource of biodiversity for the production of new beers and have the potential for wider application to other beverage and industrial applications. Almost all of the trials reviewed were conducted with varying fermentation parameters, which plays an important role in the outcome of the studies. To understand these impacts all trials were described with their major fermentation parameters. Copyright © 2016 The Institute of Brewing & Distilling     

Lachancea thermotolerans as an alternative yeast for the production of beer

The ability of Lachancea thermotolerans strains to ferment brewer's wort has been investigated. Initial fermentations with three L. thermotolerans strains compared the use of maltose and maltotriose, as well as production of glycerol and lactic acid and pH evolution over the course of the fermentation. The most promising strain was subsequently tested for additional traits important for beer production, including pitching rate, generational capacity, foam stability, hop tolerance, vicinal diketone production, oxygen requirement and flocculation. These tests suggest that L. thermotolerans may be a good choice for producing sour beers in a single fermentation step without the use of lactic acid bacteria. Copyright © 2016 The Institute of Brewing & Distilling     

啤酒酿造过程中萜烯醇类化合物变化规律

采用基于顶空固相微萃取-气相色谱质谱技术(HS-SPME/GC-MS)建立的啤酒中酒花物质的检测方法,跟踪了啤酒酿造过程中源自酒花的5种萜烯醇类香气化合物的变化规律,初步为啤酒厂酒花配方及工艺调整奠定了理论基础。通过对糖化过程中不同酒花配方、不同煮沸方式及酒花添加工艺对冷麦汁中萜烯醇类化合物含量影响的研究,发现冷麦汁中的萜烯醇类化合物主要受最后一次添加酒花的添加量和添加时机影响,最后一次酒花在煮终回旋前添加较煮终前10 min添加,更利于萜烯醇类化合物在冷麦汁中的保留,这与国外的late-hopping工艺相一致;与煮终前10 min最后一次添加相比,在煮终回旋前最后一次添加酒花能使冷麦汁中里那醇含量提高209.4%,α-萜品醇为91.2%,香叶醇为31.4%,橙花醇175.0%。通过研究发酵过程中萜烯醇类化合物的变化规律,发现5种萜烯在发酵过程中呈上升趋势,且它们之间可能被酵母相互转化。     

The effects of three mash separation systems on the isomerisation of hop alpha-acids

This study investigates the effects of wort composition from three lautering systems on hop utilisation at different hop boiling and dosing times. A response surface methodology was applied with 60 single tests at a 5 litre scale. The parameters, which were varied, were lautering system, boiling time without hops, boiling time with hops and α-acid dosage. It was shown that the wort composition from the different lautering systems requires different boiling times or enables the reduction in boiling time with hops. Although the pH and original gravity of the lauter tun and mash filter worts were similar, different boiling times were necessary to achieve the same concentration of iso-α-acids. Further, there were variations in fatty acid composition of the worts. In order to be able to assess the effects on a larger scale, six brews were performed in a 10 hL pilot brewery. The utilisation of hop bitter substances differed despite the same boiling time and the same α-acid dosage in relation to the total quantity of wort. In addition, no significant losses of hop bitter substances were observed in the wort from a continuous mash filtration system due to the process related higher dosage of α-acid. Both sets of experiments showed that the boiling times depend on the wort composition and increased as follows: novel continuous mash filtration system < mash filter < lauter tun. The results lay the foundation for calculating the optimal parameter settings for each brewery to optimise the hop isomerisation rate. © 2020 The Authors. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

Screening for new brewing yeasts in the non‐Saccharomyces sector with Torulaspora delbrueckii as model

This study describes a screening system for future brewing yeasts focusing on non‐Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off‐flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by‐products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre‐fermentation as a bio‐flavouring agent for beers that have been post‐fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour‐forming properties. Copyright © 2015 John Wiley & Sons, Ltd.     

PITCHING RATE AND YEAST ACTIVITY DURING FERMENTATION OF BREWER'S WORT

Departure from a normal pitching rate during batch fermentation of brewer's wort by strains of Sacchoromyces cerevisiae results in disproportionate changes in the time needed for fermentation. Variations in pitching rate alter the ability of the yeast to utilize maltose and this mainly determines the rate of fermentation of the wort. These changes in yeast activity modify the effect of altered yeast concentration due to pitching rate and are responsible for the disproportionate changes in fermentation time.     

Part I: the influence of serial repitching of Saccharomyces pastorianus on the uptake dynamics of metal ions and fermentable carbohydrates during the fermentation of barley and gluten‐free buckwheat and quinoa wort

Recently, research has been focusing on the use of alternative raw materials for brewing purposes and gluten‐free beer‐like beverages from malted buckwheat and quinoa are of commercial interest. A common commercial process involves the serial repitching of the yeast biomass, but this has not been described using buckwheat and quinoa wort fermentations. Our research studies (Parts I–III) explored the serial repitching of the yeast strain Saccharomyces pastorianus TUM 34/70 on the composition of a barley, buckwheat and quinoa fermentation medium. The present paper focuses on the fermentation performance and the uptake dynamics of metal ions and fermentable carbohydrates. Both pseudocereals showed high variations in all of the attributes examined during successive fermentations. In buckwheat the differences between successive fermentations were similar to those observed with barley, whereas differences in quinoa varied quite significantly from those observed with barley and showed a directional trend, suggesting a general weakening of the yeast from the sixth successive fermentation onward. In particular, the assimilation of the fermentable carbohydrates lessened and metal ion uptake appeared poorly controlled. It was concluded that buckwheat showed good potential for serial repitching of S. pastorianus TUM 34/70, whereas serial repitching of a quinoa wort appeared to be limited to five or six fermentations. Copyright © 2015 The Institute of Brewing & Distilling     

Reducing power of hop cultivars and beer ageing

Since lipid auto-oxidation during wort boiling is a determining for the appearance of staling flavour in aged beers, we have investigated the reducing power of hops added in the boiling kettle. An assay based on the inhibition of linoleic acid oxidation in the presence of an initiator [2,2′-azobis(2-amidino-propane) dihydrochloride=AAPH] enabled us to distinguish hop varieties and conditionings. Large differences in hop flavanoid contents explained the higher antioxidant activity of low-α-acid samples versus bitter varieties and CO₂ hop extracts. As expected, adding hop pellets to the kettle effectively increased the overall reducing activity of wort. Supercritical CO₂ hop extracts had no significant effect due to their extremely low level of polyphenols. The concentration of the very well-known marker of beer ageing, trans-2-nonenal, was lower in boiled wort exhibiting a better reducing power. The AAPH reducing power test applied to hops or worts was thus efficient to predict the nonenal synthesis during boiling. Hop varieties and conditionings emerged from this work as key-parameters for improving the reducing power of wort and the flavour stability of the final product.     

Characteristics of yeast flora in Chinese strong‐flavoured liquor fermentation in the Yibin region of China

The yeast community in the Chinese strong‐flavoured liquor region of Yibin was investigated and the ethanol producing abilities and extracellular enzymes activities of the isolates were tested. A total of 110 yeast were isolated on Wallerstein Laboratory medium and through 26S rRNA D1/D2 region sequence analysis identified as 13 yeast species. These were Wickerhamomyces anomalus, Debaryomyces hansenii, Issatchenkia orientalis, Lodderomyces elongisporus, Clavispora lusitaniae, Saccharomyces cerevisiae, Pichia fermentans, Pichia manshurica, Pichia membranifaciens, Torulaspora delbrueckii, Trichosporon insectorum, Trichosporonoides megachiliensis, Zygosaccharomyces bailii, and one uncertain species. These yeast species, composed of various strains, formed the special yeast community in the Yibin region. Approximately 73.6% of the strains belong to the four dominant species: W. anomalus, D. hansenii, I. orientalis and L. elongisporus. The 110 yeast strains produced 0.6–9.0% (v/v) alcohol (average of 5.4%, v/v) in a grain medium, and 0.2–7.2% (v/v) alcohol (average value of 2.9%, v/v) in a yeast extract–peptone–dextrose medium. Furthermore, the 49 strains that produced pectinase, lipase, cellulase, amylase or protease generally showed better ethanol‐producing ability than those strains that do not produce extracellular enzymes. This work profiles the ethanol‐producing ability and the organic matter utilization of the yeast community in Chinese strong‐flavoured liquor produced in the Yibin region and provides a better understanding of Chinese strong‐flavoured liquor fermentation. Copyright © 2016 The Institute of Brewing & Distilling     

Hop proanthocyanidins for the fining of beer

Fining agents are used in the clarification of beers; they help to reduce the time required to sediment suspended yeast cells and ensure the clarity and colloidal stability of beer. Following an adventitious observation during dry‐hopping experiments, we identified a fining activity associated with Saaz hops. Extracts of hop cones were subsequently shown to have the capacity to flocculate yeast and result in their sedimentation. This activity has since been identified in extracts of many different hop varieties and, significantly in spent hops, the co‐product resulting from commercial extraction of hops with either CO₂ or ethanol. Here we illustrate the activity of the novel finings extracted from spent hops following CO₂ extraction of Galena hops. The sediments formed on fining were compact, relative to those obtained when commercial isinglass was used to fine the same beers. The hop extracts were also effective in reducing 90° haze in beers under conditions designed to mimic both cask ale (12 °C) and lager (4 °C) type applications. The compounds responsible for the fining activity appear to be large (30–100 kDa, or more) polyphenols. Analysis of the polyphenols using colourimetric tests indicated the presence of proanthocyanidins. On acidic hydrolysis these generated cyanidin, which would be derived from a polymer composed of catechin and epicatechin subunits. The presence of these materials in spent hops offers the possibility to develop commercial products, with desirable fining properties, from an existing co‐product stream. Furthermore, the finings are derived from a traditional ingredient of the brewing process. Copyright © 2015 The Institute of Brewing & Distilling     

Fed‐batch l‐(+)‐lactic acid fermentation of brewer's spent grain hydrolysate

Brewer's spent grain (BSG) hydrolysates were used for l ‐(+)‐lactic acid (LA) fermentation by Lactobacillus rhamnosus ATCC 7469. The aim of this study was to evaluate fed‐batch LA fermentation of BSG hydrolysate with the addition of glucose, glucose and yeast extract, and wort during LA fermentation and its effect on fermentation parameters such as LA concentration, its volumetric productivity and yield, and L. rhamnosus cell viability. The highest LA yield, volumetric productivity and concentration of 93.3%, 2.0 g/L/h, and 116.1 g/L, respectively, were achieved with glucose and yeast extract addition during fermentation. In fed‐batch fermentation with glucose and yeast extract addition significantly higher LA concentration, yield and volumetric productivity (by 194.8; 2.2, and 20.7%, respectively) were achieved compared with batch fermentation. The results indicated that fed‐batch fermentation could be used to increase LA fermentation efficiency. Copyright © 2017 The Institute of Brewing & Distilling