Why was the work done: Whisky fermentations are typically performed using specific strains of Saccharomyces cerevisiae, which produce a wash with high ethanol yields but a limited diversity of aromas and flavours. Innovation is an important theme for alcoholic beverages and incorporating non-conventional yeasts in their production can enhance sensory complexity and enable regional differentiation. This study investigated the potential of native yeasts from Argentine Patagonia to produce whiskies with unique flavour profiles. How was the work done: Eight native Patagonian yeasts (Saccharomyces uvarum, Saccharomyces eubayanus, Hanseniaspora smithiae, Lachancea nothofagi, Lachancea cidri) were tested in pure and mixed fermentations using distillery wort. A commercial strain of Saccharomyces cerevisiae (Safspirit M1™) was included for comparison. Fermentation kinetics, sugar consumption, and ethanol production were analysed, while sensory evaluation and chemical analyses of volatile compounds were performed on the first distillate or ‘low wines’ (20-23% ABV). What are the main findings: Native yeasts produced lower ethanol yields than the commercial strain but resulted in low wines with enhanced complexity of flavour and aroma. Mixed fermentations increased ester and phenolic notes. S. eubayanus and S. uvarum were identified as promising yeasts for their ability to generate distinctive flavour compounds while maintaining good fermentation performance. Low wines made with native yeasts were preferred by a sensory panel over the control made with a commercial yeast. Why is the work important: This research highlights the potential of Patagonian yeasts as a tool for whisky innovation, enabling flavour diversity. It also contributes to industry efforts to explore microbial biodiversity as a route to enhancing product differentiation and local whisky production.

Scotch grain whisky is produced with a substantial proportion of unmalted grains, which can result in nitrogen deficiency for yeast in the fermentable grain mash. This study examined nitrogen source availability and utilisation by three commercial whisky strains (Saccharomyces cerevisiae) during Scotch grain whisky fermentation, focusing on oligopeptides. Peptide uptake kinetics in synthetic whisky mash with defined peptides showed that oligopeptides of up to nine amino acids were taken up by the strains, albeit with some variability between the strains. The study found that peptides with appropriate molecular weights could replace free amino acids without negatively affecting fermentation kinetics. Moreover, fermentation performance improved when additional nitrogen was provided via peptides rather than diammonium phosphate. Analysis of industrial grain mash indicated that despite low initial yeast assimilable nitrogen, residual proteolytic activity from malt increased nitrogen availability during fermentation. Approximately 30% of the nitrogen consumed by yeast during grain mash fermentation was derived from peptides. LC-HRMS peptide analysis revealed complex dynamics of peptide formation, degradation, and utilisation. This study highlights the importance of oligopeptides in ensuring optimal fermentation efficiency in Scotch grain mash and similar substrates.

Yeast format and pitching rate are variables which can be easily manipulated in a distillery environment but are seldom altered. Fermentations using dried and liquid yeast were studied at laboratory scale and compared by application of a 4-parameter logistic model to measurements of the decline in apparent extract during fermentation. Congener analysis of new make distillate allowed comparison between compounds of interest produced during fermentation. The liquid yeast format demonstrated a significant reduction (p<0.05) in lag time, which was 68% shorter than dried yeast. Despite this, longer overall fermentation times were observed due to a slower exponential phase as compared to the dried yeast format. Alteration of the yeast pitching rate using dried and liquid formats, demonstrated that high rates resulted in reduced lag times. The maximum fermentation rates (Vmax) were assessed from the fermentation models and no consistent trend could be identified. Dried yeast was observed to achieve its maximum fermentation rate when underpitched. Substantially higher Vmax ­values were obtained with overpitched liquid yeast fermentations, resulting in faster fermentations, compared to underpitched liquid yeast fermentations. The liquid yeast format created more esters compared to dried yeast. The concentration of ethyl esters generally trended downwards for fermentations pitched with less yeast which fully attenuated. By combining the results of these studies, distillers can make informed decisions to optimise spirit character, quality, and distillery production.

In addition to ethanol yield, the production of flavour congeners during fermentation is a major consideration for Scotch whisky producers. Experimental whisky fermentations can provide useful information to the industry, and this is the focus of this paper. This study investigated the impact of wort pretreatments (boiled, autoclaved, filtered) on fermentation performance and flavour development in Scotch whisky distillates as an alternative to freezing wort for storage. Our study showed that no significant sensorial differences were detected in low wines (first distillates), while the chemical compositions showed clear changes in increased levels of esters and higher alcohols in boiled and autoclaved wort. In contrast, filtered wort comprised overall lower levels of congeners. Regarding alcohol yield, all three pretreatments resulted in decreased yields. In practice, the pretreatment of wort prior to fermentation requires additional process operations, while freezing requires large storage units. The pretreatments adopted in this study significantly influence the composition of the malt wort used for experimental whisky fermentations, and this results in a poorer fermentation performance compared with untreated wort. We recommend the use of fresh or frozen wort as the best options for small-scale fermentation trials.

Lactobacillus helveticus is a well characterized lactobacillus for dairy fermentations that is also found in malt whisky fermentations. The two environments contain considerable differences related to microbial growth, including the presence of different growth inhibitors and nutrients. The present study characterized L. helveticus strains originating from dairy fermentations (called milk strains hereafter) and malt whisky fermentations (called whisky strains hereafter) by in vitro phenotypic tests and comparative genomics. The whisky strains can tolerate ethanol more than the milk strains, whereas the milk strains can tolerate lysozyme and lactoferrin more than the whisky strains. Several plant-origin carbohydrates, including cellobiose, maltose, sucrose, fructooligosaccharide and salicin, were generally metabolized only by the whisky strains, whereas milk-derived carbohydrates, i.e. lactose and galactose, were metabolized only by the milk strains. Milk fermentation properties also distinguished the two groups. The general genomic characteristics, including genomic size, number of coding sequences and average nucleotide identity values, differentiated the two groups. The observed differences in carbohydrate metabolic properties between the two groups correlated with the presence of intact specific enzymes in glycoside hydrolase (GH) families GH1, GH4, GH13, GH32 and GH65. Several GHs in the milk strains were inactive due to the presence of stop codon(s) in genes encoding the GHs, and the inactivation patterns of the genes encoding specific enzymes assigned to GH1 in the milk strains suggested a possible diversification manner of L. helveticus strains. The present study has demonstrated how L. helveticus strains have adapted to their habitats.

Recently there has been an increased interest in characterising the rates of alcoholic fermentations. Sigmoidal models have been used to predict changes such as the rate of density decline. In this study, three published sigmoidal models were assessed and fit to industrial fermentation data. The first is the four-parameter logistic model described in the ASBC Yeast-14 method. The second model is a nested form of the four-parameter logistic function, adding an extra parameter, creating the 5-parameter logistic equation., where an additional parameter was added to allow for asymmetry. The final model is a three-parameter logistic equation which describes the change in the Apparent Degree of Fermentation with time. The three models were compared by fitting them to industrial data from Australian and Canadian lagers, American and Scottish ales and Scotch Whisky fermentations. The model fits were then compared to one another with a technique developed by Akaike and a nested F-test. The Akaike information criterion compares the models and accounts for both the goodness of fit, and the number of parameters in the model. Finally, consideration was given to the establishment of prediction bands (that enclose the area that one can be 99% sure contains the true datapoints). Calculation of these bands was “challenging” but successful as the industrial fermentation data was heteroscedastic.

Whisk(e)y is a major global distilled spirit beverage. Whiskies are produced from cereal starches that are saccharified, fermented and distilled prior to spirit maturation. The strain of Saccharomyces cerevisiae employed in whisky fermentations is crucially important not only in terms of ethanol yields, but also for production of minor yeast metabolites which collectively contribute to development of spirit flavour and aroma characteristics. Distillers must therefore pay very careful attention to the strain of yeast exploited to ensure consistency of fermentation performance and spirit congener profiles. In the Scotch whisky industry, initiatives to address sustainability issues facing the industry (for example, reduced energy and water usage) have resulted in a growing awareness regarding criteria for selecting new distilling yeasts with improved efficiency. For example, there is now a desire for Scotch whisky distilling yeasts to perform under more challenging conditions such as high gravity wort fermentations. This article highlights the important roles of S. cerevisiae strains in whisky production (with particular emphasis on Scotch) and describes key fermentation performance attributes sought in distiller’s yeast, such as high alcohol yields, stress tolerance and desirable congener profiles. We hope that the information herein will be useful for whisky producers and yeast suppliers in selecting new distilling strains of S. cerevisiae, and for the scientific community to stimulate further research in this area.

Scotch whisky fermentations typically employ high-gravity fermentation practices to maximize product formation and to minimize both energy and water inputs. This approach increases ethanol concentrations at the end of fermentation, creating stressful conditions for the yeast. In this work we examined the relative tolerance of four Saccharomyces cerevisiae distilling yeast strains, supplied in dried, creamed, cake or slurry format, to ethanol under CO2-induced anaerobic conditions. The cells were assessed for their capacity to recover and grow on inhibition spot plates and to maintain cell viability in ethanol-dosed suspensions. Variations in ethanol tolerance were observed between strains and between the same strain supplied in different formats. The creamed yeast format typically exhibited a higher tolerance to ethanol. One possible explanation for this observation is that cells surviving the dehydration and rehydration process might incur sub-lethal genome damage. Thus the genetic integrity of the most ethanol-tolerant strain was assessed as a function of supply format (two dried and one creamed). The mitochondrial DNA was examined using mitochondrial restriction fragment length polymorphism and the chromosomal DNA using pulsed field gel electrophoresis and polymerase chain reaction with both ITS and delta-specific primers. In one dried yeast sample, genetic integrity was compromised, highlighting the requirement for yeast intake quality assurance programmes. Copyright © 2012 The Institute of Brewing & Distilling

Traditionally, distilling companies in Scotland have employed a very limited number of yeast strains in the production of alcohol for Scotch whiskies. Recent changes such as the decline in availability of brewers' yeast as a secondary yeast strain and the availability of yeast in different formats (e.g., dried and cream yeast as alternatives to compressed yeast) have promoted interest in alternative Scotch whisky distilling yeasts. In previous work, we investigated different strains of yeasts, specifically Brazilian yeasts which had been isolated from and used in fuel alcohol distilleries. One of the Brazilian yeasts (CAT 1) showed a comparable fermentation performance and superior stress tolerance compared with a standard commercial Scotch whisky distilling yeast (M Type). The Brazilian CAT 1 yeast isolate was further assessed in laboratory scale fermentations and subsequent new make spirit was subjected to sensory analyses. The spirits produced using the Brazilian strain had acceptable flavour profiles and exhibited no sensory characteristics that were atypical of Scotch whisky new make spirit. This study highlights the potential of exploiting yeast biodiversity in traditional Scotch whisky distillery fermentation processes.

Analysis of fermentation samples from the Speyside malt whisky distillery by denaturing gradient gel electrophoresis (DGGE) revealed numerous lactic acid bacteria including Lactobacillus fermentum, Weissella confusa, and strains related to Weissella kimchii. Fermentation samples from the Strathclyde grain distillery also contained L. fermentum in addition to a bacterium related to L. sanfrancisco. Comparison of these results with data from a previous study of the Glenkinchie malt whisky distillery confirmed that bacterial growth during the fermentations occurred in three phases. There was an initial phase during the first 50 hr that was characterized by declining diversity as rapid yeast growth ensued. This was followed by heterofermentative bacteria predominating, in particular L. fermentum in all three distilleries and weissellas in the Speyside distillery. Finally, homofermentative bacteria related to L. acidophilus and L. delbrueckii emerged in the malt whisky fermentations that practiced an extended fermentation period. This study extends the diversity of lactic acid bacteria associated with whisky fermentations and indicates new species that have yet to be obtained in laboratory culture.

The development of the lactic acid bacterial community in a commercial malt whisky fermentation occurred in three broad phases. Initially, bacteria were inhibited by strong yeast growth. Fluorescence microscopy and environmental scanning electron microscopy revealed, in this early stage, both cocci and rods that were at least partly derived from the wort and yeast but also stemmed from the distillery plant. Denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA genes and sequence analysis revealed cocci related to Streptococcus thermophilus or Saccharococcus thermophilus, Lactobacillus brevis, and Lactobacillus fermentum. The middle phase began 35 to 40 h after yeast inoculation and was characterized by exponential growth of lactobacilli and residual yeast metabolism. Lactobacillus casei or Lactobacillus paracasei, L. fermentum, and Lactobacillus ferintoshensis were detected in samples of fermenting wort examined by DGGE during this stage. Bacterial growth was accompanied by the accumulation of acetic and lactic acids and the metabolism of residual maltooligosaccharides. By 70 h, two new PCR bands were detected on DGGE gels, and the associated bacteria were largely responsible for the final phase of the fermentation. The bacteria were phylogenetically related to Lactobacillus acidophilus and Lactobacillus delbrueckii, and strains similar to the former had previously been recovered from malt whisky fermentations in Japan. These were probably obligately homofermentative bacteria, required malt wort for growth, and could not be cultured on normal laboratory media, such as MRS. Their metabolism during the last 20 to 30 h of fermentation was associated with yeast death and autolysis and further accumulation of lactate but no additional acetate.

Sixty-four strains of Lactobacillus were isolated from fermentation samples from 23 malt whisky distilleries located in the major whisky producing regions of Scotland. The strains were assigned to 26 ribotype patterns. Strains of some ribotype patterns were widely distributed and recovered from distilleries throughout Scotland, while strains representing other ribotypes were particular to a specific region or even a certain distillery. Repeated sampling of a single distillery over a 12 month period showed that the range of bacteria present, as indicated by ribotyping, was stable, but was influenced by changes in malt supply and the period of closure for annual maintenance. Partial 16S rDNA sequence analysis of ribotype representatives revealed Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus paracasei and Lactobacillus pentosus as the major species present in the distilleries; however, four isolates could not be identified by this procedure. Determination of the full 16S rDNA gene sequence from one of these isolates (strain R7-84) revealed >98.5% similarity to Lactobacillus buchneri and its phylogenetic neighbours. DNA from two other strains showed greater than 70% hybridization to DNA from R7-84 under non-stringent renaturation conditions and DNA from strain R7-84 shared less than 65% hybridization with members of the L. buchneri group. It is proposed that these three strains should be placed in a new species for which the name Lactobacillus ferintoshensis represented by the type strain R7-84(T) is suggested.

Seven strains of Lactobacillus isolated from malt whisky fermentations and representing Lactobacillus brevis, L. crispatus, L. fermentum, L. hilgardii, L. paracasei, L. pentosus, and L. plantarum contained genes for hydroxycinnamic acid (p-coumaric acid) decarboxylase. With the exception of L. hilgardii, these bacteria decarboxylated p-coumaric acid and/or ferulic acid, with the production of 4-vinylphenol and/or 4-vinylguaiacol, respectively, although the relative activities on the two substrates varied between strains. The addition of p-coumaric acid or ferulic acid to cultures of L. pentosus in MRS broth induced hydroxycinnamic acid decarboxylase mRNA within 5 min, and the gene was also induced by the indigenous components of malt wort. In a simulated distillery fermentation, a mixed culture of L. crispatus and L. pentosus in the presence of Saccharomyces cerevisiae decarboxylated added p-coumaric acid more rapidly than the yeast alone but had little activity on added ferulic acid. Moreover, we were able to demonstrate the induction of hydroxycinnamic acid decarboxylase mRNA under these conditions. However, in fermentations with no additional hydroxycinnamic acid, the bacteria lowered the final concentration of 4-vinylphenol in the fermented wort compared to the level seen in a pure-yeast fermentation. It seems likely that the combined activities of bacteria and yeast decarboxylate p-coumaric acid and then reduce 4-vinylphenol to 4-ethylphenol more effectively than either microorganism alone in pure cultures. Although we have shown that lactobacilli participate in the metabolism of phenolic compounds during malt whisky fermentations, the net result is a reduction in the concentrations of 4-vinylphenol and 4-vinylguaiacol prior to distillation.

Use of a yeast strain expressing glucoamylase in malt whisky fermentations

Some effects of lactic acid bacteria on laboratory-scale yeast fermentations

Forty‐six presumptive Leuconostoc strains and 13 lactobacilli isolated from malt and grain whisky fermentations together with 14 reference strains of Leuconostoc and Lactobacillus were examined for 99 characters. Data were analysed using a variety of similarity coefficients and clustering algorithms. Two aggregate clusters representing Leuconostoc and Lactobacillus were consistently recovered. Many of the presumptive leuconostocs from whisky fermentations were identified as Leuc. mesenteroides. A second large cluster of strains could not be positively identified. Although recovered within the Leuconostoc aggregate cluster, some of these strains hydrolysed arginine, a characteristic of lactobacilli. The study demonstrates a more diverse flora of lactic acid bacteria existing in the distillery fermentation than had been previously recognized.

Use of a small scale insulated fermentation to simulate the scotch malt whisky fermentation

Effect of temperature and pH on the formation of higher alcohols, fatty acids and esters in the malt whisky fermentation

The effect of inoculum level on the formation of higher alcohols, fatty acids and esters in the malt whisky fermentation

A technique has been developed to produce up to 101 of fresh wort in laboratory conditions for use in small scale whisky fermentations. Neither boiled or sterilised wort can be used for this process. Using this wort experiments have been carried out to investigate the physiology of the malt whisky fermentation. The fermentation was found to have an initial acceleration phase, a linear phase and a decline phase. The level of higher alcohols, fatty acids and esters was measured at different stages of the fermentation using a carbon disulphide extract of the cell free wash to prepare the samples for GLC analysis. The formation of the higher alcohols follows the same pattern as ethanol, as did the acetate esters of higher alcohols. However medium chain fatty acids and their ethyl esters were produced throughout the fermentation.