Isolation and Characterization of Shochu Yeasts with Superior Brewing Ability from Shochu Mashes

Seven yeasts with superior ability in terms of alcohol fermentation and aromatic ingredient generation were isolated from 272 wild yeasts obtained from shochu mashes of shochu breweries. These seven yeasts were examined for their fermentation ability with rice and sweet potato using small scale of fermentation tests. Moreover, their thermotolerance was evaluated by growth tests and fermentation tests with barley koji. Among the isolated seven yeasts, the shochu yeast “MF062” was superior in the characteristics of fast fermentation, high alcohol production, aromatic ingredient generation and thermotolerance. It was named the “Heisei Miyazaki Yeast”. From the results of analyses of physiological and genetic characteristics of MF062, it was shown to be a Saccharomyces cerevisiae, but it showed different characteristics from the industrial yeasts used in shochu production. Using a sweet potato shochu brewing test, with 60 kg of raw materials and yeast MF062, it was confirmed that a high alcohol production yield and a high quality shochu could be obtained.     

STRAINS OF YEAST LETHAL TO BREWERY YEASTS

Strains of yeast that are lethal to brewery ale and lager yeasts have been isolated from production-scale two-stage stirred continuous fermentors. These strains release a “killer” factor which is highly active in the pH range 3.8–4.2. When the level of infection reaches 2% the concentration of killer factor is sufficient to give a selective advantage in continuous fermentation, whereupon the proportion of killer yeasts rises and the brewery yeast is rapidly killed. The beer acquires a characteristic off-flavour which has been described as “herbal/phenolic”. Both flocculent and non-flocculent killer strains have been found and these show the characteristics of Saccharomyces cerevisiae but appear to ferment additional wort sugar(s), have an abormally small cell-size and are pleomorphic in mixed culture.     

CAPRYLIC FLAVOUR AS A FEATURE OF BEER FLAVOUR

Caprylic flavour is part of the distinctive overall flavour of a large proportion of beers in trade. The flavour occurs in the majority of lager beers and in about 20% of ales. It is correlated with the levels of octanoic acid and decanoic acid present in beer. Lager yeasts (S. uvarum) tend to liberate larger amounts of these two fatty acids during fermentation than do ale yeasts (S. cerevisiae). The flavour significance of these acids has been determined by correlating the results of sensory and instrumental analyses and also by adding the purified acids to beer. Implications of these two methods for assessing the flavour significance of chemical constituents of a complex product are discussed.     

SOME QUANTITATIVE ASPECTS OF THE FERMENTATION OF MALTOTRIOSE BY YEASTS

The comparative fermentation velocities with glucose and maltotriose of a series of different strains of top ale yeast show marked variation in the ratio of this function. There is a tendency for more flocculent yeasts to show a relatively lower maltotriose fermentation rate, although this is not absolute. The maltotriose fermentation rate is influenced by the nitrogen content of the cell in a similar manner to that demonstrated by Thorne (this Journal 1954, 227) for glucose. Certain factors affecting the rate of maltotriose fermentation have been investigated. Media and conditions have been elaborated to yield a reproducible determination of the maltotriose fermenation efficiency and this characteristic strain parameter has been shown to be of value in strain evaluation.     

PRODUCTION OF DIACETYL, 2-ACETOLACTATE AND ACETOIN BY YEASTS DURING FERMENTATION

Results are given for the production of diacetyl, 2,3-pentandione- I-acetolactate, 2-acetohydroxy butyrate, acetoin and 3-hydroxy-2-pentanone for 13 yeasts belonging to the genus Saccharomyces. The data presented indicate that in general the yeasts do not produce significant amounts of vicinal diketones during unstirred laboratory fermentations. The observed maximum levels of diacetyl ranged from 0·02–0·32 ppm averaging 0·09 ppm (0·07 ppm if S. carlsbergensis (strain H) is omitted). During active fermentation all the yeasts tested produced 2-acetohydroxy acids. The 2-acetohydroxy acids and the 3-hydroxy-2-ketones are determined gas chromatographically by stepwise conversion of the four compounds to diacetyl and 2,3-pentandione. The maximum amounts of 2-acetolactate produced by the yeasts tested averaged 0·7 ppm, ranging from 0·08–1·65 ppm.     

GROWTH OF AEROBIC WILD YEASTS

Wild yeasts of the genera Debaryomyces, Hansenula and Pichia are commonly considered to be associated with spoilage only under aerobic conditions. However, in pure cultures in either wort or a synthetic medium of yeast nitrogen base + 10% glucose, yeasts of these genera grew as well as a brewing strain of Saccharomyces cerevisiae under anaerobic conditions. Growth of S. cerevisiae was increased by the addition of unsaturated fatty acid (Tween 80) or ergosterol to the medium for anaerobic culture. No equivalent requirement was observed for the wild yeasts examined. Indeed, growth of the wild yeasts was often reduced by the addition of Tween 80, which as a surfactant prevented formation of the surface film of growth. Even under anaerobic conditions, these yeasts grew best with a surface pellicle. Although capable of good anaerobic growth in pure culture, growth of the wild yeasts was suppressed under anaerobic conditions in mixed culture with S. cerevisiae, simulating a contaminated brewery fermentation. However, the contaminants competed successfully with S. cerevisiae under aerobic conditions. There was no evidence of a “killer” effect, but prevention of pellicle formation, or production of inhibitory levels of pH or ethanol under anaerobic conditions could explain the suppression of wild yeasts under anaerobic fermentation conditions.     

Selection and modification of yeasts and lactic acid bacteria for wine fermentation

Historically, the fermentation of grape juice to wine has been carried out by indigenous yeasts found on the berry. However, in newer wine regions, e.g. the USA, inoculation with selected wine yeast strains is employed. Grape juice is high in nutritional factors and difficulties in fermentation usually arise from the inhibitory effects of the high concentration of sugar initially present and the ethanol produced. A secondary fermentation, brought about by indigenous or added lactic acid bacteria, converts malic acid to lactic acid and carbon dioxide and often occurs. This ‘malolactic’ fermentation is usually slow. For both yeast and bacterial fermentations strain selection is based more on fermentation performance than on sensory characteristics of the wine, with increased tolerance of the yeast to ethanol and of the bacteria to low pH being emphasized. Attempts to increase the malolactic fermentation rate have been made by cloning and transferring the malolactic gene from Lactobacillus to wine yeast. In early attempts to produce wines with enhanced or novel sensory characteristics a leucine-less mutant of a homothallic wine yeast has been obtained which does not produce isoamyl alcohol.     

Influence of different yeasts on the growth of lactic acid bacteria in wine

The influence of various yeasts on the growth of lactic acid bacteria in wine was tested by inoculating Lactobacillus hilgardii, L. brevis and two strains of Leuconostoc mesenteroides into experimental wines made with twelve different yeasts of the genus Saccharomyces. Wines made from juice which had been infected with several spoilage yeasts and then fermented with a wine yeast were also tested in this way. It was found that the yeasts differed considerably in their effects on bacterial growth. In some of the experimental wines bacterial growth was delayed or failed altogether. Generally, the unfavourable influence of any yeast on bacterial growth was much reduced if the wines were left in contact with the yeast cells for some weeks after the fermentation. The significance of these results in relation to the occurrence of malo-lactic fermentation in commercial wineries is discussed.     

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     

Contribution of wild yeasts to the formation of volatile compounds in inoculated wine fermentations

The aim of this work was to study the contribution of wild yeasts to the volatile composition of wine in inoculated fermentations. To do so, Parellada must, sterilized and inoculated with Saccharomyces cerevisiae strain Na33 (pure inoculated fermentation), inoculated Parellada must (mixed inoculated fermentation) and Parellada must that fermented with its wild yeasts (control fermentation) were used. From the results obtained in the pure inoculated fermentation it can be seen that S. cerevisiae produced appreciable quantities of isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate. However, the wild yeasts also contributed to the synthesis of esters since the total concentration of these substances was higher in the mixed inoculated fermentation than in the pure inoculated fermentation. 2-Phenylethyl acetate was only synthesized by wild yeasts when they did not compete with S. cerevisiae. The concentration of total alcohols was similar in the three samples; the important production of isobutanol and 2-phenylethanol in the control fermentation is noteworthy. As regards the acids, the greatest concentration corresponded to the mixed inoculated fermentation. The wild yeasts contributed to the synthesis of these compounds to a significant extent and S. cerevisiae synthesized appreciable amounts of short-chain fatty acids.     

Characteristics of scalded dough fermented by co-cultures of Saccharomyces cerevisiae Y10, Wickerhamomyces anomalus Y13 and Torulaspora delbrueckii Y22

Scalding process could change properties of dough. Co-culture of yeasts could improve the quality of product. In this study, characteristics of scalded dough with co-cultures of Saccharomyces cerevisiae Y10, Wickerhamomyces anomalus Y13 and Torulaspora delbrueckii Y22 were investigated. Most starch granules lost shape and crystal pattern after scalding, meanwhile, gluten network had become weak, suggesting the gelatinisation of starch and damage of gluten protein. After fermentation, Y13 and Y22 cell populations increased rapidly. However, the cell number of Y10 decreased from 6.91 Lg CFU per gram dough to 5.42 Lg CFU per gram dough. Gluten protein aggregated into chunks after 8 h of fermentation, meanwhile, the water molecules in dough were bound more tightly. These results indicated that scalded dough provide a unique environment for the growth of yeasts, and further affect the characteristics of dough during fermentation.     

Inferring the role of microorganisms in water kefir fermentations

Water kefir is a slightly alcoholic, lactic and acetic beverage fermented by yeasts, lactic acid bacteria and acetic acid bacteria that are associated with the polysaccharide of the water kefir grains. In this study, the three main metabolic products of microorganisms were evaluated during a traditional 192‐h water kefir fermentation and also after inoculating the microorganisms in fresh medium or sterilised broth from different fermentation stages. The first process to occur was alcoholic fermentation, carried out in particular by Saccharomyces cerevisiae. After 24 h, lactic and acetic acid accumulation was generated by Lactobacillus hilgardii and Acetobacter tropicalis. By the end of fermentation, ethanol had been almost entirely consumed and oxidised to acetic acid, possibly by a dissimilatory route of Acetobacter species. An original hypothetical diagram is proposed for the carbon flux from sucrose, and the metabolic role of the main yeasts and bacteria is assigned for the distinct stages of water kefir fermentation.     

APPLICATION OF SEROLOGICAL METHODS TO THE EXAMINATION OF YEAST VARIATION AND BEHAVIOUR

The technique of micro immuno-electrophoresis has been used to study variation within single yeast strains. Investigation of brewing yeasts and their variants demonstrated that a change of a fermentation property was accompanied by a change in the antigenic structure. Small variations in fermentation characteristics, as well as a larger changes such as respiratory deficiency, can be detected by this serological method.     

CONTROLLED PRODUCTION OF CIDER BY INDUCTION OF ALCOHOLIC FERMENTATION AND MALOLACTIC CONVERSION

Characterization experiments involving lactic bacteria allowed a strain of Leuconostoc oenos to be selected in terms of growth capacity at variable pH, temperature, ethanol and sulphite concentrations, malic to lactic conversion yield, acetic acid uptake and dextran production capacity. Cider was produced under controlled conditions where the effect of Kloeckera apiculata yeasts on the development of Saccharomyces cerevisiae and production of major non-volatile compounds influencing end product quality was studied. The apiculate yeast was found to produce large amounts of acetic acid and use the other organic acids; also, it hindered fermentation to a certain extent. A study of the effect of the inoculation time with L oenos as an inducer of malolactic fermentation revealed sequential inoculation of the lactic bacterium once most major sugars in the must had been depleted to be the most favourable. Using yeast cell walls boosted fermentation development, as well as degradation of malic acid and synthesis of succinic and acetic acid.     

THE EFFECT OF YEAST TREHALOSE CONTENT AT PITCHING ON FERMENTATION PERFORMANCE DURING BREWING FERMENTATIONS

The effect of yeast trehalose content at pitching on the fermentation performance during brewing fermentations was studied using a commercial strain of lager yeast, Saccharomyces cerevisiae (AJL 2155). Pitching yeasts with different trehalose contents were obtained by collecting cells in suspension after 96 h and 144 h of fermentation in EBC tubes in 10.8°P brewers wort at 14°C. The trehalose content of the pitching yeast had no effect on growth, specific gravity and ethanol production during the subsequent fermentation. A high trehalose content of the pitching yeast, however, sustained cell viability during the initial stage of fermentation, increased the carbohydrate utilisation rate and increased the production of isoamyl alcohol and isobutanol. For these aspects of fermentation performance, the trehalose content of the pitching yeast may prove useful in evaluating the vitality of pitching yeasts within the brewery .     

Resistance to aerobic deterioration of total mixed ration silage inoculated with and without homofermentative or heterofermentative lactic acid bacteria

Wet brewers grains were stored as a total mixed ration (TMR) in laboratory silos with lucerne hay, cracked maize, sugar beet pulp, soya bean meal and molasses at 5:1:1:1:1:1 on fresh weight basis. The TMR mixture was inoculated with or without Lactobacillus casei or Lactobacillus buchneri to obtain silages with differing fermentation and stability after exposure to air. In the first experiment, ensiling was stopped at 10, 20 and 60 days, and the stability was tested for the following 7 days. Ethanol and lactic acid were the main products in untreated TMR silage, while addition of L. casei and L. buchneri increased lactic and acetic acid, respectively. No silages deteriorated in the presence of air over 7 days, regardless of inoculation, ensiling period and the level of yeasts determined at unloading. In the second experiment, silos were opened at 14 days and then subjected to aerobic stability test for 14 days. Resistance to deterioration was sustained in the untreated control, even with a high population (>10⁴ cfu g^?1) of yeasts throughout the 14‐day test. Spoilage was found in L. casei‐treated silage at about 5 days, while increase of yeasts preceded the distinct heating (degradation). In L. buchneri‐treated silage, no yeasts were detected at unloading or after exposure to air. These results suggest that substantial stability can be expected in TMR silage with or without inoculation of lactic acid bacteria. This property is not associated with the counts of yeasts at loading and the characteristics of silage such as alcoholic and lactic acid fermentation. Copyright © 2007 Society of Chemical Industry     

Characteristics of aromatic compound production using new shochu yeast MF062 isolated from shochu mash

The effect of temperature on the production of aromatic compounds using Heisei Miyazaki yeast MF062 was compared between 10 industrial yeasts. All yeasts tested produced characteristic patterns of alcohols and esters in fermentation tests with rice‐koji at 20, 28 and 38°C. The concentration and composition in mature moromi with rice‐koji at 20, 28 and 38°C were almost the same as those with barley‐koji. Therefore, it was suggested that fermentation temperature is an important factor in the production of aromatic compounds. MF062 produced almost the same concentration of β‐phenethyl alcohol at both 38 and 28°C. The concentration was higher than that generated by the other 10 yeasts. MF062 produced higher concentrations of i‐butyl alcohol than the other yeasts at higher fermentation temperatures. Moreover, compared with the other yeasts, MF062 produced a lower concentration of acetate, which can give an off‐flavour in excess concentrations in shochu. The production of acetoin was divided into two groups – a high producing group and a low producing group – at all temperatures. MF062 belonged to the latter group and showed preferred characteristics in the production of shochu, resulting in a high concentration of preferred aromatic compounds and a low concentration of compounds that impart an off‐flavour. Copyright © 2013 The Institute of Brewing & Distilling     

FATTY ACIDS AND ESTERS PRODUCED DURING THE SPONTANEOUS FERMENTATION OF LAMBIC AND GUEUZE

Lambic and gueuze are Belgian beers obtained by spontaneous fermentation of wort. During previous studies it was found that they result from the successive development of enterobacteria, Kloeckera and Saccharomyces yeasts, bacteria of the genus Pediococcus, and Brettanomyces yeasts. The beers are characterized by high concentrations of acetic and lactic acid, ethyl acetate and ethyl lactate. This study of the content of the higher fatty acids during a 20 month fermentation period confirms the succession of the different micro-organisms. Pure cultures of isolated yeasts and bacteria produced fatty acids which were also found in the fermenting wort at periods when these organisms were active. Lambic and Gueuze are especially rich in caprylic (C₈) and capric (C₁₀) acids. These are probably produced by Saccharomyces and Brettanomyces. Important amounts of ethyl caprylate and ethyl caprate were also found. As ethyl caprate is almost absent in other beers, it might be considered as another typical aroma component of lambic and gueuze.     

Mixed Culture Fermentation of Cucumber Juice with Lactobacillus plantarum and Yeasts

Saccharomyces cerevisiae and Saccharomyces rosei were tested for use in mixed culture fermentation of cucumber juice with Lactobacillus plantarum. Extent of sugar utilization and the ratio of products formed (lactic acid:ethanol) were influenced by time of inoculation and cell numbers of each microorganism. Sugar fermentation was complete within 6 days at 28°C when similar numbers of bacteria and yeasts were added simultaneously, or when yeasts were added before the bacteria. Sugar remained when only L. plantarum was added, or when yeasts were added in low numbers. Glycerol was produced when yeasts were present, the concentration being directly related to NaCl concentration. Results suggest advantageous uses of yeasts to complete fermentation and to modify acidity.     

Preservation of non-Saccharomyces yeasts: Current technologies and challenges

There is a recent and growing interest in the study and application of non-Saccharomyces yeasts, mainly in fermented foods. Numerous publications and patents show the importance of these yeasts. However, a fundamental issue in studying and applying them is to ensure an appropriate preservation scheme that allows to the non-Saccharomyces yeasts conserve their characteristics and fermentative capabilities by long periods of time. The main objective of this review is to present and analyze the techniques available to preserve these yeasts (by conventional and non-conventional methods), in small or large quantities for laboratory or industrial applications, respectively. Wine fermentation is one of the few industrial applications of non-Saccharomyces yeasts, but the preservation stage has been a major obstacle to achieve a wider application of these yeasts. This review considers the preservation techniques, and clearly defines parameters such as culturability, viability, vitality and robustness. Several conservation strategies published in research articles as well as patents are analyzed, and the advantages and disadvantages of each technique used are discussed. Another important issue during conservation processes is the stress to which yeasts are subjected at the time of preservation (mainly oxidative stress). There is little published information on the subject for non-Saccharomyces yeast, but it is a fundamental point to consider when designing a preservation strategy.