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

The levels of higher alcohols, fatty acids and esters formed in a small-scale whisky fermentation which had been inoculated with different amounts of yeast was investigated. The total level of higher alcohols increased with increasing inoculum levels. However, the relative levels of propanol, isobutanol, amyl alcohols and 2-phenyl ethanol were unaffected. The levels of octanoic, decanoic and dodecanoic acids and their ethyl esters were depressed at inoculum levels above 2 × 10⁷ cells ml^?1. Varying the inoculum levels did not have a consistent effect on the acetate esters of the higher alcohols. However, the highest values were obtained at inoculum levels 4 × 10⁷ cells ml^?1.     

THE INFLUENCE OF PANTOTHENATE CONCENTRATION AND INOCULUM SIZE ON THE FERMENTATION OF A DEFINED MEDIUM BY SACCHAROMYCES CEREVISIAE

Laboratory fermentations were carried out using defined media containing from 0.02 to 0.40 mg pantothenate 1^?1 and a brewing strain of Saccharomyces cerevisiae at inocula of 1 × 10⁶ to 1 × 10⁷ cells ml^?1, Cell size, increase in cell number, increase in yeast weight, production of ethanol. n-propanol, isobutanol, isoamyl alcohols, acetaldehyde and ethyl acetate were measured. All the properties were influenced to some extent by pantothenate concentration and/or inoculum size but no general correlations were found. At high inoculum levels variation in the pantothenate concentration above 0.1 mg 1^?1 did not have a major effect on the total quantity of volatile compounds produced but did alter the relative proportions of the individual compounds.     

Populations and potential association of Saccharomyces cerevisiae with lactic acid bacteria in naturally fermented Korean rice wine

The microbial populations focused on predominant yeast species and lactic acid bacteria (LAB) in 15 commercial makgeolli brands, where a fungal starter nuruk was used were examined. Viable yeast counts were obtained on yeast potato dextrose (YPD) and MRS agar containing sodium azide. MRS-C (0.1% cycloheximide supplemented) was used for selective counts of LAB. Saccharomyces cerevisiae was found to be predominant in the 15 samples tested, with an average count of 4.6×10⁷ CFU/mL. Contrary to the earlier studies, Lactobacillus plantarum and Weissella cibaria were shown as predominant LAB species with an average count of 1.7×10⁷ CFU/mL. Surprisingly, as many as 7 log viable cells/mL were present at the ethyl alcohol concentration of 6–7%. The data from real-time PCR also indicated that the yeast populations remains almost constant during the refrigerated storage of 12 days, while that of LAB decrease slightly first 9 days and increase after then, despite the overall increase in acidity. Data from the differential microbial counts suggest that yeast S. cerevisiae might be associated with 2 LAB species, L. plantarum and W. cibaria, under ethyl alcohol stress during the turbid rice wine fermentation.     

SEMICONTINUOUS ETHANOL FERMENTATION WITH SHOCHU DISTILLERY WASTE*

An effective utilization system using distillery waste discharged from Japanese traditional shochu factory was developed. Mugi (barley) shochu distillery waste discharged from a novel vacuum distillation procedure (35–40°C) contained a large number of viable yeast (7 × 10⁶ cells/ml), glucoamylase activity (19.7 units/ml), acid protease activity (940 units/ml), and neutral protease activity (420 units/ml). Ethanol fermentation was achieved with a mash composed of glucose as the sola carbon source and mugi shochu distillery waste. After ethanol fermentation was completed the fermented broth was again distilled at 35–40°C in vacuo and the non volatile residue used in the next ethanol fermentation. In this way, semicontinuous ethanol fermentation system of more than 10 cycles was developed. Even in the distillate of the mash of the 8th fermentation cycle, 7.9% of ethanol, 33.0 ppm of ethyl acetate, 28.5 ppm of isobutyl alcohol, and other aromatic compounds were present. A semicontinuous ethanol fermentation system has been developed for shochu distillery waste which conventionally is treated as wastewater.     

The effect of pitching rate on fermentation,maturation and flavour compounds of beer produced on an industrial scale

The aim of the study was to determine the effect of the initial number of yeast cells in the wort on the process of fermentation, maturation and the content of the volatile components of beer, as well as the viability and vitality of the yeast biomass. The experiments were performed on an industrial scale, with fermentation and maturation in cylindro‐conical fermentation tanks with a capacity of 3800 hL. Yeast for pitching was collected after secondary fermentation (third passage) and wort pitching levels were 5 × 10⁶, 7 × 10⁶ and 9 × 10⁶ cells/mL. During fermentation and maturation, the changes in the content of the extract, yeast growth, yeast vitality and selected volatile components were investigated. Experiments showed that the yeast inoculum had a significant impact on the course of the fermentation and metabolic changes. With increasing numbers of cells introduced into the wort, the content of the esters and fusel alcohols increased, while the acetaldehyde concentration decreased. These changes affected the final quality of the beer. Copyright © 2015 The Institute of Brewing & Distilling     

SUPPRESSION OF α-ACETOLACTATE FORMATION IN BREWING WITH IMMOBILIZED YEAST

Immobilized yeast cells extensively produced the diacetyl precursor, α-acetolactate, during alcohol fermentation. The activity of acetohydroxy acid synthetase, which is responsible for the formation of α-acetolactate from pyruvic acid, was high in cell-free extracts of immobilized yeast cells compared with that of free yeast cells. It was suggested that the expression of AHA synthase of immobilized yeast cells was increased during growth in the carrier as compared with free yeast cells. When the initial immobilizing yeast cell concentration was changed from 1.0 × 10⁶ cells/ml to 1.0 × 10⁹ cells/ml, production of α-acetolactate was reduced from 0.94 mg/l to 0.30 mg/l. Furthermore, during continuous fermentation for 10 d, the concentration of α-acetolactate in beer was 0.30 mg/l.     

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).     

Modulation of grape wine flavor via the sequential inoculation of Williopsis saturnus and Saccharomyces cerevisiae

The effects of the inoculum ratio of Williopsis saturnus var. saturnus NCYC22 and Saccharomyces cerevisiae var. bayanus EC-1118 at 1:200 and 1:800 on the chemical and volatile compositions of grape wine were studied in sequential fermentation. The grape juice was first inoculated with Williopsis (W.) saturnus for 9 d; thereafter, Saccharomyces (S.) cerevisiae was inoculated to continue the fermentation until d 19. The cell population of W. saturnus disappeared by d 13, with S. cerevisiae dominating until the end of the fermentation in both inoculum ratios. The changes in yeast count, pH, total soluble solids, sugars, organic acids, and amino acids were similar between the two inoculum ratios. A range of volatile compounds was formed, including alcohols, esters, fatty acids, aldehydes, and terpenes. There were significant differences between both inoculum ratios for medium-chain fatty acids (C8, C10, and C12), ethyl esters of fatty acids of C6, C10, C12, and C14 as well as isoamyl octanoate, while other volatiles were statistically the same.     

Low‐temperature wine‐making using yeast immobilized on pear pieces

A biocatalyst was prepared by the immobilization of Saccharomyces cerevisiae AXAZ‐1 yeast cells on pear pieces and tested for grape must fermentation in both batch and continuous conditions. The immobilized yeast cells were stable and active even at low temperatures (<10 °C). Wine production under batch fermentation at 8 °C was completed within 15 days while at 3 °C it took 36 days. In continuous fermentation, the bioreactor was operated for 33 days, then stored for 12 days at 10 °C, and re‐run for another 15 days without any diminution of the ethanol productivity. Total acidity of the produced wines remained within the ranges usually observed in dry wines, while volatile acidity was found in rather increased levels. The concentrations of higher alcohols (1‐propanol, isobutyl alcohol and amyl alcohols) were relatively low, while ethyl acetate was detected at up to 118 mg l^?1, contributing to the fruity aroma of the wines produced. Preliminary sensory evaluations carried out in the laboratory indicated the fine quality of the produced wines. Copyright © 2004 Society of Chemical Industry     

Maximization of Fructooligosaccharides and β-Fructofuranosidase Production by Aspergillus japonicus under Solid-State Fermentation Conditions

The conditions of temperature, moisture content, and inoculum rate able to maximize the production of fructooligosaccharides (FOS) and β-fructofuranosidase (FFase) enzyme by solid-state fermentation were established. Fermentation assays were performed using the support material (coffee silverskin) moistened to 60, 70, or 80 % with a 240-g/l sucrose solution and inoculated with a spore suspension of Aspergillus japonicus to obtain 2?×?10⁵, 2?×?10⁶, or 2?×?10⁷ spores/gram dry material. The fermentation runs were maintained under static conditions at 26, 30, or 34 °C during 20 h. The moisture content did not influence the FOS and FFase production; however, temperature between 26 and 30 °C and inoculum rate of approximately 2?×?10⁷ spores/gram dry material maximized the results (FOS?=?208.8 g/l with productivity of 10.44 g/l h; FFase?=?64.12 units U/ml with productivity of 4.0 U/ml h). These results are considerably higher than those obtained under no optimized fermentation conditions and represent an important contribution for the establishment of a new industrial process for FOS and FFase production.     

CONTENTS OF CYTOCHROMES IN YEAST

The contents of cytochromes in yeast were determined quantitatively from the absorption spectra, using a solid cell paste of intact yeast. During the industrial production of baker's yeast, the contents of the cytochromes, particularly of cytochrome aa₃ at successive stages, increased gradually with increasing aeration. In semi-aerobically grown baker's yeast, the contents of cytochromes aa₃, b and c were 0·9, 2·9 and 2·9 × 10^?5 moles/litre of fresh yeast (total amount 6·7 × 10^?5 moles/litre), while in vigorously aerated commercial baker's yeast the respective values were 2·3, 4·8 and 5·2 × 10^?5 moles/litre (total amount 12·3 × 10^?5 moles/litre). In brewer's yeasts separated after the brewing process, the contents of cytochromes were markedly lower than in baker's yeast grown with limited aeration, whereas in top-fermenting yeast the total cytochrome content, aa₃ + b + c, was in some samples markedly higher, 7·1 × 10^?5 moles/litre, than in bottom-fermenting brewer's yeast, 2·4 × 10^?5 moles/litre. When brewer's bottom yeast was grown on a laboratory scale under increasing aeration, a maximum appeared in the cytochrome contents when aeration was moderate, and increased aeration inhibited the formation of cytochromes. The cytochrome contents in brewer's bottom yeast may exceed the amounts found in commercial baker's yeast. In addition to aeration, the type of metabolism influences the amounts of cytochromes in yeast.     

Degradation of 5-hydroxymethylfurfural during yeast fermentation

5-Hydroxymethyl furfural (HMF) may occur in malt in high quantities depending on roasting conditions. However, the HMF content of different types of beers is relatively low, indicating its potential for degradation during fermentation. This study investigates the degradation kinetics of HMF in wort during fermentation by Saccharomyces cerevisiae. The results indicated that HMF decreased exponentially as fermentation progressed. The first-order degradation rate of HMF was 0.693?×?10^?2 and 1.397?×?10^–2?min^?1 for wort and sweet wort, respectively, indicating that sugar enhances the activity of yeasts. In wort, HMF was converted into hydroxymethyl furfuryl alcohol by yeasts with a high yield (79–84% conversion). Glucose and fructose were utilised more rapidly by the yeasts in dark roasted malt than in pale malt (p?<?0.05). The conversion of HMF into hydroxymethyl furfuryl alcohol seems to be a primary activity of yeast cells, and presence of sugars in the fermentation medium increases this activity.     

Saccharomyces cerevisiae Biodiversity During the Brewing Process of an Artisanal Beer: A Preliminary Study

In this study we investigated the biodiversity of Saccharomyces cerevisiae during the brewing of an artisanal beer, as well as during its storage in the bottle for 107 days at 20°C. After inoculation with an active dried yeast (ADY), the yeast counts were followed during fermentation and after bottling. Yeast loads remained stable at 10⁶–10⁷ colony forming units (cfu)/mL, and only after day 21, were they were reduced to 10⁴ cfu/mL. After three months in the bottle they spanned 10²–10⁵ cfu/mL. Almost all isolated yeasts were identified as S. cerevisiae and after molecular characterization, unexpected results were obtained. The ADY did not take over the fermentation process and only after 21 days did isolates from the beer share similarities with the inoculated strain. During storage, a high diversity was found, underlining that each bottle developed its own micro‐ecosystem. This study highlighted the necessity for better investigations of S. cerevisiae population dynamics during artisanal brewing. Even when the chemical parameters measured confirmed a correct fermentation process, the inoculated strain was not the main yeast involved in the fermentation and consequently, the final product may have different sensory characteristics from the ones expected by the producers.     

Fermentation Characteristics and Aromatic Profile of Plum Wines Produced with Indigenous Microbiota and Pure Cultures of Selected Yeast

The aim of this study was to assess and compare fermentation characteristics and aromatic profile of plum wines produced with indigenous microbiota and pure cultures of different selected yeast. Experiments were carried out with plum (Prunus domestica L.) varieties of different fruit ripening times (?a?anska rana, ?a?anska lepotica, and Po?ega?a). Wine fermentations were conducted by the activity of indigenous microbiota, commercially available Saccharomyces cerevisiae and Saccharomyces bayanus yeast strains and joint activity of Schizosaccharomyces pombe and S. cerevisiae (sequential inoculation). Statistically significant differences in fermentative characteristics and the content of certain volatile compounds were observed as a result of metabolic activity of various indigenous and/or selected yeasts during fermentation of plum pomace. Minimal duration of fermentation (4 to 5 d) and fastest ethanol production rate (from 12.3 to 15.5 g/L/d) were the characteristics of the studied S. cerevisiae strains. Isobutanol, 3‐methyl‐1‐butanol, 1‐heptanol, and 1‐octanol were the most prevalent higher alcohols in the tested plum wine samples. The predominant ester in plum wines was ethyl acetate, ethyl lactate, amyl acetate, isoamyl acetate, and ethyl palmitate, esters responsible for the floral and fruity olfactory tones, were also present in large amounts. Also, the use of S. cerevisiae strains resulted in the production of plum wines with better sensory characteristics than ones produced with other investigated yeasts. Obtained results are significant since there is limited data on the compounds responsible for the unique flavor of plum wine, as well as on the impact of different yeast starter cultures application on the overall quality of fruit wines.     

Production and Characterization of Wine with Sugarcane Piece Immobilized Yeast Biocatalyst

In the present study, an economically cheap and sustainable food-grade yeast biocatalyst for wine production was successfully prepared. It was prepared through absorption technique by using fresh sugarcane (Saccharum officinarum L.) pieces as immobilizing support for yeast, Saccharomyces cerevisiae CFTRI 101. Immobilization was confirmed by scanning electron microscopy. Suitability of biocatalyst was investigated at low and room temperatures by using it in repeated batch. The fermentation rate and other parameters were compared with free yeast cells at different temperatures. In all cases, fermentation time was short (24 h at 30 °C and 98 h at 10 °C) and ethanol productivities were high as 4.2 g/l/h (3.33-fold at 30 °C and 2.31-fold at 10 °C). The volatile compounds methanol, ethyl acetate, propanol-1, isobutanol (2-methyl-1-propanol), and amyl alcohols (2-methyl-1-butanol and 3-methyl-1-butanol) that formed during fermentation were analyzed with the help of a gas chromatograph–flame ionization detector. The concentrations of ethyl acetate and methanol were not more than 100 mg/l in all cases, indicating an improvement in the product quality. Cell metabolism of immobilized yeast was not negatively affected by immobilization process. It was found that the immobilization of yeast cells on sugar pieces increased the fermentation rate and viability of yeast cells. Preliminary sensory tests recognized the fruity aroma, fine taste, and the overall improved quality of the produced wines.     

High-cell-density fermentation of Saccharomyces cerevisiae for the optimisation of mead production

Mead is a traditional drink that contains 8%–18% (v/v) of ethanol, resulting from the alcoholic fermentation of diluted honey by yeasts. Mead fermentation is a time-consuming process and the quality of the final product is highly variable. Therefore, the present investigation had two main objectives: first, to determine the adequate inoculum size of two commercial wine-making strains of Saccharomyces cerevisiae for the optimisation of mead fermentation; and second, to determine if an increase in yeast pitching rates in batch fermentations altered the resulting aroma profiles. Minor differences were detected in the growth kinetics between the two strains at the lowest pitching rate. With increasing pitching rates net growth of the strain ICV D47 progressively decreased, whereas for the QA23 the increasing inoculum size had no influence on its net growth. The time required to reach the same stage of fermentation ranged from 24 to 96 h depending on the inoculum size. The final aroma composition was dependent on the yeast strain and inoculum size. Fourteen of the twenty-seven volatile compounds quantified could contribute to mead aroma and flavour because their concentrations rose above their respective thresholds. The formation of these compounds was particularly pronounced at low pitching rates, except in mead fermented by strain ICV D47, at 10⁶ CFUs/mL. The esters isoamyl acetate, ethyl octanoate and ethyl hexanoate were the major powerful odourants found in the meads. The results obtained in this study demonstrate that yeast strain and inoculum size can favourably impact mead's flavour and aroma profiles.     

THE INFLUENCE OF A COLIFORM BACTERIUM ON FERMENTATION BY YEAST

Hopped wort (20°C) was inoculated with brewing yeast alone (10⁷ cells/ml), or with Citrobacter freundii (10⁵ cells/ml) or with both organisms at the concentrations stated. The mixture grew somewhat more quickly than the pure yeast culture, the oxidation-reduction potential and S.G. fell more quickly but the pH less rapidly. The mixed culture produced more ethanol and various organic acids but less volatile carbon. Dimethyl sulphide was produced in the mixed culture but not in the pure yeast culture. It is concluded that C. freundii can have a profound influence on yeast fermentation, consistent with its pure culture characteristics.     

Microbial and chemical analysis of a kvass fermentation

Kvass is a fermented cereal beverage which produced from malt, rye flour, stale rye bread, and sucrose and is consumed mainly in Eastern Europe. Moreover, it is of interest as one of the few examples of traditional, non-alcoholic cereal-based beverages. In this study, a commercial kvass sample was characterized with respect to the fermentation microflora and the concentration of microbial metabolites. Lactobacillus casei and Leuconostoc mesenteroides were present in cell counts of 7.3 × 10⁷ and 6.0 × 10⁷ cfu mL⁻¹, respectively. Saccharomyces cerevisiae was present in cell counts of 3.0 × 10⁷ cfu mL⁻¹. PCR-DGGE analysis verified that all dominant fermentation organisms were cultivated. Microbial metabolites in kvass were ethanol, lactate, and acetate. One of the kvass isolates, Ln. mesenteroides FUA 3086 harboured a putative dextransucrase gene and formed dextran and isomalto-oligosaccharides from sucrose and maltose. Fermentation of model kvass wort confirmed that all kvass isolates grew in the fermentation substrate, moreover, formation of isomaltotriose by Ln. mesenteroides FUA 3086 was observed in model kvass fermentation.     

Effect of co-inoculation of Saccharomyces cerevisiae and Lactobacillus fermentum on the quality of the distilled sugar cane beverage cachaça

Abstract: The effect of Lactobacillus fermentum in co‐inoculation with Saccharomyces cerevisiae UFLA CA11 on the quality of cachaça (sugar cane spirit) was evaluated. The co‐inoculation was first evaluated in flask fermentation, and subsequently, yeast and bacteria were co‐cultured at approximately 10⁵ CFU/mL and 10⁸ CFU/mL, respectively, in 4 consecutive batches. L. fermentum did not affect the growth or activity (sugar consumption and fermentation rate) of S. cerevisiae UFLA CA11 during fermentation. The physicochemical analysis revealed a higher concentration (Tukey test) of aldehydes (22.07 mg/100 mL anhydrous alcohol) in cachaça produced by co‐inoculation. Analysis of volatile compounds using GC‐FID demonstrated that cachaça produced by co‐inoculation had higher concentrations of acetaldehyde (25.15 mg/L), ethyl acetate (30.17 mg/L), and 2,3‐butanedione (170.39 μ/L), while cachaça produced by UFLA CA11 contained higher concentrations of ethyl lactate (1205.98 μ/L), propionic acid (127.97 μ/L), butyric acid (2335.57 μ/L), and 1‐pentanol (469.23 μ/L). The lowest concentration of acetic acid measured by HPLC was found in cachaça obtained with UFLA CA11. The sensory analysis, performed using the Mann–Whitney test, revealed that cachaça produced by co‐inoculation differed from that produced by UFLA CA11 in taste and aroma. Practical Application: This study reports on the use of a mixed culture of Saccharomyces cerevisae and Lactobacillus fermentum to produce cachaça and shows the influence of co‐inoculation of yeast and bacteria on the quality of this beverage.