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.     

Influence of pollen addition on mead elaboration: Physicochemical and sensory characteristics

Mead (honey-wine) results from the alcoholic fermentation of diluted honey using a wine yeast strain. However, mead elaboration can be hampered by several problems, including delayed or arrested fermentation, production of an unpleasant aroma, poor quality and inconsistency of the final product. These difficulties are due to honey’s low nutrient content, its natural antifungal components, and the inability of the yeast strain to adapt to these unfavourable growth conditions. In this study, we evaluated the results of adding pollen at concentrations ranging from 10 to 50 g/l as a fermentation activator to improve the fermentation kinetic and the quality of meads. The effect of pollen addition on the honey must, fermentation kinetics, physicochemical characteristics, aroma profiles and sensorial aspects of the obtained meads were evaluated. The results showed that pollen addition improved fermentation rates, alcohol yields, and the final characteristics of meads. An increase in the volatile contents of the meads and an improved sensory profile was observed with pollen addition; however, this improvement was not correlated with the concentration of pollen. The adequate dose of pollen was determined by the final characteristics and sensory profile of the meads.     

Nitrogen addition influences formation of aroma compounds,volatile acidity and ethanol in nitrogen deficient media fermented by Saccharomyces cerevisiae wine strains

The effects of nitrogen addition into nitrogen deficient/depleted media on the release of aroma compounds post-fermentation were investigated in three commercial yeast strains of Saccharomyces cerevisiae which highlight the yeast strain effect as well as nitrogen effects. By comparing the two timings of nitrogen addition, prior to fermentation or later at stationary phase (72 h), it was shown that nitrogen addition at stationary phase significantly decreases ethanol and acetic acid formation and significantly increases the following compounds: 2-phenylethanol, ethyl isobutyrate, 2-phenylethyl acetate, ethyl 2-methylbutyrate and ethyl propionate in the three strains, and also isovaleric acid, isoamyl alcohol and ethyl isovalerate in both PYCC4072 and UCD522. The strain EC1118 produced significantly less medium chain fatty acids, hexanoic, octanoic and decanoic acids and their respective esters after nitrogen addition. Therefore, timing of nitrogen addition to a ferment media can vary the concentration of certain aroma compound and might provide a means for varying wine composition.     

Microencapsulation of curcumin in cells of Saccharomyces cerevisiae

Curcumin was successfully encapsulated in yeast cells of Saccharomyces cerevisiae as confirmed by fluorescence microscopy, Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared Spectroscopy (FT-IR). Emphasis was given on the encapsulation parameters including temperature, plasmolysis of yeast cells, presence of ethanol and mass ratio curcumin:cells that affected the amount of curcumin finally encapsulated, as expressed by the %Encapsulation Yield (%EY) and %Encapsulation Efficiency (%EE). Encapsulation was favoured at temperatures above 35 °C and preparation of microcapsules in water instead of 50% v/v ethanol increased the %EY and %EE values by at least 2-fold. Although plasmolysis of yeast cells modified membrane’s fluidity and cell wall’s composition, the microcapsules prepared with plasmolysed cells did not differ in their curcumin content when compared to those prepared with non-plasmolysed cells. Proper combination of the abovementioned parameters resulted in microcapsules that contained up to 35.8 ± 0.86% w/w curcumin. In all microcapsules prepared curcumin was integrated in the plasma membrane bilayer but also interacted with constituents of the cell wall network.     

Optimization of honey-must preparation and alcoholic fermentation by Saccharomyces cerevisiae for mead production

Mead fermentation is a time-consuming process, often taking several months to complete. Despite of the use of starter cultures several problems still persist such as lack of uniformity of the final products, slow or premature fermentation arrest and the production of off-flavors by yeast. Thus the aim of this study was to optimize mead production through the use of an appropriate honey-must formulation to improve yeast performance alcoholic fermentation and thereby obtain a high quality product. Honey-must was centrifuged to reduce insoluble solids, pasteurized at 65 °C for 10 min, and then subjected to different conditions: nitrogen supplementation and addition of organic acids. Although the addition of diammonium phosphate (DAP) reduced fermentation length, it did not guarantee the completeness of the fermentation process, suggesting that other factors could account for the reduced yeast activity in honey-must fermentations. Sixteen yeast-derived aroma compounds which contribute to the sensorial quality of mead were identified and quantified. Global analysis of aromatic profiles revealed that the total concentration of aroma compounds in meads was higher in those fermentations where DAP was added. A positive correlation between nitrogen availability and the levels of ethyl and acetate esters, associated to the fruity character of fermented beverages, was observed whereas the presence of potassium tartrate and malic acid decreased, in general, their concentration.This study provides very useful information that can be used for improving mead quality.     

Redox effect on volatile compound formation in wine during fermentation by Saccharomyces cerevisiae

Although redox state is a well-known key process parameter in microbial activity, its impact on wine volatile aroma compounds produced during fermentation has not been studied in detail. In this study we report the effect of reductive and microaerobic conditions on wine aroma compound production using different initial amounts of yeast assimilable nitrogen (YAN: 180 and 400 mg N/l) in a simil grape must defined medium and two S. cerevisiae strains commonly used in wine-making. In batch fermentation culture conditions, reductive conditions were obtained using flasks plugged with Muller valves filled with sulphuric acid; while microaerobic conditions were attained with defined cotton plugs. It was found that significant differences in redox potential were obtained using the different plugs, and with variation of over 100 mV during the main fermentation period.     

Acetic acid removal by Saccharomyces cerevisiae during fermentation in oenological conditions. Metabolic consequences

The commercial Saccharomyces cerevisiae strains used in champagne winemaking were tested for their ability to metabolise acetic acid during alcoholic fermentation. Fermentation tests were performed in conditions close to oenological ones using a Chardonnay grape juice supplemented with acetic acid. The amount of acetic acid metabolised by wine yeast increased with increasing initial acetic acid concentration and this elimination occurred during the second part of the exponential growth phase. When the initial acetic acid concentration exceeds 1 g/l, and whatever the yeast strain used, the concentration of acetic acid in the resulting wine cannot be reduced to an acceptable level according to the current legislation. Acetic acid removal modified yeast metabolism, since more acetaldehyde, less glycerol and less succinic acid were produced. Considering the reduction of the NADPH/NADP⁺ ratio following acetic acid consumption, we propose, as a new hypothesis, that acetic acid could modify yeast metabolism by reducing the activity of the NADP⁺ dependent aldehyde dehydrogenase Ald6p.     

Stability and release properties of curcumin encapsulated in Saccharomyces cerevisiae, β-cyclodextrin and modified starch

Curcumin, a polyphenol with pharmacological function and colouring power, was encapsulated in baker’s yeast (Saccharomyces cerevisiae) cells, β-cyclodextrin (β-CD) and modified starch (MS) by various methods. The encapsulation forms were evaluated for their efficiency in overcoming curcumin’s heat, light and oxygen sensitivity (storage stability). The release (dissolution) profile of curcumin in simulated gastric (SGF) and pancreatic fluid (SPF) was, also, obtained. All the encapsulation forms drastically increased curcumin’s solubility in SGF. A rapid dissolution of curcumin was observed for β-CD and MS microcapsules in SGF while, in yeast microcapsules, a slow and prolonged release occurred, along with low degradation rate in SPF. All the microcapsules tested protected curcumin against oxidation in environments of elevated relative humidity (%RH) and yeast microcapsules stabilized curcumin at RH above 75.5%, where oxidation was significantly increased. Yeast cells offered better protection to curcumin than did β-CD or MS against deleterious photochemical reactions and against heat degradation following isothermal (inert or oxidative) heating at 200 °C. Heating at lower temperatures (isothermal and non-isothermal) revealed that β-CD and MS can also enhance curcumin’s heat resistance.     

Selenium biotransformation by Saccharomyces cerevisiae and Saccharomyces bayanus during white wine manufacture: Laboratory-scale experiments

The main purpose of this laboratory-scale study was to evaluate the transformation of inorganic selenium, as sodium selenite, when added to white grape juice as part of the fermentation process of white wine. The participation of yeast, added in the fermentation of the must, is necessary to convert inorganic selenium into organoseleno species. Two different yeasts, Saccharomyces cerevisiae and Saccharomyces bayanus were used for fermentation. The effects of different Se concentrations on cells and their viability during fermentation were evaluated. The alcoholic fermentation that produced wine was not affected by the presence of selenium, regardless of the type of Saccharomyces used. After 21 days of fermentation, the white wine and residual yeast were separated and analysed by ICP-MS and LC–ICP-MS for determination of total selenium and speciation. Selenomethionine was found to be the main Se-species in the selenised white wine. The results obtained are preliminary but they could be considered for future studies using both pilot and full-scale vinification processes.     

Alkaline pH enhances farnesol production by Saccharomyces cerevisiae

External environments affect prenyl alcohol production by squalene synthetase-deficient mutant Saccharomyces cerevisiae ATCC 64031. Cultivation of the yeast in medium with an initial pH ranging from 7.0 to 8.0 increased the amount of secreted farnesol (FOH). In contrast, acidic medium with a pH below 4.0 increased the intracellular FOH and its isomer nerolidol. These effects of alkaline pH were also observed on constant pH cultivation in a jar fermenter. On cultivation for 133 h, the FOH production reached 102.8 mg/l.     

Multifactorial analysis of acetaldehyde kinetics during alcoholic fermentation by Saccharomyces cerevisiae

Acetaldehyde is the terminal electron acceptor in the alcoholic fermentation by Saccharomyces cerevisiae. Quantitatively the most important carbonyl by-product, it has relevance for ethanol production yields as well as product stabilization and toxicology. The aim of this study was to investigate the effect of various enological parameters on acetaldehyde kinetics during alcoholic fermentations. Two commercial yeast strains were tested in two grape musts and the pH, temperature, SO₂ and nutrient addition were varied. All incubations had uniform kinetics where acetaldehyde reached an initial peak value followed by partial reutilization. Peak acetaldehyde concentrations and residual concentrations after 15 days of fermentations ranged from 62 to 119 mg l^− 1 and 22 to 49 mg l^− 1, respectively. A positive linear relationship was found between peak and final acetaldehyde levels in Gewürztraminer, but not Sauvignon Blanc fermentations, where sluggish fermentations were observed. Several factors had a significant effect on peak and/or final acetaldehyde levels. SO₂ addition, grape cultivar and fermentation nutrition were important regulators of peak acetaldehyde production, while final acetaldehyde concentrations were correlated with SO₂ addition, grape cultivar and temperature. The results allowed to estimate the acetaldehyde increase caused by SO₂ addition to 366 ??g of acetaldehyde per mg of SO₂ added to the must. The course of the final fermentation phase was shown to determine acetaldehyde residues. Comparison of acetaldehyde and hexose kinetics revealed a possible relationship between the time of occurrence of peak acetaldehyde concentrations and the divergence of glucose and fructose degradation rates.     

Effect of Saccharomyces cerevisiae fermentation product on ruminal fermentation and nutrient utilization in dairy cows

The goal of this experiment was to investigate the effect of yeast culture (Saccharomyces cerevisiae) on rumen fermentation, nutrient utilization, and ammonia and methane emission from manure in dairy cows. Eight ruminally cannulated Holstein cows were allocated to 2 dietary treatments in a crossover design. Treatments were control (no yeast culture) and XP (yeast culture, fed at 56 g/head per day; XP, Diamond V Mills Inc., Cedar Rapids, IA). Dry matter intake, milk yield, milk composition, and body weight were similar between treatments. Milk urea nitrogen concentration was also not affected by treatment. Rumen pH was similar between the control and XP treatments, but rumen ammonia concentration tended to be lower with XP than with the control. Treatment had no effect on concentrations of total or individual volatile fatty acids, protozoal counts, polysaccharide-degrading activities (except amylase activity that tended to be increased by XP), or methane production in the rumen. Urinary N losses did not differ significantly between treatments, but allantoin and total purine derivative excretions and the estimated microbial N outflow from the rumen tended to be increased by XP compared with the control treatment. Total-tract apparent digestibility of dietary nutrients was not affected by XP. Milk fatty acid composition was also not altered by XP supplementation. Cumulative (253 h) ammonia and methane emissions from manure, measured in a steady-state gas emission system, were slightly decreased by XP. Overall, the yeast culture tested had little effect on ruminal fermentation, digestibility, or N losses, but tended to reduce rumen ammonia concentration and increase microbial protein synthesis in the rumen, and decreased ammonia and methane emissions from manure.     

Influence of autochthonous Saccharomyces cerevisiae strains on volatile profile of Negroamaro wines

Saccharomyces cerevisiae is the yeast species predominating the alcoholic fermentation of grape must. The aim of this research was to evaluate the impact of indigenous S. cerevisiae strains biodiversity on the aroma of wines from Negroamaro grapes. Grapes collected in two different Negroamaro producing micro districts in Salento (Southern Italy), were subjected to natural fermentation and two indigenous S. cerevisiae populations were isolated. Fifteen strains for each of the two populations were selected and tested by micro fermentation assay in order to evaluate their specific contribute to the volatiles composition and sensory impact of the produced wines. The aromatic profile of wines obtained by each selected strain was characterized by different contents of acetates, ethyl esters of fatty acids, higher alcohols, thus showing to be related to the strains geographical origin. The sensorial analysis of wines produced by the six best performing strains confirmed that they are good candidates as industrial starter cultures, This study indicates that the use of a “microarea-specific” starter culture is a powerful tool to enhance the peculiarity of wines deriving from specific areas.     

Thermal and surface behavior of yeast protein fractions from Saccharomyces cerevisiae

An easy and inexpensive method of fractionation of a yeast homogenate was proposed and it is based on differential centrifugation steps of insoluble components and subsequent precipitations of soluble fractions. In this fractionation, the effect of addition of protease inhibitor was studied. The procedure, which was performed in mild conditions in order to minimize protein denaturation, produced four fractions that proceed from distinct parts of the yeast cell and with a different chemical composition: Fr I, Fr II, Fr III and Fr IV. Thermal and surface behavior of these samples was also analyzed. Fr I and Fr II, mainly composed by cell wall debris and membrane cell components, respectively, exhibited an adsorption rate (Δγ/Δt^1/2) ten-fold higher than Fr III and Fr IV, composed by nucleoproteins and cytoplasmic proteins. All fractions exhibited a unique DSC endotherm with different peak temperature (T_p) and enthalpy values (ΔH). Fr IV exhibited the highest T_p value (74 °C) and less affected by inhibitor absence. Fr I and Fr II showed the highest ΔH values (27-47 J/g protein) but they were markedly affected reducing their enthalpy values and increasing their surface properties in absence of protease inhibitor.     

Biogenic amine production by Oenococcus oeni during malolactic fermentation of wines obtained using different strains of Saccharomyces cerevisiae

Twenty-six wild Oenococcus oeni strains were investigated for their ability to form biogenic amines during malolactic fermentation in synthetic medium and in wine. Eight strains produced histamine and tyramine in screening broth at concentrations of 2.6-5.6 mg/L and 1.2-5.3 mg/L, respectively. Based on their ability to form biogenic amines, five strains were selected to inoculate three wines obtained by the fermentation of three different Saccharomyces cerevisiae strains (A, B, and C). All bacterial strains could perform malolactic fermentation for short periods in wine C, whereas only one strain performed complete malolactic fermentation in wines A and B. Two O. oeni strains (261 and 351) produced histamine and tyramine in wine C. Time-course analysis of these compounds showed that for both strains, histamine and tyramine production began at day 10 and finished on day 25, after the end of malolactic fermentation. These results indicate that the ability of O. oeni to produce histamine and tyramine is dependent on the bacterial strain and on the wine composition, which in turn depends on the yeast strain used for fermentation, and on the length of bacteria-yeast contact time after the completion of malolactic fermentation.