Alcoholic Fermentation Of Grape Research Articles

Simultaneous improvement of fructophilicity and ethanol tolerance of Saccharomyces cerevisiae strains through a single Adaptive Laboratory Evolution Strategy

Saccharomyces cerevisiae is the main yeast used in the winemaking industry. Its innate glucophilicity provokes a discrepancy in glucose and fructose consumption during alcoholic fermentation of grape must, which, combined with the inhibitory effect of ethanol accumulated in the fermentation broth, might lead to stuck or sluggish fermentations. In the present study, we realized an Adaptive Laboratory Evolution strategy, where an alcoholic fermentation of a 20 g/L fructose broth was followed by cell selection in a high ethanol concentration environment, employed in two different S. cerevisiae strains named CFB and BLR. The evolved populations originated from each strain after 100 generations of evolution exhibited diverse fermentative abilities. One evolved population, originated from CFB strain, fermented a synthetic broth of 100 g/L glucose and 100 g/L fructose to dryness in 170 h, whereas the parental strain did not complete the fermentation even after 1000 h of incubation. The parameters of growth of the parental and evolved populations of the present study, as well as of the ethanol tolerant populations acquired in a previous study, when grown in a synthetic broth of 100 g/L glucose and 100 g/L fructose, were calculated through a kinetic model, and were compared to each other in order to identify the effect of evolution on the biochemical behavior of the strains. Finally, in a 200 g/L fructose synthetic broth fermentation, only the evolved population derived from CFB strain showed improved fermentative behavior than its parental strain.

Time course of l -tryptophan metabolites when fermenting natural grape musts: effect of inoculation treatments and cultivar on the occurrence of melatonin and related indolic compounds

Background and Aims Tryptophan is involved in the formation of bioactive compounds, such as melatonin (MEL) and 3-indoleacetic acid (3-IAA), by yeast. Melatonin is a neurohormone whose occurrence in wine has been widely reported in recent years. The occurrence, however, of MEL and other indolic compounds related to tryptophan metabolism by wine yeast strains has been scarcely reported in grape musts. This work examined the occurrence of these compounds during the alcoholic fermentation (AF) of musts from seven grape cultivars, Corredera, Chardonnay, Moscatel, Palomino Fino, Sauvignon Blanc, Tempranillo and Vijiriega. Methods and Results Must was fermented with three Saccharomyces cerevisiae strains and then in two cases an additional sequential inoculation with the non-Saccharomyces yeast Torulaspora delbrueckii was carried out. Fermented must samples were analysed by UHPLC/HRMS to determine the concentration of: l-tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, MEL, 3-IAA, tryptamine, tryptophol and l-tryptophan ethyl ester. The profile of indolic compounds during AF with the Aroma White strain depended on the cultivar. The yeast strain did not influence the profile of indolic compounds; instead, fermentation time was found to be a more influential factor. Conclusions The production of indolic compounds during the AF depends largely on the cultivar used and the day of fermentation on natural grape musts. Significance of the Study This is the first study that quantifies 5-hydroxytryptophan and N-acetyl-5-hydroxytryptamine during the AF of grape must. The occurrence of compounds with bioactive potential, for example 3-IAA and ML, during fermentation with commercial yeast strains is also described.

CO2 recovery from wine production: Possible implications on the carbon balance at territorial level

The Province of Siena (Tuscany, Italy) is characterized by two features: 1) the carbon neutrality of its territory and 2) the great production of quality wine. The GHG balance of the Province of Siena, realized in time series according to the IPCC guidelines and validated ISO 14064-1 under the REGES Project, testifies the carbon neutrality of the territory (the percentage abatement of gross emissions passed from 72% in 2006 to 106% in 2015). This ambitious goal was achieved in 2011 after the activation of specific environmental policies (concerning energy and waste sectors, forests, transport and education) with local benefits in the short run. This paper presents a systemic investigation of the effects of the application of technologies for carbon dioxide (CO2) capture/storage on the GHG balance of the provincial area; in particular, recovery of biogenic CO2 from alcoholic fermentation of grapes could be seen as a new strategy to consolidate the status of carbon neutrality. The hypothetical installation of recovery plants in Siena wine farms could approximately capture more than 6000 t CO2, corresponding to an improvement of the GHG balance of the territory (i.e. increase of net absorption of almost 8%). CO2 can be captured and stocked in high-pressure containers and reused in production activities, both within and outside the area, avoiding the use of gas obtained in chemical industries by fossil fuel-driven production processes. The economic evaluation confirms good performances obtained by the environmental assessment. These results can inspire policies at the farm level for reducing impacts on GHG emissions from a life cycle thinking viewpoint and at the territorial (or producer consortia) level, in order to decrease impacts by means of dedicated projects, generating positive effects on the local carbon balance and relevant consequences at global scale.

The inhibition ofSaccharomyces cerevisiaepopulation during alcoholic fermentation of grape must by octanoic, decanoic and dodecanoic acid mixture

The inhibition of alcoholic fermentation by octanoic, decanoic and dodecanoic acid mixture was investigated. Middle chain fatty acids (MCFA) mixture contained 10 grams of C8 :C10 :C12 in a ratio of 2:7:1 was dissolved in 100 ml of 70% ethanol, and in such form, it was subsequently applied into the fermenting must samples. A flow cytometry test showed that the 10 mg/L dose of MCFA mixture had a toxic effect on Saccharomyces cerevisiae (45.9% of viable cells) compared with the control variant (74.35%). In combination with 60 mg/L of SO2 , it had a higher efficiency (3.1% of viable cells) than using a dose of SO2 alone (13.9%). Direct counting of yeast cells confirmed a higher concentration of dead cells with a higher concentration of MCFA. A dose of 10 mg/L of MCFA mixture caused the highest percentage of dead yeast after 24 hours (about 60%) compared to the control variant without MCFA dosage (about 24%). The results of residues showed that there is no significant quantitative difference between the treated and untreated musts because of fixing of MCFA inside the yeast cells. This method can effectively reduce the cost of production technology for wines with residual sugar and, in general, reduce the dosage of SO2 .

Production of White Wine from Grapes Using Sacharomyces cervisiae

Wine is an alcoholic beverage often made of fermented grape juice. The natural chemical balance of grapes is such that they can ferment without the addition of sugars, acids, enzymes or other nutrients. Wine is produced by fermenting crushed grapes using various types of yeast which consume the sugars found in the grapes and convert them into alcohol. Different varieties of grapes and strains of yeasts are used depending on the types of wine being produced. Although other fruits such as apples and berries can also be fermented, the resultant wines are normally named after the fruit from which they are produced (for example, apple wine or elderberry wine) and are generically known as fruit wine or country wine. Wine is an undistilled product obtained after normal alcoholic fermentation of grapes by yeast. Wine is basically the transformation of sugars of grapes by yeast under anaerobic conditions in to ethanol, co2, and small amounts of byproducts like D-Glucose, D-fructose. In our project, for the extraction of juice grapes must be stemed, cleaned, and crushed and then sodium or potassium metabiosulphate is added to check undesirable organisms. The crushed grape is called must. Fermentation is carried out by adding 2–5% of wine yeast namely sacharomyces cervisiae, anaerobic conditions favors the alcoholic fermentation liberating ethanol, co2. Main aim of this work is to produce white wine from grapes. A detailed study on grapes and different methods to produce wine is also reported. By analysis of wine, we reported the percentage purity, alcohol percentage in produced wine and different compound compositions.

A new method for monitoring the extracellular proteolytic activity of wine yeasts during alcoholic fermentation of grape must

The existing methods for testing proteolytic activity are time consuming, quite difficult to perform, and do not allow real-time monitoring. Proteases have attracted considerable interest in winemaking and some yeast species naturally present in grape must, such as Metschnikowia pulcherrima, are capable of expressing this activity. In this study, a new test is proposed for measuring proteolytic activity directly in fermenting grape must, using azocasein, a chromogenic substrate. Several yeast strains were tested and differences in proteolytic activity were observed. Moreover, analysis of grape must proteins in wines revealed that protease secreted by Metschnikowia strains may be active against wine proteins.

A proteomic and metabolomic approach for understanding the role of the flor yeast mitochondria in the velum formation

Saccharomyces cerevisiae “flor” yeast shows a strong tolerance to high ethanol concentrations and develops a velum (biofilm) on the wine surface after the alcoholic fermentation of grape must. This velum remains along several years during the so called “biological aging” process in the elaboration of some special wines carried out in specific regions around the world and it contributes to the typical organoleptic characteristics of these wines. In order to grow in this condition, flor yeast has to elaborate a response where the mitochondrial function is essential. The objective of this study is to elucidate the role of the mitochondria in the response of a flor yeast, S. cerevisiae G1, growing in a controlled velum formation condition. For this purpose, proteome and metabolome were characterized by comparing data with those from an initial fermentative condition used as reference. The obtained proteomic profiles show more mitochondrial proteins related with the ethanol resistance (13), cell respiration (18), mitochondrial genome maintenance (13), and apoptosis (2) detected under the velum formation condition. Also, the finger-printing obtained by means of the exo-metabolites directly related with the quality of fermented beverages and quantified in the velum condition shows important differences from those obtained in the reference condition.

Alcoholic fermentation induces melatonin synthesis in orange juice

Melatonin (N-acetyl-5-methoxytryptamine) is a molecule implicated in multiple biological functions. Its level decreases with age, and the intake of foods rich in melatonin has been considered an exogenous source of this important agent. Orange is a natural source of melatonin. Melatonin synthesis occurs during alcoholic fermentation of grapes, malt and pomegranate. The amino acid tryptophan is the precursor of all 5-methoxytryptamines. Indeed, melatonin appears in a shorter time in wines when tryptophan is added before fermentation. The aim of the study was to measure melatonin content during alcoholic fermentation of orange juice and to evaluate the role of the precursor tryptophan. Identification and quantification of melatonin during the alcoholic fermentation of orange juice was carried out by UHPLC-QqQ-MS/MS. Melatonin significantly increased throughout fermentation from day 0 (3.15 ng/mL) until day 15 (21.80 ng/mL) reaching larger amounts with respect to other foods. Melatonin isomer was also analysed, but its content remained stable ranging from 11.59 to 14.18 ng/mL. The enhancement of melatonin occurred mainly in the soluble fraction. Tryptophan levels significantly dropped from 13.80 mg/L (day 0) up to 3.19 mg/L (day 15) during fermentation. Melatonin was inversely and significantly correlated with tryptophan (r = 0.907). Therefore, the enhancement in melatonin could be due to both the occurrence of tryptophan and the new synthesis by yeast. In summary, the enhancement of melatonin in novel fermented orange beverage would improve the health benefits of orange juice by increasing this bioactive compound.

An innovative tool reveals interaction mechanisms among yeast populations under oenological conditions

Alcoholic fermentation of grape must is a complex process, involving several yeast genera and species. The early stages in fermentation are dominated by non-Saccharomyces yeasts that are gradually replaced by the Saccharomyces cerevisiae species, which takes over the fermentation. Quantitative studies have reported the influence of non-Saccharomyces yeast species on wine quality and evaluated their biotechnological interest. The industrial yeast market, which, until recently, exclusively focused on S. cerevisiae, now offers S. cerevisiae/non-Saccharomyces (including Torulaspora delbrueckii) multi-starters. The development of these new mixed industrial starters requires a better understanding of the interaction mechanisms between yeast populations in order to optimize the aromatic impact of the non-Saccharomyces yeast while ensuring complete alcoholic fermentation thanks to S. cerevisiae. For this purpose, a new double-compartment fermentor was designed with the following characteristics: (1) physical separation of two yeast populations, (2) homogeneity of the culture medium in both compartments, (3) fermentation kinetics monitored by weight loss due to CO2 release, and (4) independent monitoring of growth kinetics in the two compartments. This tool was used to compare mixed inoculations of S. cerevisiae/T. delbrueckii with and without physical separation. Our results revealed that physical contact/proximity between S. cerevisiae and T. delbrueckii induced rapid death of T. delbrueckii, a phenomenon previously described and attributed to a cell-cell contact mechanism. In contrast, when physically separated from S. cerevisiae, T. delbrueckii maintained its viability and its metabolic activity had a marked impact on S. cerevisiae growth and viability. The double fermentor is thus a powerful tool for studying yeast interactions. Our findings shed new light on interaction mechanisms described in microorganism populations.

Dynamics and diversity of non- Saccharomyces yeasts during the early stages in winemaking

This detailed study observed the yeasts present in the ecological niche of “wine must”. The dynamics and identity of non- Saccharomyces yeasts during the cold maceration and alcoholic fermentation of grape must were investigated under real production conditions in the Bordeaux region. Furthermore, we studied the impact of two oenological parameters on the development and diversity of non- Saccharomyces yeasts during cold maceration: temperature management and the timing of dried yeast addition. The non- Saccharomyces community underwent constant changes throughout cold maceration and alcoholic fermentation. The highly diverse non- Saccharomyces microflora was present at 10 4–10 5 CFU/mL during cold maceration. The population increased to a maximum of 10 6–10 7 CFU/mL at the beginning of alcoholic fermentation, then declined again at the end. The population at this point, evaluated at around 10 3–10 4 CFU/mL, was shown to be dependent on the timing of yeast inoculation. The choice of temperature was the key factor for controlling the total yeast population growth, as well as the species present at the end of cold maceration. Hanseniaspora uvarum was a major species present in 2005 and 2006, while Candida zemplinina was very abundant in 2006. A total of 19 species were isolated.

Low potential detection of NADH with Prussian Blue bulk modified screen-printed electrodes and recombinant NADH oxidase from Thermus thermophilus

A biosensor for the determination of the reduced coenzyme nicotinamide adenine dinucleotide (NADH) has been assembled using a recombinant enzyme NADH oxidase from Thermus thermophilus covalently immobilized on Prussian Blue bulk-modified screen-printed electrodes. Flow injection analysis (FIA) coupled with amperometric detection was used to detect NADH. Various parameters such as cofactor (FMN, flavin mononucleotide) concentration (2 mM), flow rate (0.35 mL/min), buffers (citrate–phosphate, phosphate and glycine–KOH), pH dependence (range 3.0–10.5), response time (12 s) and operational stability (120 injections) were evaluated and optimised. At pH 5.0, for which the biosensor showed the highest response, the detection and quantification limits were 1.1 × 10 −7 and 3.6 × 10 −7 M, respectively, and the linear working range was comprised between 1 and 400 μM. The proposed biosensor was stable for 2 months (preserved in 50 mM phosphate buffer, pH 6.8, at 4 °C). The possibility to co-immobilize glycerol dehydrogenase (GDH) and the NADH oxidase in order to measure glycerol, a key target analyte during the alcoholic fermentation of grapes, was also investigated. Different dilutions of a complex matrix such as wine were tested to assess the interferences, the probe recovery and stability.

Investigation of volatiles evolution during the alcoholic fermentation of grape must using free and immobilized cells with the help of solid phase microextraction (SPME) headspace sampling.

A biocatalyst was prepared by immobilization of Saccharomyces cerevisiae strain AXAZ-1 on delignified cellulosic material (DCM). Repeated batch fermentations were conducted using these biocatalysts and free cells, separately, at temperatures of 20, 15, and 10 degrees C. Solid phase microextraction (SPME) was used in monitoring the formation of volatile alcohols, acetate esters, and ethyl esters of fatty acids. The kinetics of volatile production were similar for free and immobilized cells. In all cases immobilized cells showed a better rate of volatile production, which was directly connected to sugar consumption. The main difference observed was in propanol production, which increased with temperature decrease for the immobilized cells, whereas it remained constant for the free ones. In the case of immobilized cells significant amounts of esters were also produced. It is well-known that esters contribute to the fruity aroma of wine. It was also established that SPME is a very sensitive, accurate, and reliable technique and can be used without any reservation in the characterization of volatile constituents of wine.

Influence of pre-fermentative treatment on the fatty acid content of Saccharomyces cerevisiae (M 330-9) during alcoholic fermentation of grape must

The effect of skin maceration and must filtration on the growth and fermentative capacity of one strain of Saccharomyces cerevisiae was studied. Skin maceration increased the fatty acid concentration of the must, thus affecting the lipid composition of the yeast cell membranes. Sterile filtration resulted in a reduction of the fatty acid content of the must extracted during maceration, but increased the fatty acid content of the yeast membranes, thus increasing the fermentative capacity without affecting either growth or cell viability.

Genetic and biochemical characterization of Saccharomyces cerevisiae mutants resistant to trifluoroleucine

Eighteen mutants resistant to 5',5',5'-trifluoroleucine (TFL), a leucine analog, were isolated in Saccharomyces cerevisiae strains YNN281 and YNN282. The mutants were characterized genetically and clustered in two groups, one comprising all the dominant (TFL1) and the other one all the recessive (tfl2) mutations. Genetic and biochemical data suggested that the dominant mutations are located on the LEU4 gene, coding for alpha-isopropylmalate synthase I. These mutations resulted in accumulation of leucine as a consequence of the synthesis of an enzyme insensitive to the feedback inhibition by leucine. Leucine excretion in the TFL1 mutants appeared to be affected by the genetic background of the strain and was greatly influenced by lysine metabolism. The measurement of intra- and extracellular amino acid concentrations in prototrophic strains carrying TFL1 or tfl2 genes showed that both were leucine overproducers. Some of the TFL-resistant mutants were tested in alcoholic fermentation of grape must: analysis of the fermentation secondary metabolites showed that the major effect of the TFL-resistant strains was an increased production of isoamyl alcohol compared to that of the parental strain.

Influence of the fermentation yeast strain on the composition of wine spirits

AbstractFifteen indigenous strains of Saccharomyces cerevisiae, differentiated by their genetic polymorphism, were compared in standard conditions for their respective ability to produce volatile compounds during the alcoholic fermentation of grape must. The results were processed using analysis of variance and of principal components. They exhibited significant differences between strains, in particular for the higher alcohols, fatty acid esters, acetates or acetaldehyde. On the basis of their analytical profile, five strains were selected and compared on the pilot scale. Overall, the pilot results confirmed laboratory findings. As the strain effect appears to be determinant on the aromatic composition of wine spirits, a programme of selection based on both intraspecific molecular characterisation and chromatographic analysis is carried out to obtain specific yeast strains for high‐quality spirits.

Changes in the composition of alcohols and aldehydes of C6 chain length during the alcoholic fermentation of grape must

The goal of this work was the study of the changes occurring in the concentrations of alcohols and aldehydes of C 6 chain length during alcoholic fermentation, in the presence or in the absence of SO 2 . To this aim, grape must or synthetic media and Saccharomyces cerevisiae as a fermentative agent were used

Možnosti stimulácie alkoholového kvasenia hroznového muštu preparátom z bunkových stien kvasiniek.

Možnosti stimulácie alkoholového kvasenia hroznového muštu preparátom z bunkových stien kvasiniek.