Influence of the timing of nitrogen additions during synthetic grape must fermentations on fermentation kinetics and nitrogen consumption

Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. In the present study, we have supplemented nitrogen-deficient fermentations with a mixture of ammonium and amino acids at various stages throughout the alcoholic fermentation. The timing of the nitrogen additions influenced the biomass yield, the fermentation performance, the patterns of ammonium and amino acid consumption, and the production of secondary metabolites. These nitrogen additions induced a nitrogen-repressed situation in the cells, and this situation determined which nitrogen sources were selected. Glutamine and tryptophan were the main amino acids consumed in all the fermentations. Ammonium is the preferred nitrogen source for biomass production but was hardly consumed when it was added in the final stages of the fermentation. The higher ammonium consumption in some fermentations correlated with a greater synthesis of glycerol, acetate, and acetaldehyde but with a lower synthesis of higher alcohols.     

戴尔有孢圆酵母调控晚采小芒森葡萄酒乙酸和香气

为降低甜型葡萄酒中的挥发酸,进一步增加其香气复杂度,该研究利用本土戴尔有孢圆酵母R12与酿酒酵母NX11424同时接种和顺序接种发酵,研究其对贺兰山东麓产区晚采小芒森葡萄酒乙酸及香气成分的调控。与酿酒酵母单菌发酵相比,本土戴尔有孢圆酵母与酿酒酵母按照5:1、20:1、50:1的不同菌体数量比例同时接种和间隔5d顺序接种发酵,均可以显著降低晚采小芒森葡萄酒的乙酸含量,其乙酸产率分别降低19.1%、21.2%、38.2%和48.9%。此外,按20:1比例同时接种发酵,可显著提高晚采小芒森葡萄酒中萜烯类和降异戊二烯类等品种香气物质含量,和乙酸异戊酯、丁酸乙酯及苯乙醇等发酵香气物质的含量。该研究表明合理的酿酒酵母接种发酵能有效降低高糖原料酿造葡萄酒挥发酸含量,为贺兰山东麓产区葡萄酒新产品的开发提供了的技术参考。     

Wine production using yeast immobilized on apple pieces at low and room temperatures

A biocatalyst was prepared by immobilization of Saccharomyces cerevisiae strain AXAZ-1 on apple pieces. It was examined by electron microscope and studied during the fermentation of grape must for batch wine-making. The immobilized yeast showed an important operational stability without any decrease of its activity even at low temperatures (1-12 degrees C). Specifically, at 6 degrees C the biocatalyst favored wine production within 8 days, which is less time than is required for the natural fermentation of grape must. At 1 degrees C wine production was effected in 1 month. This speeding up of the fermentation could be accepted and adopted by the industry for scaling up the wine-making process. Total and volatile acidities were similar to those found in dry wines. The concentrations of higher alcohols (propanol-1 and isobutyl alcohol) were low. The presence of amyl alcohols proved to be temperature dependent and decreased with the temperature decrease. The values of ethyl acetate concentrations were relatively high, up to 154 mg/L. This probably contributes to the fruity aroma and excellent taste of the produced wines. There was no indication of vinegar odor in the product given that the volatile acidity was <0.47 g of acetic acid/L. From the GC-MS analysis it was concluded that cell immobilization did not create serious changes in the product (wine) with regard to the qualitative composition of the aroma compounds.     

Influence of a Saccharomyces cerevisiae selected strain in the volatile composition of rosé wines. Evolution during fermentation

There has been considerable controversy about the use of selected pure strains in wine fermentation. For that reason it is important to determine the influence of this vinification technique in the composition of wine because it arises from the type of yeast and the subsequent evolution during fermentation. This study researches the volatile composition of rosé wines from the Garnacha must, inoculated with one selected NA33 strain of Saccharomyces cerevisiae. The inoculated yeast did not predominate in all of the samples. These samples showed a behavior intermediate between those of the control and samples in which NA33 did predominate. The greatest concentration of higher alcohols was in the control wine, and its evolution was similar in all fermentations. The esters formed at the end of the fermentation and their concentrations were higher in the control than in the inoculated samples. In the control, acids were produced above all, in the first half of fermentation, and decreased from then onward. In the sample in which the yeast predominated, the synthesis occurred later and to a lesser extent than in the control.     

Coinoculated fermentations using saccharomyces yeasts affect the volatile composition and sensory properties of Vitis vinifera L. cv. sauvignon blanc wines

Alcoholic fermentation using Saccharomyces wine yeast is an effective means of modulating wine aroma. This study investigated the impact of coinoculating commercial yeast strains (Vin7, QA23, Vin13) on the volatile composition and sensory profile of Sauvignon Blanc wines. Small-scale replicated fermentations were conducted using single-strain and coinoculations of Vin7 with QA23 and with Vin13. The results showed that the chemical and sensory profiles of the coinoculated wines were different from both the single-strain wines and equal blends of the single-strain wines. Volatile thiol analysis indicated that the Vin7/QA23 coinoculated wines were highest in 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), although this pattern was not observed for the Vin7/Vin13 yeast combination. The negative white vinegar aroma and high volatile acidity measured in the Vin7 single-strain wines were not present in the coinoculated wines. This study demonstrates that coinoculations can modify the aroma profile of wines, when complementary yeasts are used.     

Selection of yeast starter culture strains for the production of marula fruit wines and distillates

Juice of the Sclerocarya birrea subsp. caffra (marula) fruit was fermented by indigenous microflora and different commercial Saccharomyces cerevisiae yeast strains at different temperatures, namely, 15 and 30 degrees C. Volatile acids, esters, and higher alcohols were quantified in the wine and distillates, and the results were interpreted using a multivariate analysis of variance and an average linkage cluster analysis. Significant differences between 15 and 30 degrees C and also among yeasts with respect to volatile compounds were observed. Yeast strains VIN7 and FC consistently produced wines and final distillates significantly different from the other strains. A panel of tasters and marula and brandy producers was asked to select wines and distillates that had an acceptable and typical marula "nose". They were also asked to detect the differences among wines and distillates fermented with the same yeast strain at different temperatures.     

优选非酿酒酵母胞外酶增香酿造干白葡萄酒效果

为了研究非酿酒酵母胞外酶促进葡萄酒发酵产香的效果,该文在爱格丽葡萄汁酒精发酵12 h后,分别添加优选胶红酵母(Rhodotorula mucilaginosa,RM)胞外酶液,优选发酵毕赤酵母(Pichia fermentans,PF)胞外酶液和商业糖苷酶制剂(almondβ-glucosidase,AG)(10 mU/mL),以不添加酶制剂的酿酒酵母纯发酵为对照,发酵过程中每24 h取样,采用HS-SPME-GC/MS(headspace solid-phase microextraction-gas chromatography/mass spectrometry)监测挥发性成分的生成变化。葡萄酒含糖量低于2 g/L时终止发酵,低温满罐密封储存,次年4月,进行葡萄酒香气仪器和感官量化分析。胞外酶液共测得6种风味酶活性,其中RM酶液中的β-D-葡萄糖苷酶、α-L-鼠李糖苷酶、β-D-木糖苷酶活性均显著(P<0.05)高于PF中的,而α-L-阿拉伯糖苷酶和酯酶在PF酶液中活性更高(P<0.05)。发酵过程中,胞外酶处理显著促进了(P<0.05)品种香气和发酵香气物质的生成,其中RM酶液显著提高了萜烯类物质和苯乙基类化合物含量,其作用效果分别比商业酶处理高41.7%和31.8%,PF酶液显著促进了发酵香气化合物的生成,尤其是脂肪酸乙酯含量约为对照的2倍。酒样感官分析结果显示,两株酵母的胞外酶处理表现出各自增香酿造的优势,其中RM酶液促进温带酸果类香气的效果突出,PF酶液显著提升了柑橘类香气。研究结果为酵母风味酶应用于葡萄酒的增香酿造提供了理论和实践指导。     

Influence of aeration on the physiological activity of flor yeasts.

The effect of periodic aeration on the physiological activity of a strain of Saccharomyces cerevisiae yeast during development of velum (flor) and biological aging of Sherry wine of the Fino type was investigated. L-Proline amino acid was the main nitrogen source for yeasts cells during the biological aging, and its exhaustion may be the cause of the production and consumption of other compounds that are involved in the aroma of wines. Aeration was found to increase adenylate energy charge, growth, and viability of the yeast cells. Also, it affected the intracellular redox equilibrium and the consumption and production of compounds including acetoin, acetaldehyde, higher alcohols, ethanol, glycerol, and acetic acid. Acetaldehyde reached its highest level after the second aeration, which coincided with the exhaustion of the nitrogen source in the medium. The enzyme activity of alcohol dehydrogenases I and II decreased immediately after each aeration, subsequently increasing once all of the dissolved oxygen in the wine had been consumed by yeast cells. Aldehyde dehydrogenase activity was detected only after the first aeration, and it may be related to the production and consumption of acetic acid in the wine.     

Use of nitrogen compounds in spontaneous and inoculated wine fermentations.

In this paper the use of nitrogen compounds in garnacha must inoculated with active dry wine yeast Saccharomyces cerevisiae subsp. cerevisiae strain Na33 has been studied. The results are compared to garnacha must fermented with indigenous yeasts (control must). In the samples where the inoculated yeast predominated, no qualitative differences were appreciated in the use of amino acids with respect to the control samples, although there were quantitative differences. In the musts where the Na33 strain dominated, a lesser quantity of amino acids were consumed at the beginning of fermentation than in the control samples. For that reason, probably, this yeast showed problems in competing for the nitrogen nutrients of the must; this could have made its implantation in one of the inoculated samples more difficult. At the end of fermentation the Na33 strain continued to consume amino acids at high concentrations of ethanol. Its high tolerance to this toxic could be favored by the production and rehydration of dry wine yeast in the presence of air.     

Malolactic fermentation in wine with Lactobacillus casei cells immobilized on Delignified cellulosic material

In this work Lactobacillus casei ATCC 393 cells immobilized on delignified cellulosic material (DCM) were used for malolactic fermentation (MLF) of wine. Wine was produced using yeast cells immobilized on DCM at 20 degrees C, and after alcoholic fermentation, MLF at 27 degrees C followed using immobilized L. casei ATCC 393 cells. A total of 11 repeated alcoholic and subsequent MLF batches were performed within a period of 1 month. As the repeated MLF batches proceeded, the MLF activity of the immobilized biocatalyst was reduced. Malic acid degradation was reduced from 80 to 2%, pH was reduced by 0.5-0.1 unit, acetic acid concentrations were slightly reduced or remained stable (0.002 g/L), the higher alcohols 1-propanol, isobutyl alcohol, and amyl alcohol were decreased by 84, 23, and 11%, respectively, and ethyl acetate concentration was increased by approximately 56%. Wine samples were analyzed by GC-MS before and after MLF, revealing some qualitative differences.     

Hydrolysis and transformation of grape glycosidically bound volatile compounds during fermentation with three Saccharomyces yeast strains

The ability of three Saccharomyces wine yeasts (S. cerevisiae AWRI 838, S. cerevisiae AWRI 1537, and S. bayanus AWRI 1375) to liberate volatile compounds from sugar-bound aroma precursors was investigated using synthetic and grape glycosides under different experimental conditions. In model systems involving the incubation of yeast cells with either synthetic or grape-derived glycosides under conditions more favorable for glycosidase activities and less favorable for acid-catalyzed hydrolysis (pH 5.0 and 30 degrees C), all yeast strains studied proved to be capable of hydrolyzing glycosides, with S. bayanus AWRI 1375 displaying greater hydrolytic activity than S. cerevisiae AWRI 838 and AWRI 1537. During the fermentation of a chemically defined grape juice-like medium containing glycosidic precursors extracted from Vitis vinifera cv. White Frontignac (synonym Muscat à Petit Grains Blanc), all yeasts promoted a significant hydrolysis of different precursors, which varied according to the chemical structures of both the sugar and the aglycon moieties, as determined by GC-MS analysis of trifluoroacetylated derivatives. Hydrolysis of the White Frontignac derived glycosidic precursors during fermentation resulted in the release of monoterepene alcohols, terpene oxides, terpene diols, and 3-oxo-alpha-ionol, demonstrating the significant potential of these yeast strains to contribute to wine varietal volatile composition during alcoholic fermentation.     

Hydroxycinnamic acid ethyl esters as precursors to ethylphenols in wine

A method for determining ethyl coumarate and ethyl ferulate in wine using GC-MS with deuterium-labeled analogues has been developed and used to measure the evolution of these two esters during the production of two commercial monovarietal red wines, cv. Grenache and Shiraz. During fermentation, the concentration of ethyl coumarate rose from low levels to 0.4 mg/L in Grenache and 1.6 mg/L in Shiraz wines. These concentrations then increased further during barrel aging to 1.4 and 3.6 mg/L, respectively. The concentration of ethyl ferulate was much lower, reaching a maximum of only 0.09 mg/L. Conversion of ethyl coumarate and ethyl ferulate to their corresponding ethylphenols was observed during fermentations of a synthetic medium with two strains of Dekkera bruxellensis (AWRI 1499 and AWRI 1608), while a third (strain AWRI 1613) produced no ethylphenols at all from these precursors. Strains AWRI 1499 and 1608 produced 4-ethylphenol from ethyl coumarate in 68% and 57% yields, respectively. The corresponding yields of 4-ethylguaiacol from ethyl ferulate were much lower, 7% and 3%. Monitoring of ethyl coumarate and ethyl ferulate concentration during the Dekkera fermentations showed that the selectivity for ethylphenol production according to yeast strain and the precursor was principally a result of variation in esterase activity. Consequently, ethyl coumarate can be considered to be a significant precursor to 4-ethylphenol in wines affected by these two strains of Brettanomyces/Dekkera yeast, while ethyl ferulate is not an important precursor to 4-ethylguaiacol.     

Release of lipids during yeast autolysis in a model wine system

The release of lipids during the aging of sparkling wines in contact with yeast can influence wine sensory attributes and, especially, foam characteristics. Model systems allow study of the autolysis process in a reasonable period of time compared to natural conditions, at which it can last several months. In this paper, the release of the different classes of lipids during the autolysis of three commercial yeast strains in a model wine medium has been monitored. Due to the absence of accurate quantitative methods, an HPLC method for separating and quantifying the different neutral and polar yeast lipid classes was developed. Lipids were eluted through a YMC PVA-Sil column with a complex solvent mixture. Detection was carried out with a light-scattering detector. The yeasts were suspended in the model wine buffer and incubated at 30 degrees C for up to 12 days. A release of triacylglycerols, 1,3-diacylglycerols, 2-monoacylglycerols, free fatty acids, sterol esters, and sterols was observed over the first 2 days, a period that corresponded to the maximum loss of yeast viability. A decrease in most of these lipids was observed from day 2, possibly indicative of the release of yeast hydrolytic enzymes due to the breakdown of the cell wall. Phospholipids were not detected in any of the autolysates. The mean lipid content in the autolysates as a percentage of the total lipid content in the yeasts was 8.6% for sterol esters, 3.8% for sterols, 2% for triacylglycerols, and <2% for 1,3-diacylglycerols and free fatty acids.     

Oenological influences on the D/H ratios of wine ethanol

Within the European Union the determination of chaptalization of wine involves the comparison of the D/H ratios of ethanol with the ratios of authentic wine samples that are similar to the suspect wine in terms of geographical origin, grape variety, and vintage. In the frame of a databank project comparison, wines are produced under official control on a small scale. To clarify the influence of the different production conditions between commercial wines and these databank wines, wines that were produced under varying conditions were investigated by the (2)H NMR method. None of the parameters under investigation, such as yeast strain, fermentation temperature, or wine fining, showed a significant influence on the (D/H)(I) ratio of wine ethanol, which is the most indicative parameter for the determination of the addition of extraneous sugar to wine. For the (D/H)(II) ratio, different values were found for different yeast strains used for fermentation and a slight decrease was observed with increasing fermentation temperature. At increasing points of fermentation yield an increase of the D/H ratios was found in the present alcohol. The total increase of the (D/H)(I) ratio throughout the fermentation was approximately 1 ppm, so that with a fermentation yield of more than 50% no statistical difference could be observed.     

Influence of prefermentative treatments to the major volatile compounds of Assyrtiko wines

A study of the volatile fraction of Assyrtiko wines, using gas chromatography coupled with olfactometry, was realized. Twenty-seven volatile compounds were identified as potent odorants, most of them originating from the fermentation process. Quantification of the major volatile compounds was realized developing a rapid analytical method based on fractionation of a 50 mL wine aliquot using C 18-reversed phase adsorbent. After elution of the volatile compounds with pentane-diethyl ether and concentration under nitrogen, the final wine extract was injected in a gas chromatography-flame ionization detection system. The method allows satisfactory determination of more than 15 volatile compounds of wine. The linearity of the method gave a typical r (2) between 0.990 and 0.999, while reproducibility ranged from 5.1 to 12.2% (as relative standard deviation) with 9.5% as the average. The method was applied to wines produced by Assyrtiko grapes (AOC Santorini), for two consecutive years, to compare the effect of skin contact prior to fermentation and the must clarification process. Direct press and skin contact wines were differentiated analytically; however, highly significant differences were not. Inversely, the differences found between direct press/clarified and nonclarified wines were significant. Wines produced by direct press and clarified must presented significantly higher levels of ethylic esters and fusel alcohol acetates but lower fusel alcohol levels, leading probably to more fruity wines. This difference, between clarified and nonclarified grape musts, was not significant in the case of the wines produced by skin contact of Assyrtiko berries. These findings were validated by preference sensory analysis tests.     

Low-temperature alcoholic fermentation by delignified cellulosic material supported cells of kefir yeast.

A novel system for low-temperature alcoholic fermentation of glucose is described. This system consists of kefir yeast immobilized on delignified cellulosic materials. Batch fermentations were carried out at various pH values, and the effect of temperature on kinetic parameters, in the range of 5-30 degrees C, was examined. At pH 4.7 the shortest fermentation time was obtained. The formation of volatiles indicates that the concentration of amyl alcohols (total content of 2-methylbutanol-1 and 3-methylbutanol-1) is reduced as the temperature becomes lower. Propanol-1 and isobutyl alcohol formation drops significantly below 15 degrees C. The percentage of ethyl acetate increases as the temperature is diminished. At 5 degrees C the content of total volatiles in the product was only 38% of the volatiles formed during fermentation at 30 degrees C.     

Low-temperature brewing using yeast immobilized on dried figs

Dried figs, following exhaustive extraction of their residual sugars with water, were used for immobilization of Saccharomyces cerevisiae AXAZ-1. The immobilized biocatalyst was used in repeated batch fermentations of glucose at 30 degrees C, where significant reduction of the fermentation time was observed, falling from 65 h in the first batch to 7 h after the sixth batch. Repeated fermentations of wort at room and low temperatures resulted in fermentation times that fell from 26 to 20 h and from 27 to 24 days at 18 and 3 degrees C, respectively. Ethanol and beer productivities were high, showing suitability of the biocatalyst for low-temperature brewing. Diacetyl concentrations were low (0.3-0.5 mg/L), and polyphenols were lower than in commercial products and decreased as the fermentation temperature was decreased (126-50 mg/L). Ethyl acetate concentrations increased from 53 to 88 mg/L as the temperature was decreased, while the concentration of amyl alcohols at 3 degrees C (58 mg/L) was lower than half of that at 18 degrees C (125 mg/L). The beers produced at the end of the main fermentation had a fine clarity and a special fruity figlike aroma and taste, distinct from commercial products and more intense than beers produced by cells immobilized on other food-grade supports (gluten pellets or delignified cellulosic materials). GC-MS analysis did not show significant differences in the qualitative composition of the aroma compounds of the beers produced by immobilized and free cells.     

Effect of carbohydrate substrate on fermentation by kefir yeast supported on delignified cellulosic materials

The suitability of delignified cellulosic (DC) material supported kefir yeast to ferment raw materials that contain various single carbohydrates, for the production of potable alcohol and alcoholic drinks, is examined in this investigation. Results are reported of fermentations carried out with sucrose, fructose, and glucose in synthetic media. Repeated batch fermentations at various initial sugar concentrations of sucrose, fructose, and glucose were performed at 30 degrees C in the presence of the aforementioned biocatalyst. The results clearly show feasible yields in the range of 0.38-0.41 g/g, alcohol concentrations of 7.6-8.2% v/v, fermentation time of 90-115 h, and conversion of 92-96%. DC material supported kefir fermented 11-fold more rapidly than free cells and 9-fold more rapidly in comparison to kissiris supported kefir. The main volatile byproducts such as amyl alcohols (mixture of 2-methyl-1-butanol and 3-methyl-1-butanol), ethanal, and ethyl acetate were formed in all sugar fermentation products. The formation of 65-110 ppm of ethyl acetate is as high and even higher than that obtained with traditional wine yeasts. The increase of the initial concentration of sugar in the fermentation media resulted in an increase in contents of volatiles. The fine aroma that was obtained in the product of fructose could be attributed to the high percentage of ethyl acetate on total volatiles. The efficiency of DC material supported kefir was the same for the fermentations of individual sugars or a mixture of fructose, sucrose, and glucose. When whey with raisin extracts was fermented, lower yields were obtained but the aroma of the product was even better.     

Corn Endosperm Fermentation Using Endogenous Amino Nitrogen Generated by a Fungal Protease

Fractionating the corn kernel to separate endosperm from germ and pericarp improves corn ethanol processing by increasing fermentation throughput and generating salable coproducts. One fractionation technology, dry fractionation (DF), suffers from loss of germ‐derived nutrients and amino acids, resulting in poor fermentation performance. Such deficiencies may be addressed by increasing nitrogen and other nutritional supplementation. As an alternative to exogenous nitrogen source, we investigated the use of a fungal protease to generate free amino nitrogen (FAN) from corn endosperm. Incubation of endosperm with protease did not affect subsequent liquefaction and saccharification. FAN supplementation through proteolysis resulted in fermentation being 99% complete in 48 hr, compared to 93% maximum with urea supplementation. Viable cell growth rates were similar in FAN and urea‐supplemented fermentations. Urea and FAN addition resulted in similar fermentation characteristics and similar FAN consumption rates as with FAN alone, which was indicative that FAN was assimilated preferentially. Increased amounts of maltose remaining after fermentation were correlated with initial FAN concentrations in mash. This observed trend was implicated in ethanol yield reduction of 2 g/L at high protease loading (generating 1.6 mg of FAN/g of glucose substrate) compared to a urea control. Using a glucose and maltose solution, we confirmed higher residual maltose in fermentations supplemented with high FAN concentrations. Use of protease to generate optimal FAN concentration in mash (1.2 mg of FAN/g of glucose substrate) could improve economics of dry fractionated corn ethanol production by increasing fermentation rates and, consequently, reducing fermentation time.