Recovery of aroma compounds from a wine-must fermentation by organophilic pervaporation

This study investigates the recovery of a wine-must aroma profile, formed by Saccharomyces cerevisiae during a muscatel wine-must fermentation, using organophilic pervaporation. Experiments were carried out along two independent, but organoleptically similar, fermentations. The wine-must samples and the aroma concentrates obtained were characterized organoleptically by a sensory panel and analytically with regard to eight major wine-must components: four alcohols; three esters; and one monoterpenic compound. Pervaporation performance was studied under fermentation conditions, and the permeate concentration, partial fluxes, and enrichment of the respective compounds were determined. The muscatel wine-must aroma profile was recovered purely and faithful to its origin between wine-must densities of 1075 and 1055 g L. At the beginning of the fermentation, too few aromas were present in the must for recovery. Toward the end of the fermentation, high ethanol concentrations in the wine-must caused a dramatic enrichment of two esters in the permeate, whereas other components investigated seemed unaffected. This shift resulted in an unbalanced aroma. In conclusion, it was shown that organophilic pervaporation can be highly suitable for the continuous recovery of very complex and delicate aromatic profiles produced during microbial fermentation. Copyright 1999 John Wiley & Sons, Inc.     

Wine flavor and aroma

The perception of wine flavor and aroma is the result of a multitude of interactions between a large number of chemical compounds and sensory receptors. Compounds interact and combine and show synergistic (i.e., the presence of one compound enhances the perception of another) and antagonistic (a compound suppresses the perception of another) interactions. The chemical profile of a wine is derived from the grape, the fermentation microflora (in particular the yeast Saccharomyces cerevisiae), secondary microbial fermentations that may occur, and the aging and storage conditions. Grape composition depends on the varietal and clonal genotype of the vine and on the interaction of the genotype and its phenotype with many environmental factors which, in wine terms, are usually grouped under the concept of “terroir” (macro, meso and microclimate, soil, topography). The microflora, and in particular the yeast responsible for fermentation, contributes to wine aroma by several mechanisms: firstly by utilizing grape juice constituents and biotransforming them into aroma- or flavor-impacting components, secondly by producing enzymes that transform neutral grape compounds into flavor-active compounds, and lastly by the de novo synthesis of many flavor-active primary (e.g., ethanol, glycerol, acetic acid, and acetaldehyde) and secondary metabolites (e.g., esters, higher alcohols, fatty acids). This review aims to present an overview of the formation of wine flavor and aroma-active components, including the varietal precursor molecules present in grapes and the chemical compounds produced during alcoholic fermentation by yeast, including compounds directly related to ethanol production or secondary metabolites. The contribution of malolactic fermentation, ageing, and maturation on the aroma and flavor of wine is also discussed.     

Ranking the significance of fermentation conditions on the volatile organic compounds of Tuber melanosporum fermentation system by combination of head-space solid phase microextraction and chromatographic fingerprint similarity analysis

Tuber melanosporum is highly appreciated in culinary contexts due to its unique and characteristic aroma. T. melanosporum fermentation has been established as a promising alternative for fruiting bodies to produce volatile organic compounds (VOCs). In this work, a technique using a combination of chromatographic fingerprint similarity analysis, head-space solid phase microextraction and gas chromatography was developed to rank the significance of fermentation conditions on the VOCs profile during T. melanosporum fermentation. Omission tests indicated that the absence of major carbon source (i.e., sucrose) in the fermentation media had the most significant effect on the profile of VOCs, followed by the absence of yeast extract or peptone. Consideration of the culture conditions revealed that VOCs produced was the most significantly affected by temperature. These results indicated that it is possible to adjust the aroma of truffles via fermentation process control.     

Microbiological,rheological and aromaticcharacteristics of fermented Uji (an East African Sour Porridge)

Three methods for fermentation of uji were compared in laboratory trials: spontaneous, backslopping (using an inoculum from a previous fermentation) and use of a starter culture of lactic acid bacteria. Spontaneous fermentation resulted in the slowest decrease in pH, while the use of starter culture led to the lowest final pH (3.5). Coliforms were eliminated in less than 8 h using backslopping or starter culture, but increased in numbers during spontaneous fermentation. The viscosity of uji was only marginally affected by the method of fermentation. The aroma profile following spontaneous fermentation contained esters with fruity notes and ethanol and higher alcohols, while mainly organic acids was produced by fermentation with the starter culture. Backslopping led to the lowest production of almost all volatiles identified.     

A novel approach for the production of natural aroma compounds using agro-industrial residue

Experiments were carried out to study the production of aroma compounds from coffee husk by a fungal culture of C. fimbriata in solid state fermentation. Hot water treated coffee husk seemed to be useful substrate for aroma production in comparison to whole coffee husk or its water extract. Raw data were integrated in order to calculate the total volatiles (TV) accumulated during the fermentation using the Gompertz model. Glucose addition between 20-35% increased significantly the yields of aroma compounds. Supplementation of leucine further improved the TV production significantly (about 58%), specially the esters. When soybean oil was added, TV production was similar to the control, showing that the fungus was not able to use soybean oil for its primary metabolism, nor it acted as precursor for the synthesis of methyl ketones as reported for other fungi. Addition of saline solution drastically decreased the volatile production. Under optimized conditions, a total of 13 compounds were produced which included alcohols (2), aldehyde (1), ketones (2) and esters (8). Ethyl acetate was the prominent compound, followed by ethanol.     

Optimization of the production of aroma compounds by Kluyveromyces marxianus in solid-state fermentation using factorial design and response surface methodology

Studies were carried out for the production of aroma compounds in solid-state fermentation using factorial design and response surface methodology (RSM) experiments. Five agro-industrial residues were evaluated as substrate for cultivating a strain of Kluyveromyces marxianus. The results proved the feasibility of using cassava bagasse and giant palm bran (Opuntia ficus indica) as substrates to produce fruity aroma compounds by the yeast culture. In order to test the influence of the process parameters on the culture to produce volatile compounds, two statistical experimental designs were performed. The parameters studied were initial substrate pH, addition of glucose, cultivation temperature, initial substrate moisture and inoculum size. Using a 2(5) factorial design, addition of glucose and initial pH of the substrate was found statistically significant for aroma compounds production on palm bran. Although this experimental design showed that addition of glucose did not have a significant role with cassava bagasse, 2(2) factorial design revealed that glucose addition was significant at higher concentrations. Head-space analysis of the culture by gas chromatography showed the production of nine and eleven compounds from palm bran and cassava bagasse, respectively, which included alcohols, esters and aldehyde. In both the cases, two compounds remained unidentified and ethyl acetate, ethanol and acetaldehyde were the major compounds produced. Esters produced were responsible for the fruity aroma in both the cases. With palm bran, ethanol was the compound produced in highest concentration, and with cassava bagasse (both supplemented with 10% glucose), ethyl acetate was produced at highest concentration, accumulating 418 and 1395μmoll(-1) head-spaceg(-1) substrate in 72h, respectively.     

Use of pervaporation process for the recovery of aroma compounds produced by P. fermentans in sugarcane molasses

Bioprocess and Biosystems Engineering - Natural fruity aroma was produced during submerged fermentation by Pichia fermentans using sugarcane molasses as a cultivation broth. The aroma compounds...     

Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation

The multi-yeast strain composition of wine fermentations has been well established. However, the effect of multiple strains of Saccharomyces spp. on wine flavour is unknown. Here, we demonstrate that multiple strains of Saccharomyces grown together in grape juice can affect the profile of aroma compounds that accumulate during fermentation. A metabolic footprint of each yeast in monoculture, mixed cultures or blended wines was derived by gas chromatography - mass spectrometry measurement of volatiles accumulated during fermentation. The resultant ion spectrograms were transformed and compared by principal-component analysis. The principal-component analysis showed that the profiles of compounds present in wines made by mixed-culture fermentation were different from those where yeasts were grown in monoculture fermentation, and these differences could not be produced by blending wines. Blending of monoculture wines to mimic the population composition of mixed-culture wines showed that yeast metabolic interactions could account for these differences. Additionally, the yeast strain contribution of volatiles to a mixed fermentation cannot be predicted by the population of that yeast. This study provides a novel way to measure the population status of wine fermentations by metabolic footprinting.     

一种紫斑牡丹花茶的研制及其主要香气成分分析

以紫斑牡丹(Paeonia suffruticosa var. papaveracea)花瓣为原料,采用隔离窨制,对牡丹花茶窨制过程中花坯、配花量等主要影响因子进行研究。结果表明,密闭箱桶温度21 ℃、相对湿度90%、窨制时间48 h条件下经一窨一提获得的花茶,感官评审花与茶叶的协调度高、香气高锐持久、茶汤滋味醇正鲜爽。采用气相色谱-质谱联用仪分析花茶香气成分,新鲜花瓣与茶叶配比5:1窨制48 h的花茶苯乙醇、香叶醇、橙花醇含量较高,其香气高扬、茶汤醇正鲜爽,配比2.5:1的花茶上述成分含量和感官其次,而拌和型茶品透素欠鲜爽。发酵或发酵揉捻花瓣窨制的花茶乙醇、环氧芳樟醇及高级烷烃含量较高,其主要赋香物质低于新鲜花瓣含量,渥味明显,茶汤有浊气欠鲜爽。     

Microbial Species Diversity,Community Dynamics,and Metabolite Kinetics of Water Kefir Fermentation

Water kefir is a sour, alcoholic, and fruity fermented beverage of which the fermentation is started with water kefir grains. These water kefir grains consist of polysaccharide and contain the microorganisms responsible for the water kefir fermentation. In this work, a water kefir fermentation process was followed as a function of time during 192 h to unravel the community dynamics, the species diversity, and the kinetics of substrate consumption and metabolite production. The majority of the water kefir ecosystem was found to be present on the water kefir grains. The most important microbial species present were Lactobacillus casei/paracasei, Lactobacillus harbinensis, Lactobacillus hilgardii, Bifidobacterium psychraerophilum/crudilactis, Saccharomyces cerevisiae, and Dekkera bruxellensis. The microbial species diversities in the water kefir liquor and on the water kefir grains were similar and remained stable during the whole fermentation process. The major substrate, sucrose, was completely converted after 24 h of fermentation, which coincided with the production of the major part of the water kefir grain polysaccharide. The main metabolites of the fermentation were ethanol and lactic acid. Glycerol, acetic acid, and mannitol were produced in low concentrations. The major part of these metabolites was produced during the first 72 h of fermentation, during which the pH decreased from 4.26 to 3.45. The most prevalent volatile aroma compounds were ethyl acetate, isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate, which might be of significance with respect to the aroma of the end product.     

Determination of the intracellular concentration of ethanol in Saccharomyces cerevisiae during fermentation.

Considerable controversy exists concerning the intracellular concentration of ethanol in Saccharomyces cerevisiae during fermentation. This controversy results from problems in the measurement of the intracellular concentration of compounds like ethanol, which are being produced rapidly by metabolism and potentially diffuse rapidly from the cell. We used a new method for the determination of intracellular ethanol based on the exclusion of [14C]sorbitol to estimate the aqueous cell volume. This method avoided many of the technical problems in previous reports. Our results indicate that the extracellular concentrations of ethanol in fermenting suspensions of S. cerevisiae are less than or equal to those in the intracellular environment and do not increase to the high levels previously reported even during the most active stages of batch fermentation.     

Determination of the intracellular concentration of ethanol in Saccharomyces cerevisiae during fermentation

Considerable controversy exists concerning the intracellular concentration of ethanol in Saccharomyces cerevisiae during fermentation. This controversy results from problems in the measurement of the intracellular concentration of compounds like ethanol, which are being produced rapidly by metabolism and potentially diffuse rapidly from the cell. We used a new method for the determination of intracellular ethanol based on the exclusion of [14C]sorbitol to estimate the aqueous cell volume. This method avoided many of the technical problems in previous reports. Our results indicate that the extracellular concentrations of ethanol in fermenting suspensions of S. cerevisiae are less than or equal to those in the intracellular environment and do not increase to the high levels previously reported even during the most active stages of batch fermentation.     

Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates

The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid concentration decreased by more than half. These changes in the wine and distillate composition had a pronounced effect on the solvent or chemical aroma (associated with ethyl acetate and iso-amyl acetate) and the herbaceous and heads-associated aromas of the final distillate and the solvent or chemical and fruity or flowery characters of the Chenin blanc wines. This study establishes the concept that the overexpression of acetyltransferase genes such as ATF1 could profoundly affect the flavor profiles of wines and distillates deficient in aroma, thereby paving the way for the production of products maintaining a fruitier character for longer periods after bottling.     

Influence of wine fermentation temperature on the synthesis of yeast-derived volatile aroma compounds

The yeast Saccharomyces cerevisiae synthesises a variety of volatile aroma compounds during wine fermentation. In this study, the influence of fermentation temperature on (1) the production of yeast-derived aroma compounds and (2) the expression of genes involved in aroma compounds’ metabolism (ADH1, PDC1, BAT1, BAT2, LEU2, ILV2, ATF1, ATF2, EHT1 and IAH1) was assessed, during the fermentation of a defined must at 15 and 28°C. Higher concentrations of compounds related to fresh and fruity aromas were found at 15°C, while higher concentrations of flowery related aroma compounds were found at 28°C. The formation rates of volatile aroma compounds varied according to growth stage. In addition, linear correlations between the increases in concentration of higher alcohol and their corresponding acetates were obtained. Genes presented different expression profiles at both temperatures, except ILV2, and those involved in common pathways were co-expressed (ADH1, PDC1 and BAT2; and ATF1, EHT1 and IAH1). These results demonstrate that the fermentation temperature plays an important role in the wine final aroma profile, and is therefore an important control parameter to fine-tune wine quality during winemaking.     

Volatile compounds of Soumbala, a fermented African locust bean (Parkia biglobosa) food condiment

AIMS: To determine the profile of volatile compounds responsible for the aroma of Soumbala produced spontaneously and with pure and mixed cultures of Bacillus subtilis and Bacillus pumilus. METHODS AND RESULTS: Traditional and controlled fermentation trials of African locust bean with pure and mixed starter cultures of B. subtilis (B7, B9 and B15) and B. pumilus (B10) were performed. Aroma volatiles were analysed using Likens-Nikerson method coupled with gas chromatography and mass spectrophotometry. Sensory analysis of Soumbala as well as rice dishes prepared with each type of Soumbala were carried out by 10 panellists. In total 116 compounds were identified. They included pyrazines, aldehydes, ketones, esters, alcohols, acids, alkanes, alkenes, amines, pyridines, benzenes, phenols, sulphurs, furans and other compounds. Using principal component analysis for comparison, the aroma profiles of the Soumbala samples could be separated into three groups. The sensory evaluation showed variable acceptability. However, it was noticed that Soumbala samples produced with starter cultures were scored higher than traditionally prepared Soumbala. CONCLUSIONS: Aroma volatiles and organoleptic properties of Soumbala vary according to the Bacillus isolates involved in the fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus starter cultures for controlled production of Soumbala.     

多粮浓香型白酒中特征酵母菌与耐酸乳杆菌的关系

【背景】多菌种协同代谢是浓香型白酒发酵的根本特征之一。【目的】探究多粮浓香型白酒发酵过程中功能微生物菌株间的相互协作关系,为实现发酵过程优化提供理论基础。【方法】采用传统分离培养法获得了多粮浓香型白酒发酵过程中的特征酵母菌和耐酸乳杆菌,并探讨了共培养过程中菌株的相互关系以及主要挥发性代谢产物的变化。【结果】共分离到3株优势特征性酵母菌(KazachstaniahumilisZ1、PichiakudriavzeviiZ2、CandidaethanolicaZ3)和1株耐酸乳杆菌(Lactobacillus acetotolerans W)。K. humilis Z1和L. acetotolerans W共培养体系中耐酸乳杆菌数量明显少于纯培养L. acetotolerans W的菌体数量;3株酵母菌均一定程度抑制L. acetotolerans W产乳酸,K. humilis Z1能抑制L. acetotolerans W不产乳酸;K. humilis Z1纯培养与Z1W共培养体系的挥发性代谢产物的构成相似;纯培养P. kudriavzevii Z2与Z2W共培养体系的挥发性代谢产物差异明显,主要表现为共培养时乙酸乙酯和乳酸乙酯含量显著增加。【结论】在共培养体系条件下,产乙醇酵母菌对耐酸乳杆菌的乳酸代谢有抑制作用,而耐酸乳杆菌又对酵母菌的乙醇代谢有一定影响,这对多粮浓香型白酒品质调控和菌群间相互关系具有重要意义。     

Production of ethanol,organic acids and hydrogen: an opportunity for mixed culture biotechnology?

Anaerobic fermentation of biodegradable organic materials is usually carried out to obtain the final product, methane, a valuable energy source. However, it is also well known that various intermediates are produced in this process, e.g. ethanol, volatile organic acids and hydrogen. All these species have applications and value as fuels or chemicals. This paper shows a critical analysis of the potential of using anaerobic fermentation by mixed cultures to produce intermediates, e.g. ethanol, acetic, lactic and butyric acid and hydrogen, rather than methane. This paper discusses the current processes to produce these chemicals and compares them with the alternative approach of using open mixed cultures to produce them simultaneously via fermentation from renewable resources. None of these chemicals is currently produced via mixed culture fermentation: ethanol and lactic acid are usually produced in pure culture fermentation using food crops, e.g. corn or sugar cane, as starting materials; hydrogen, acetic and butyric acids are mainly produced via chemical synthesis from fossil fuel derived starting materials. A possible flow-sheet for the production of these chemicals from organic waste using mixed culture fermentation is proposed and the advantages and disadvantages of this process compared to current processes are critically discussed. The paper also discusses the research challenges which need to be addressed to make this process feasible.     

Modulating aroma compounds during wine fermentation by manipulating carnitine acetyltransferases in Saccharomyces cerevisiae

The wine yeast Saccharomyces cerevisiae is central in the production of aroma compounds during fermentation. Some of the most important yeast-derived aroma compounds produced are esters. The esters ethyl acetate and isoamyl acetate are formed from alcohols and acetyl-CoA in a reaction catalysed by alcohol acetyltransferases. The pool of acetyl-CoA available in yeast cells could play a key role in the development of ester aromas. Carnitine acetyltransferases catalyse the reversible reaction between carnitine and acetyl-CoA to form acetylcarnitine and free CoA. This reaction is important in transferring activated acetyl groups to the mitochondria and in regulating the acetyl-CoA/CoA pools within the cell. We investigated the effect of overexpressing CAT2, which encodes the major mitochondrial and peroxisomal carnitine acetyltransferase, on the formation of esters and other flavour compounds during fermentation. We also overexpressed a modified CAT2 that results in a protein that localizes to the cytosol. In general, the overexpression of both forms of CAT2 resulted in a reduction in ester concentrations, especially in ethyl acetate and isoamyl acetate. We hypothesize that overproduction of Cat2p favours the formation of acetylcarnitine and CoA and therefore limits the precursor for ester production. Carnitine acetyltransferase expression could potentially to be used successfully in order to modulate wine flavour.     

Characterization of kinetic parameters and the formation of volatile compounds during the tequila fermentation by wild yeasts isolated from agave juice

The production of aroma compounds during tequila fermentation using four native yeast strains isolated from agave juice was quantified at controlled (35 degrees C) and uncontrolled temperatures (room temperature) by gas chromatography (FID). Three of the four strains were identified as Saccharomyces cerevisiae (MTLI 1, MALI 1 and MGLI 1) and one as Kloeckera apiculata (MALI 2). Among the aroma compounds produced, acetaldehyde has the highest accumulation at the controlled temperature and before 50% of sugar was consumed. The S. cerevisiae strains produced ethyl acetate in almost the same quantity at a concentration of 5 mg/L and the K. apiculata produced six-times more (30 mg/L) than the S. cerevisiae strains, independent of the fermentation temperature. The rate and amount of 1-propanol, amyl alcohols and isobutanol production were affected by the type of yeast used. The K. apiculate strain produced 50% less of the higher alcohols than the Saccharomyces strains. The results obtained showed that indigenous isolated yeasts play an important role in the tequila flavor and suggest that mixtures of these yeasts may be used to produce tequila with a unique and desirable aroma.