Characterization of β-glucosidases from Hanseniaspora sp. and Pichia anomala with potentially aroma-enhancing capabilities in juice and wine

The properties of intracellular β-glucosidases produced from two yeast isolates identified as Hanseniaspora sp. BC9 and Pichia anomala MDD24 were characterized. β-Glucosidase from Hanseniaspora sp. BC9 was not inhibited by both 20% w/v fructose and 20% w/v sucrose and was slightly inhibited by glucose (> 40% relative β-glucosidase activity with 10% w/v glucose). β-Glucosidase from P. anomala MDD24 was inhibited by glucose, fructose and sucrose. In the presence of 4–12% v/v ethanol, β-glucosidase from P. anomala MDD24 was stimulated in range 110–130% relative activity whereas β-glucosidase from Hanseniaspora sp. BC9 was substantially inhibited in the presence of ethanol. Finally, juice and wine of the Muscat-type grape variety, Traminette, were selected to determine sugar-bound volatile aroma release, particularly terpenes, by the activity of those β-glucosidases. The results showed that high concentration of free aroma compounds were detected from Traminette juice treated with β-glucosidase from Hanseniaspora sp. BC9 and Traminette wine treated with β-glucosidase from P. anomala MDD24. The preliminary results with proposed an application of these enzymes in commercial wine production lead to more efficient of β-glucosidase from Hanseniaspora sp. BC9 in releasing desirable aromas during an early stage of alcoholic fermentation while β-glucosidase from P. anomala MDD24 is suitable at the final stage of alcoholic fermentation.     

Early discrimination of fungal species responsible of ochratoxin A contamination of wine and other grape products using an electronic nose

An electronic nose (e-nose) system using an array of metal oxide sensors (Fox 3000, Alpha MOS) was used to detect and discriminate two ochratoxigenic fungal species, Aspergillus carbonarius (Bain.) Thom and A. niger Van Tieghem, that are responsible for the contamination of wine and other wine grape products, using their volatile production patterns. Two well-known ochratoxigenic strains were used in this study: A. carbonarius A941 and A. niger A75. These strains were grown on three culture media, Czapek Dox modified (CDm) agar, yeast extract sucrose (YES) agar and white grape juice (WGJ) agar, and the volatile organic compounds produced in the headspace by these species were evaluated over periods of 48–120 h. The e-nose system was able to differentiate between the two species within 48 h of growth on YES and WGJ agar using principal component analysis (PCA), which accounted for 99.9% and 97.2% of the data respectively, in principal components 1 and 2, based on the qualitative volatile profiles. This differentiation was confirmed by cluster analysis of data. However, it was not possible to separate these species on CDm agar. Our results show that the two closely related ochratoxigenic species responsible for the contamination of wine and other wine grape products can be discriminated by the use of qualitative volatile fingerprints. This approach could have potential for rapid identification of A. carbonarius and A. niger on wine grape samples, thereby significantly reducing the time of detection of these ochratoxin A producing species.     

Analysis of volatile aroma constituents of wine produced from Indian mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) by GC-MS

Volatile aroma compounds are synthesized by wine yeast during wine fermentation. In this study the volatile aroma composition of two varieties of mango wine were determined to differentiate and characterize the wines. The wine was produced from the fruits of two varieties of mango cultivars namely Banginapalli and Alphonso. The volatile compounds formed in mango wine were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). Thirty-two volatile compounds in wines were determined of which four were new and unidentified present in lower concentration. Apart from the ethanol (8.5 ± 0.28 and 7.2 ± 0.28% v/v), 1-propanol (54.11 ± 0.33 and 42.32 ± 0.57 mg/l), isobutyl alcohol (102 ± 1.57 and 115.14 ± 2.88 mg/l) and isoamyl alcohol (123 ± 2.88 and 108.40 ± 0.23 mg/1) were found to be the major flavouring higher alcohols in the mango wines produced from the fruits of Banginapalli and Alphonso respectively. Ethyl acetate (35 ± 0.57 and 30.42 ±1.15 mg/l) was the major ester component in both wines produced. Besides, other esters like ethyl octonoate, ethyl hexanoate and ethyl decanoate were also present in the wines. Cyclohexane methanol (1.45 ± 0.11 mg/l) was present only in wine made from Banginapalli and β-phenylethyl butanoate (0.62 ± 0.01 mg/1) was found only in Alphonso wine. The results demonstrate that the wine prepared from Banginapalli variety had better aroma composition and good taste than that from the Alphonso variety.     

The production of hydrogen sulphide and other aroma compounds by wine strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in synthetic media with different nitrogen concentrations

The aim of this study was to evaluate the combined effect of initial nitrogen content on the production of hydrogen sulphide and other volatile compounds during alcoholic fermentation. For that propose, three commercial wine strains of Saccharomyces cerevisiae were used to ferment synthetic grape juice media under different nitrogen concentrations. H₂S was measured throughout fermentations and other aroma compounds were analyzed at the end of the experiments. The trigger levels at which an inverse relationship between the initial nitrogen present in media and total H₂S production varied among the three strains tested. For UCD522 and PYCC4072, the highest H₂S levels were produced in media with 267 mg N l⁻¹ of initial nitrogen, whereas the lowest levels were detected with nitrogen limitation/starvation conditions (66 mg N l⁻¹). Moreover, 21 other aroma compounds belonging to different chemical classes were identified and quantified by solid phase micro-extraction (SPME) coupled to gas chromatography–mass spectrometry (GC–MS). The initial nitrogen concentration more than yeast strain had a decisive effect on the final aroma composition, suggesting that modulation of nutrients emerges as a useful tool for producing desired flavour and odour compounds.     

Use of interdelta polymorphisms of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains to monitor population evolution during wine fermentation

The industrial use of starter cultures containing a consortium of different strains from the same species is nowadays seen as a possible strategy to enhance the organoleptic complexity of wines. To assess the relative contribution of each strain to the final product it is essential to quantify population evolution during the wine fermentation process, which requires strain-specific methods to identify and differentiate each strain. In the present study, a molecular method based on analysis of the polymorphisms exhibited by the PCR-amplification of the delta regions of three Saccharomyces cerevisiae strains was developed. A set of three pairs of primers (delta1–delta2, delta12–delta2, delta12–delta21) was used for each strain, and analysis of the resulting polymorphism patterns showed that the delta12–delta2 primer pair exhibited the highest resolution and discriminatory power. Thus, this pair of primers was selected to monitor the population evolution of a laboratory-scale wine fermentation performed in synthetic grape juice that was inoculated with similar amounts of each strain. The results showed that all strains grew together during the exponential growth phase (2–3 days) and maintained high cell density values (10⁶–10⁷ cfu ml⁻¹) throughout the stationary growth phase without significantly changing their relative population proportion, thus indicating that each strain can influence the chemical composition and final flavor of wine, albeit at different levels. This study also showed that PCR-amplification of DNA delta sequences of S. cerevisiae strains is a reproducible, strain-specific and simple method that can be used successfully to monitor yeast strain population dynamics during wine fermentations.     

Stability ofAlternaria toxins in fruit juices and wine

Alternaria alternata is a common fungal parasite on fruits and other plants and produces a number of mycotoxins, including alternariol (3,7,9-trihydroxy-1-methyl-6H-dibenzo [b,d]pyran-6-one), alternariol monomethyl ether (3,7-dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one), and the mutagen altertoxin I {[1S-(1α,12aβ,12bα)] 1,2,11,12,12a, 12b-hexahydro-1,4,9,12a-tetrahydroxy-3,10-perylenedione}. Alternariol and alternariol monomethyl ether have previously been detected in some samples of fruit beverages. Stability studies of these toxins as well as altertoxin I added to fruit juices and wine (10–100 ng/mL) were carried out. To include altertoxin I in the analysis, cleanup with a polymer-based Varian Abselut solid phase extraction column was used, as recoveries from C-18 columns were low. The stabilities of alternariol and alternariol monomethyl ether in a low acid apple juice containing no declared vitamin C were compared with those in the same juice containing added vitamin C (60 mg/175 ml); there were no apparent losses at room temperature over 20 days or at 80°C after 20 min. in either juice. Altertoxin I was moderately stable in pH 3 buffer (75% remaining after a two week period). Furthermore, altertoxin I was stable or moderately stable in three brands of apple juice tested over 1–27 day periods and in a sample of red grape juice over 7 days. It is concluded that altertoxin I is sufficiently stable to be found in fruit juices and should be included in methods for alternariol and alternariol monomethyl ether.     

Analysis of wines, grape juices and cranberry juices forAlternaria toxins

Sixty six samples of red and white wine from Ontario (VQA), British Columbia (VQA), Québec (“vins artisanaux”), imported wines (from Italy, South America and USA) and Canadian and US grape and cranberry juices were analysed for theAlternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME). After cleanup on aminopropyl SPE columns, AOH and AME were initially determined by reversed phase LC with UV detection. Positive sample extracts were re-analysed by LC-tandem negative ion electrospray mass spectrometry (MS/MS) in multiple reaction mode. Overall mean method recoveries measured by LC-UV were 93% for AOH and 81% for AME. Limits of detection in wine (and juice) by LC-UV for AOH were 0.8 (0.4) ng/ml and for AME were 0.5 (0.4) ng/ml; they were below 0.01 ng/ml by LC-MS/MS. As determined by LC-MS/MS, AOH was found in 13/17 Canadian red wines at levels of 0.03 to 5.02 ng/ml and in 7/7 imported red wines at 0.27–19.4 ng/ml, usually accompanied by lower concentrations of AME. Red grape juices (5 positive/10 samples) contained only sub ng/ml levels of AOH or AME except for one sample (39 ng AME/ml). White wines (3/23 samples), white grape juices (0/4 samples) and cranberry juices (1/5 samples) contained little AOH/AME (≤1.5 ng/ml). Presented at the World Mycotoxin Forum, Noordwijk, The Netherlands, November 10–11, 2005     

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.     

Yeast identification in grape juice concentrates from Argentina

Aims: The purpose of this study was to identify yeast species present in spoiled and unspoiled grape juice concentrates from Argentine industries. Methods and Results: Osmophilic and osmotolerant yeasts were isolated from spoiled – visually effervescent – and unspoiled – without any visible damage – grape juice concentrates by the spread‐plate technique in two culture media. Yeast identification was done by classical and molecular methods. Zygosaccharomyces rouxii was the only species isolated from spoiled grape juice concentrates. In unspoiled samples, five different species were identified: Z. rouxii was isolated at a higher frequency, followed in decreasing order by Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia anomala and Kluyveromyces delphensis. Conclusions: Yeasts isolated from grape juice concentrates were characterized by a limited taxonomic diversity, where Z. rouxii was the main species isolated. Significance and Impact of the Study: Grape production in Argentina is mainly devoted to the industry where wine and grape juice concentrates represent major types of commercial products. Little information on common yeast contaminants is available for grape juice concentrates. This study constitutes the first report of osmophilic yeast species present in spoiled and unspoiled grape juice concentrates elaborated in Argentina.     

In Vivo PCR-DGGE Analysis of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Oenococcus oeni</Emphasis> Populations in Red Wine

In order to monitor Lactobacillus plantarum and Oenococcus oeni in red wine produced with Italian grape (variety “Primitivo di Puglia”), a polymerase chain reaction– denaturing gradient gel electrophoresis (PCR-DGGE) approach using the rpoB as gene target was established. Wine was treated or not with potassium metabisulphite and supplemented with a commercial bacterial starter of O. oeni to encourage malolactic fermentation. Samples were taken from the vinification tanks at 4, 10, 16, 22, and 28 days after the start of alcoholic fermentation. Genomic DNA was directly isolated from wine and identification of lactic acid bacteria was performed using primers rpoB1, rpoB1O, and rpoB2 able to amplify a region of 336 bp corresponding to the rpoB gene. Amplified fragments were separated in a 30–60% DGGE gradient, and the ability of the PCR-DGGE analysis to distinguish L. plantarum and O. oeni was assessed. The results reported suggest that the PCR-DGGE method, based on the rpoB gene as molecular marker, is a reproducible and suitable tool and may be of great value for wine makers in order to monitor spoilage microorganisms during wine fermentation.     

Nutrient utilization profile of Saccharomyces Cerevisiae from palm wine in tropical fruit fermentation

The nutrient utilization pattern of Saccharomyces cerevisiae from palm wine was studied using tropical fruits as substrate. Starter cultures were prepared by growing 15-18 h old stock cultures of the yeast in successively larger bottles containing pasteurized fruit must. Microvinification, substrate utilization and assay of yeast activity were performed. Soluble solute (SS) content of the juices ranged from 10-18 Brix. Pinapple must had the highest SS content (18 Brix) while pawpaw had a low SS value of 10 Brix. These SS values were low compared to that of grape juice. The wines produced from the fruit must had percentage alcohol levels ranging from 10.6 to 12.6. Volatile activity ranged from 0.25 to 0.32 while crude protein values ranged from 0.58 to 0.68%. Palm wine yeast and all the other yeast strains fermented and utilized the fruit must for growth with specific growth rates ranging from 0.18 to 0.22. Sugar loss in Brix was gradual for all the fruit musts from 20.0-24.0 Brix to a range of 4.8 to 6.0 Brix. Pineapple was highly preferred for tropical wine making. Mango, cashew and pawpaw had equal ranking for commercial scale fermentation though more sugar will be needed to ameliorate cashew and pawpaw than mango juice. Palm wine yeast (OW-11) compared favourably with the other wine yeasts (CBS 8066 and ATCC 4126) both in nutrient utilization pattern and growth performance. A high degree of adaptability was observed in palm wine yeast recommands it for industrial wine production.     

The development of techniques for tissue culture of mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) var. Carabao and successful transfer of <Emphasis Type="Italic">ex vitro</Emphasis>-grafted plants to soil and the field

The mango, a virtually neglected fruit before the advent of nitrate-induced flowering, is now the third leading export fruit in the Philippines, with the ‘Carabao’ mango being the only variety exported. Delay or control of ripening, as well as the ability to program market availability, would further increase the value of mango. Prolonging its shelf life and improving other characteristics can be achieved through biotechnology, requiring a reliable tissue culture regeneration protocol, and a successful transfer-to-soil system. Between 2004 and 2008, immature fruits were collected and cultured in vitro. Primary somatic embryo (SE) induction ranged from 16–100% depending on the strain, collection time, and tree source. A single SE was required to initiate SE proliferation. When subcultured, the SEs proliferated, germinated, and produced plantlets. Initial shoot formation was 8–64%, while succeeding true leaf formation was reduced to 0–36%. In some cultures, abscission and browning were observed, but gas chromatography assays ruled out ethylene as the cause. Regenerated plants survived transfer to soil, but at low percentages. While it took 1 y to develop the tissue culture protocol, it took 3 y to develop the technique for successful transfer of plantlets to soil, and to the field. This is the first report, of a polyembryonic mango, variety Carabao, for which successful tissue culture and field transplantation systems were developed. The technology is equally important as a way to rapidly propagate uniform rootstocks of superior ‘Carabao’ strains or other varieties, for generation of variability, genetic transformation, and for germplasm conservation and exchange.     

A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice

E thiraj , S. & S uresh , E.R. 1985. A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice. Journal of Applied -Bacteriology 59 , 239–242.
A strain of Leuconostoc oenos was isolated from a blown can of mango juice. Various tests to identify and characterize the bacterium suggested that it could be a strain of L. oenos . This is the first report of L. oenos as a spoilage organism in fruit products other than wine.     

Evidence for exopolysaccharide production by Oenococcus oeni strains isolated from non‐ropy wines

Aims: The aim of this study was to assess the exopolysaccharide (EPS) production capacities of various strains of Oenococcus oeni, including malolactic starters and strains recently isolated from wine . Methods and Results: Fourteen O. oeni strains displaying or not (PCR check on genomic DNA) the gtf gene generally associated with β‐glucan formation and ropiness were grown on grape juice medium, dialysed MRS‐derived medium or synthetic medium. The soluble polysaccharides (PS) remaining in the culture supernatant were alcohol precipitated, and their concentration was quantified by the phenol‐sulfuric method. Most of the O. oeni strains studied produced significant amounts of EPS, independently of their genotype (gtf+ or gtf?). The EPS production was not directly connected with growth and could be stimulated by changing the growth medium composition. The molecular weight distribution analysis and attempts to determine the PS chemical structure suggested that most strains produce a mixture of EPS. Conclusion: Oenococcus oeni strains recently isolated from wine or cultivated for many generations as a malolactic starter are able to produce EPS other than β‐glucan. Significance and Impact of the Study: These EPS may enhance the bacteria survival in wine (advantage for malolactic starters) and may contribute to the wine colloidal equilibrium.     

Mycotoxin production from fungi isolated from grapes

AIMS: In order to assess the potential for producing mycotoxins, fungi were isolated from wine producing grapes. METHODS AND RESULTS: The isolates were identified and Penicillium expansum, the most well recognized mycotoxin producer, was analysed for mycotoxin production by TLC. Many of the strains produced patulin and/or citrinin, often depending on whether they were grown on a grape or yeast extract sucrose media. CONCLUSION: Citrinin was produced by all strains grown in the yeast extract sucrose medium, but only one strain (from 51) was able to produce this compound in grape juice medium. Patulin was produced in the yeast extract medium by 20 strains and in grape juice medium by 33 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of mycotoxins in wine producing grapes is discussed. Grapes contamination with patulin seems not to contribute to wine contamination, and no ochratoxin producing fungi was identified.     

Killer Toxin of Kluyveromyces phaffii DBVPG 6076 as a Biopreservative Agent To Control Apiculate Wine Yeasts

The use of Kluyveromyces phaffii DBVPG 6076 killer toxin against apiculate wine yeasts has been investigated. The killer toxin of K. phaffii DBVPG 6076 showed extensive anti-Hanseniaspora activity against strains isolated from grape samples. The proteinaceous killer toxin was found to be active in the pH range of 3 to 5 and at temperatures lower than 40°C. These biochemical properties would allow the use of K. phaffii killer toxin in wine making. Fungicidal or fungistatic effects depend on the toxin concentration. Toxin concentrations present in the supernatant during optimal conditions of production (14.3 arbitrary units) exerted a fungicidal effect on a sensitive strain of Hanseniaspora uvarum. At subcritical concentrations (fungistatic effect) the saturation kinetics observed with the increased ratio of killer toxin to H. uvarum cells suggest the presence of a toxin receptor. The inhibitory activity exerted by the killer toxin present in grape juice was comparable to that of sulfur dioxide. The findings presented suggest that the K. phaffii DBVPG 6076 killer toxin has potential as a biopreservative agent in wine making.     

Fermentation behaviour and volatile compound production by agave and grape must yeasts in high sugar Agave tequilana and grape must fermentations

Few studies have been performed on the characterization of yeasts involved in the production of agave distilled beverages and their individual fermentation properties. In this study, a comparison and evaluation of yeasts of different origins in the tequila and wine industries were carried out for technological traits. Fermentations were carried out in high (300 g l⁻¹) and low (30 g l⁻¹) sugar concentrations of Agave tequilana juice, in musts obtained from Fiano (white) and Aglianico (red) grapes and in YPD medium (with 270 g l⁻¹ of glucose added) as a control. Grape yeasts exhibited a reduced performance in high-sugar agave fermentation, while both agave and grape yeasts showed similar fermentation behaviour in grape musts. Production levels of volatile compounds by grape and agave yeasts differed in both fermentations.     

Enhanced Production of β-Carotene by Recombinant Industrial Wine Yeast Using Grape Juice as Substrate

In this study, both recombinant Saccharomyces cerevisiae T73-63 and FY-09 derived from the industrial wine yeast T73-4 and laboratory yeast FY1679-01B, respectively, were constructed and compared for their β-carotene production in real grape juice. The results showed that highest β-carotene content (5.89 mg/g) was found in strain T73-63, which was 2.1 fold higher than that of strain FY-09. Although the cell growth was inhibited by the metabolic burden induced by the production of heterogeneous β-carotene, the pigment yield in T73-63 was still 1.7 fold higher than that of FY-09. Furthermore, high contents of ergosterol and fatty acid were also observed in T73-63. These results suggest that industrial wine yeast has highly active metabolic flux in mevalonate pathway, which leads to more carbon flux into carotenoid branch compared to that of laboratory yeast. The results of this study collectively suggest that in the application of recombinant strains to produce carotenoid using agro-industrial by-products as substrate, the suitable host strains should have active mevalonate pathway. For this purpose, the industrial wine yeast is a suitable candidate.     

Comparison of soluble proteins in juice and wine from Koshu grapes

Proteins were separated from Koshu grape juice and wine by precipitation with ammonium sulfate. The protein fractions were further fractionated by gel electrophoresis and gel isoelectric focusing, followed by amino acid analyses. The juice contained more than eleven protein fractions with molecular weights between 13,000 and 65,000, and their isoelectric points were between 3.6 and 10.5. The wine also contained more than eleven protein fractions with molecular weights between 21,000 and 65,000, while their isoelectric points were between 3.6 and 11.0. All the juice proteins and some major wine proteins were glycoproteins. The same three protein fractions were present in both juice and wine. The other juice proteins were lost during wine-making and thus, were not detected in the wine. About half of the proteins detected in the wine were not observed in the juice. Some juice proteins were bound to the flavonoid phenolics extracted from the wine and were removed as insoluble precipitates. There was specific interaction between wine flavonoids and juice proteins.     

Killer toxin of Kluyveromyces phaffii DBVPG 6076 as a biopreservative agent to control apiculate wine Yeasts.

The use of Kluyveromyces phaffii DBVPG 6076 killer toxin against apiculate wine yeasts has been investigated. The killer toxin of K. phaffii DBVPG 6076 showed extensive anti-Hanseniaspora activity against strains isolated from grape samples. The proteinaceous killer toxin was found to be active in the pH range of 3 to 5 and at temperatures lower than 40 degrees C. These biochemical properties would allow the use of K. phaffii killer toxin in wine making. Fungicidal or fungistatic effects depend on the toxin concentration. Toxin concentrations present in the supernatant during optimal conditions of production (14.3 arbitrary units) exerted a fungicidal effect on a sensitive strain of Hanseniaspora uvarum. At subcritical concentrations (fungistatic effect) the saturation kinetics observed with the increased ratio of killer toxin to H. uvarum cells suggest the presence of a toxin receptor. The inhibitory activity exerted by the killer toxin present in grape juice was comparable to that of sulfur dioxide. The findings presented suggest that the K. phaffii DBVPG 6076 killer toxin has potential as a biopreservative agent in wine making.