A Tobacco necrosis virus D isolate from Olea europaea L.: viral characterization and coat protein sequence analysis

A virus isolated from Olea europaea L. grown in Portugal, was identified as a member of the species Tobacco necrosis virus D (TNV-D, genus Necrovirus, family Tombusviridae), based on the molecular and serological properties of the purified virus particles. The genomic region encoding the coat protein (CP) of this isolate (named GP isolate) was amplified by RT-PCR and the cDNA was cloned and sequenced. The CP gene encodes a predicted protein of 269 amino acids showing high identity (86.2%) to TNV-D coat protein sequence. Phylogenetic analysis based on necroviruses CP sequences, confirmed GP as a TNV-D isolate. The alignment with homologous TNV-D CP sequences revealed four conserved amino acids involved in Ca2+ binding as well as the plant virus icosahedral capsid protein "S" signature. Based on the determined nucleotide sequence, specific primers were designed and successfully used in RT-PCR for virus diagnosis in naturally infected olive trees

First characterization of infectious cDNA clones of Olive mild mosaic virus

Full-length cDNA clones of an Olive mild mosaic virus (OMMV) isolate were constructed in order to find infectious cDNA clones. The sequencing of three individual full-length clones revealed some differences between them. In vitro transcription of these clones was performed and the effect of spontaneous mutations in the biological behaviour of the in vitro transcripts was evaluated by symptomatology, RNA accumulation and virus replication in inoculated plants. In vitro synthesized RNA from one of these clones was found to mimic the wild-type OMMV, making it useful in future studies on protein structure and function by site directed mutagenesis of individual genes. This is the first report on constructing full-length cDNA clones of OMMV from which infectious RNAs can be transcribed in vitro

Biological and Molecular Characterization of Olive latent virus 1

Olive latent virus 1 (OLV-1) belongs to the Necrovirus genus, Tombusviridae family and is pathogenic to olive, citrus and tulip plants. It is easily mechanically transmissible to indicator plants causing necrotic lesions and can be transmitted through the soil into the plant roots in the absence of biological vectors. Infected cells contain virus aggregates, inclusions made up of excess of viral coded peptides and extensive vesiculation in the cytoplasm. The virions are isometric with ca. 30 nm, possess a monopartite single-stranded positive-sense RNA genome sized 3700 nt with 5 open reading frames (ORFs) and small inter cistronic regions. ORF 1 encodes a polypeptide with a molecular weight of 23 kDa and the read through of its amber stop codon results in ORF 1 RT that encodes the virus RNA dependent RNA polymerase with 82 kDa. ORF2 and ORF3 encode two small peptides, with 8 kDa and 6 kDa, respectively, which appear to be involved in the virus cell-to-cell movement. ORF 4 is located in the 3′-terminal and encodes a protein with 30 kDa identified as the viral coat protein. The complete genomic sequences of two well characterized OLV-1 isolates (obtained from citrus and olive) are similar, revealing an overall nucleotide sequence identity of 95%. The electrophoretic profile of the dsRNAs recovered from infected tissues exhibits three major species with ca. 3.7, 1.5, and 1.3 kbp. Application of molecular techniques based on PCR and on dot blot hybridization has been successfully used for routine diagnosis of OLV-1 infections

Alcoholic beverage from cheese whey: identification of volatile compounds

A spirit was produced from cheese whey continuous fermentation by Kluyveromyces marxianus, and the volatile compounds present in this alcoholic drink were identified. Fermentation was performed in a 1000 L reactor at 30 ºC, with initial lactose concentration and hydraulic residence time of 50 g/L and 5 h (dilution rate 0.2 h-1), respectively. The raw spirit (35.4% v/v ethanol) was obtained by distilling the fermentation broth in a pot still. Volatile compounds were quantified by gas chromatography, either by direct injection (with flame ionization detector) or after dichloromethane extraction (coupled with mass spectrometry). Higher alcohols were quantitatively the most abundant group of volatile compounds in this drink, with isoamyl alcohol, isobutanol, and 1-propanol being the most abundant (886.6 mg/L, 542.1 mg/L and 266 mg/L, respectively). Among the total esters, ethyl acetate showed the highest concentration (138.2 mg/L). Other components, such as the terpenes linalool, α-terpineol and geraniol were also identified. Considering that the quality of an alcoholic drink can be evaluated by the ratio between isoamyl alcohol/2-methyl-1-propanol and 2-methyl-1-propanol/1-propanol, which have to be higher than unity, it can be concluded that a novel spirit of acceptable organoleptic character could be produced by whey fermentation with K. marxianus

Characterisation of volatile compounds in an alcoholic beverage produced by whey fermentation

An alcoholic beverage (35.4% v/v ethanol) was produced by distillation of the fermented broth obtained by continuous whey fermentation with a lactose-fermenting yeast Kluyveromyces marxianus. Forty volatile compounds were identified in this drink by gas chromatography. Higher alcohols were the most abundant group of volatile compounds present, with isoamyl, isobutyl, 1-propanol, and isopentyl alcohols being found in highest quantities (887, 542, 266, and 176 mg/l, respectively). Ethyl acetate had the highest concentration (138 mg/l) among the esters. Besides higher alcohols and esters, other components, including aldehydes, acids and terpenes were also identified in the whey spirit. Considering that the quality of an alcoholic beverage can be evaluated by the relation between isoamyl alcohol/2-methyl-1-propanol and 2-methyl-1-propanol/1-propanol, which have to be higher than unity, it was concluded that a novel spirit of acceptable organoleptic characteristics can be produced by cheese whey continuous fermentation with K. marxianus

Effect of the support material and storage conditions of immobilized lactic acid bacteria on malolactic fermentation of white wine

In this work, the lactic acid bacterium Oenococcus oeni was immobilized on three different natural materials (namely corn cobs, grape skins and grape stems) and used to induce malolactic fermentation in white wine. Additionally, the biocatalyst reuse after different periods of storage in cold or in hot environments was also evaluated. The resistance of the immobilized lactic acid bacterium against inhibitors was determined by performing the MLF in presence of high SO2 concentration. Immobilization occurred in situ during the fermentation, which was performed in 500 mL Erlenmeyer flasks containing 6 g of support material, 1 g/L cells of O. oeni and 200 mL of complex medium. Fermentations were carried out in duplicate, and samples were taken periodically for the estimation of glucose, fructose and malic acid consumption, and lactic acid production. At the end of the fermentation (16 h), since all the assays presented similar results, different strategies were adopted. One of them consisted on the recovery of the corn cobs with immobilized cells, and subsequent storage of this biocatalyst at 5 ºC during 31 d. The flasks containing grape skins and stems were directly stored at 25 ºC during 27 d and 37 d, respectively. After these periods, the support materials with immobilized cells were recovered, washed with sterilized distilled water and added to 200 mL of white wine for conducting MLF, which was performed during 18 d. Subsequently, the biocatalysts were recovered, washed and added to 200 mL of white wine for conducting MLF in presence of 32 mg/L of free SO2, which was maintained during 17 d. Malic acid consumption and lactic acid production was observed during all the MLF, independently of the used support material. However, fermentation runs with cells immobilized on grape skins gave the best results, providing the highest lactic acid concentration and also high conversion of malic acid. The presence of high SO2 concentration (32 mg/L) did not affect the conversion of malic acid for cells immobilized in grape skins and stems, and gave similar results of produced lactic acid (3.60 g/L and 2.90 g/L, for grape skins and stems, respectively). The presence of high SO2 concentration strongly affected the conversion of malic acid by cells immobilized on corn cobs. Oenococcus oeni immobilized on grape skins and grape stems can be successfully used on MLF even after long periods of storage at 25 oC, and in the presence of high SO2 concentration

Influence of concentration and type of support material on the immobilization of Oenococcus oeni

The malolactic fermentation (MLF) in winemaking is a complex and difficult to control biological process . The implementation of MLF is very important for wines produced in cold regions as it reduces the acidity, brings biological stability and may improve the organolept ic characteristics of the product. MLF normally occurs spontaneously during storage of a new wine and is usually a very slow process that can undergo for we eks and even months, and not always give a satisfactory result. The use of immobilized lactic acid bacteria during MLF helps to accelerate the process and also simplifies the control of its extension. However, t he material to be used as immobilization support must be carefully chosen in order to not negatively affect the final product, and should also be cheap, abundant i n nature, and of food grade purity. The aim of the present work was to evaluate differe nt low cost natural materials of food grade purity (na mely corn cobs, grape stems and grape skins) and their concentration, for immobilization of Oenococcus oeni for use on MLF

Selection of natural materials for Saccharomyces cerevisiae immobilization

Immobilized cells systems have been considered as a promising alternative to improve the performance of biotechnological processes, since in these systems, immobilized cells completely maintain their biological functions with increased stability that may often lead to increased cell productivity. However, the correct selection of immobilization carrier is essential to design an effective system to each particular purpose. The objective of the present study was to find a cheap and abundant natural material suitable for immobilization of Saccharomyces cerevisiae yeast, which is usually used in fermentation processes for wine production. Four different carriers were evaluated, namely grape seeds, grape skin, grape stems and corn cobs. To be used in the experiments, the carriers were washed with water and dried to constant weight. The inoculum was prepared by cultivating the yeast in YPD medium under static conditions for 24 h and 30 ºC. Fermentation runs were performed in semi-defined medium composed by: glucose (120 g/L), yeast extract (4 g/L), (NH4)2SO4 (1 g/L), KH2PO4 (1 g/L), and MgSO4 (5 g/L). The assays were carried out in 500 mL Erlenmeyer flasks containing 200 mL of medium and 2 g of material carrier, statically incubated at 30 C for 24 h. Samples were taken periodically for estimation of biomass, glucose consumption and ethanol production. Corn cobs and grape skins were the best material carriers for S. cerevisiae immobilization, since they immobilized the highest amount of cells (22.2 ± 0.9 mg/g and 25.1± 10.8 mg/g, respectively) and fermentation with these immobilized cells gave elevated ethanol yield (YP/S = 0.51 and 0.49 g/g, respectively) and productivity (QP = 3.35 and 3.41 g/(Lh), respectively). These results are of great interest since the material did not require any pre-treatment to be used as immobilization carrier

Malolactic fermentation with Oenococcus oeni immobilized on natural materials

Bioprocesses with immobilized cells is a fast expanding field of investigation due to the various advantages of this system when compared to conventional free cells fermentations. One of the advantages of this system is the easier control of the fermentation process, which is of great importance especially for processes that are complex and difficult to control, like the malolactic fermentation (MLF) in winemaking. The implementation of MLF is very important for wines produced in cold regions as it reduces the acidity, brings biological stability and may improve the organoleptic characteristics of the product. MLF normally occurs spontaneously during storage of a new wine and is usually a very slow process that can undergo for weeks and even months, and not always give a satisfactory result. The use of immobilized lactic acid bacteria during MLF helps to accelerate the process and also simplifies the control of its extension. However, the material to be used as immobilization support must be carefully chosen in order to not negatively affect the final product, and should also be cheap, abundant in nature, and of food grade purity. The aim of the present work was to find low cost natural materials of food grade purity, suitable for immobilization of Oenococcus oeni for use on MLF. Four natural materials, including corn cobs, grape stems, grape seeds and grape skins were evaluated. Immobilization and fermentation occurred simultaneously in 500 mL Erlenmeyer flasks containing 2 g of support material and 200 mL of synthetic medium inoculated with 1 g/L cells of O. oeni. Fermentations were carried out in duplicate, and samples were taken every 2 h for estimation of free biomass, glucose, fructose and malic acid consumption, and lactic acid production. For comparison, free cells assays under the same conditions described above were also performed. Fermentations with immobilized cells gave better results than the assays containing only free cells in suspension. Among the support materials, corn cobs, grape skins and grape stems proportioned the best results. Grape skins immobilized the highest amount of cells (40.3 mg/g) followed by corn cobs (31.9 mg/g) and grape stems (30.9 mg/g). Fermentations with cells immobilized in corn cobs and grape stems achieved the highest lactic acid productivities [4.06 g/(L h) and 4.03 g/(L h), respectively]. It was concluded that such materials are suitable for use as support for immobilization of Oenococcus oeni during malolactic fermentation