Polyphasic identification of yeasts isolated from bark of cork oak during the manufacturing process of cork stoppers

A two-step protocol was used for the identification of 52 yeasts isolated from bark of cork oak at initial stages of the manufacturing process of cork stoppers. The first step in the identification was the separation of the isolates into groups by their physiological properties and RFLPs of the ITS-5.8S rRNA gene. The second step was the sequencing of the D1/D2 domains of the 26S rRNA gene of selected isolates representing the different groups. The results revealed a predominance of basidiomycetous yeasts (11 species), while only two species represented the ascomycetous yeasts. Among the basidiomycetous yeasts, members representing the species Rhodosporidium kratochvilovae and Rhodotorula nothofagi, that have been previously isolated from plant material, were the most abundant. Yeasts pertaining to the species Debaryomyces hansenii var. fabryii, Rhodotorula mucilaginosa and Trichosporon mucoides were isolated in small numbers.     

Unveiling the fungal mycobiota present throughout the cork stopper manufacturing process

A particular fungal population is present in the main stages of the manufacturing process of cork discs. Its diversity was studied using both dependent (isolation) and independent culture methods (denaturing gel gradient electrophoresis and cloning of the ITS1-5.8S-ITS2 region). The mycobiota in the samples taken in the stages before and after the first boiling seems to be distinct from the population in the subsequent manufacturing stages. Most isolated fungi belong to the genera Penicillium, Eurotium and Cladosporium. The presence of uncultivable fungi, Ascomycota and endophytes in raw cork was confirmed by sequencing. The samples taken after the first boiling contained uncultivable fungi, but in a few samples some isolated fungi were also detected. The main taxa present in the following stages were Chrysonilia sitophila, Penicillium glabrum and Penicillium spp. All applied techniques had complementary outcomes. The main factors driving the shift in cork fungal colonization seem to be the high levels of humidity and temperature to which the slabs are subjected during the boiling process.     

Enzymatic activity of micro-organisms isolated from cork wine stoppers

The production of enzymes by micro-organisms which are found on vegetal substrates is important due to their ability to decompose cellulose, lignin and other components, which guarantee the integrity of the vegetal cell. The objective of this study was to determine the enzymatic activity of filamentous fungi, yeasts and bacteria, isolated from natural cork stoppers for bottles of still and sparkling wines. Suspensions of fungal conidia, yeasts and bacterial cells of micro-organisms were established in concentrations of 10(6) CFU/ml. The enzymatic activity of these micro-organisms was evaluated by means of the API ZYM system, with which it was possible to determine and semi-quantify nineteen enzymatic activities simultaneously. The enzymes produced by all of the species were esterase (C1), esterase lipase and naphthol-AS-BI-phosphohydrolase. The micro-organisms with the greatest enzymatic activity were Monilia sitophila, Alternaria alternata, Aspergillus niger and Aeromonas sp.     

Role of Chrysonilia sitophila in the quality of cork stoppers for sealing wine bottles

The contribution of Chrysonilia sitophila in cork stopper manufacture was studied and a simulation of the industrial processing of cork stoppers was performed. Stoppers cut from slabs where mold development was inhibited were compared with others cut from slabs colonized by C. sitophila alone or with several molds, in terms of physical properties and chemical taints. C. sitophila does not produce 2,4,6-trichloroanisole, guaiacol, or 1-octene-3-ol on cork slabs incubated for 66 days. Since some chlorophenol-related compounds contaminate cork slabs during the production processes, metabolic tests were performed to investigate the capability of molds to produce 2,4,6-trichloroanisole by methylation of 2,4,6-trichlorophenol. Degradation of 2,4,6-trichlorophenol by C. sitophila resulted in a very high level of degradation without production of 2,4,6-trichloroanisole. C. sitophila restricted growth of other molds on maturing slabs for at least 30 days. These results show that C. sitophila can be exploited by industrial producers of cork stoppers since it is able to inhibit the development of other molds and it does not produce the compounds responsible for ‘cork-taint’, even in the presence of chlorophenols. Journal of Industrial Microbiology & Biotechnology (2000) 24, 256–261. Received 28 July 1999/ Accepted in revised form 05 January 2000     

Population variation and natural selection on leaf traits in cork oak throughout its distribution range

A central issue in evolutionary biology is the exploration of functional trait variation among populations and the extent to which this variation has adaptive value. It was recently proposed that specific leaf area (SLA), leaf nitrogen concentration per mass (N_mass) and water use efficiency in cork oak play an important role in adaptation to water availability in the environment. In order to investigate this hypothesis, we explored, first, whether there was population-level variation in cork oak (Quercus suber) for these functional traits throughout its distribution range; if this were the case, it would be consistent with the hypothesis that different rainfall patterns have led to ecotypic differentiation in this species. Second, we studied whether the population-level variation matched short-term selection on these traits under different water availability conditions using two fitness components: survival and growth. We found high population-level differentiation in SLA and N_mass, with populations from dry places exhibiting the lowest values for SLA and N_mass. Likewise, reduced SLA had fitness benefits in terms of growth for plants under dry conditions. However, contrary to our expectations, we did not find any pattern of association between functional traits and survival in nine-year-old saplings despite considerable drought during one year of the study period. These results together with findings from the literature suggest that early stages of development are the most critical period for this species. Most importantly, these findings suggest that cork oak saplings have a considerable potential to cope with dry conditions. This capacity to withstand aridity has important implications for conservation of cork oak woodlands under the ongoing climate change.     

Study of Enterobacteriaceae throughout the manufacturing and ripening of hard goats' cheese

The evolution of the counts and the species of Enterobacteriaceae as well as some physico-chemical parameters (pH, α_w and NaCl and moisture contents) during manufacturing and ripening of a hard Spanish goats' cheese of the Armada-Sobado variety were studied. Enterobacteriaceae (mean log counts 4.45 g^-1 in milk) increased 0.71–2.18 log units in curd and afterwards decreased until they disappeared after 2–4 weeks of ripening. This premature disappearance seems to be due to the decrease in α_w values and in moisture contents. However, the low pH values, reached from the beginning of the ripening process, could also contribute to this phenomenon.
The most abundant species in milk was Serratia liquefaciens (57.5% of isolates), followed by Morganella morganii (27.5%), Hafnia alvei (5%), Klebsiella oxytoca (5%) and Yersinia enterocolitica (5%). Yersinia enterocolitica was not subsequently isolated from either curd or in cheese. Hafnia alvei numbers increased in curd and in 1-week-old cheese where this micro-organism was the most abundant (47.5% and 75% of the isolates respectively). Escherichia coli , which was not isolated from milk, curd or 1-week-old cheese, was the predominant organism in 2-week-old cheese (57.8% of isolates). This confirms the finding of other authors who have shown that it is one of the most resistant species in ripening cheeses.     

High levels of genetic diversity throughout the range of the Portuguese wheat landrace 'Barbela'

BACKGROUND AND AIMS: Landrace populations represent an important intra-crop reservoir of biodiversity and source of novel gene alleles for use in breeding programmes. Here the aim was to measure the diversity of a wheat landrace, 'Barbela', from the north of Portugal. METHODS: DNA was extracted from 59 accessions of Barbela collected across its geographical range. Diversity was measured by microsatellite length polymorphisms using 27 primer pairs amplifying 34 polymorphic microsatellite loci. KEY RESULTS: High levels of polymorphism were found, with an average polymorphism information content of 0.52; an average of 4.77 alleles (range 2-11) were present at each locus, and half of these loci showed an additional allele in the reference variety 'Chinese Spring'. CONCLUSIONS: 'Barbela' is maintained from seeds collected by farmers, but it maintains high allelic variation, and no groupings of accessions were detected when analysed by geographical region, farm or climate, indicating that the wheat landrace is a homogeneous entity. The diversity within the farmer-maintained landrace demonstrates the importance of characterization and maintenance of landrace collections before valuable genetic combinations are lost as uniform commercial crops are introduced.     

Variability of cork from Portuguese Quercus suber studied by solid-state (13)C-NMR and FTIR spectroscopies.

A new approach is presented for the study of the variability of Portuguese reproduction cork using solid-state (13)C-NMR spectroscopy and photoacoustic (PAS) FTIR (FTIR-PAS) spectroscopy combined with chemometrics. Cork samples were collected from 12 different geographical sites, and their (13)C-cross-polarization with magic angle spinning (CP/MAS) and FTIR spectra were registered. A large spectral variability among the cork samples was detected by principal component analysis and found to relate to the suberin and carbohydrate contents. This variability was independent of the sample geographical origin but significantly dependent on the cork quality, thus enabling the distinction of cork samples according to the latter property. The suberin content of the cork samples was predicted using multivariate regression models based on the (13)C-NMR and FTIR spectra of the samples as reported previously. Finally, the relationship between the variability of the (13)C-CP/MAS spectra with that of the FTIR-PAS spectra was studied by outer product analysis. This type of multivariate analysis enabled a clear correlation to be established between the peaks assigned to suberin and carbohydrate in the FTIR spectrum and those appearing in the (13)C-CP/MAS spectra.     

Mycobiota of the date palm phylloplane: description and interactions

We have analysed the mycobiota of date palm (Phoenix dactylifera, L.) leaves using several techniques. Profusely sporulating fungi (Penicillium spp. and Cladosporium spp.) developed when plating leaf fragments and leaf washings. Fusarium oxysporum, was particularly abundant in leaves infested with the red scale insect Phoenicococcus marlatti Cockerell, 1899, but an undescribed Lecanicillium cf. psalliotae was also found. Dual and overlay cultures showed interactions between palm pathogens, entomopathogenic and saprotrophic fungi. The most significant was the strong inhibition of the palm pathogen Penicillium vermoesenii caused by the entomopathogen Beauveria bassiana. No symptoms developed when F. oxysporum isolated from scale insects or the entomopathogens B. bassiana or Lecanicillium dimorphum were wound-inoculated on P. dactylifera petioles.     

Acropora cervicornis genet performance and symbiont identity throughout the restoration process

Coral Reefs - In the Caribbean, corals are commonly cultured in ocean-based nurseries and outplanted back to reefs for population enhancement. Intraspecific diversity in host and symbiont is an...     

Degradation behavior of chitosan chains in the 'green' synthesis of gold nanoparticles

The degradation behavior of chitosan chains in the synthesis of Au nanoparticles by a 'green' method was investigated in this paper for the first time. UV-vis absorption spectra suggested the formation of Au nanoparticles and TEM images showed that their sizes were between 10 and 50nm. During the process of synthesis, the intrinsic viscosity [eta] of chitosan was observed to decrease gradually, implying that the chitosan chains degraded under the reaction conditions. Further studies showed that the degree of degradation of the chitosan chains was changed with different reaction temperatures, reactant ratios, and the molecular weights of chitosan.     

Monitoring of an antigen manufacturing process

Fluorescence spectroscopy in combination with multivariate statistical methods was employed as a tool for monitoring the manufacturing process of pertactin (PRN), one of the virulence factors of Bordetella pertussis utilized in whopping cough vaccines. Fluorophores such as amino acids and co-enzymes were detected throughout the process. The fluorescence data collected at different stages of the fermentation and purification process were treated employing principal component analysis (PCA). Through PCA, it was feasible to identify sources of variability in PRN production. Then, partial least square (PLS) was employed to correlate the fluorescence spectra obtained from pure PRN samples and the final protein content measured by a Kjeldahl test from these samples. In view that a statistically significant correlation was found between fluorescence and PRN levels, this approach could be further used as a method to predict the final protein content.     

Mixing in process vessels used in biopharmaceutical manufacturing

The use of nonbaffled vessels for mixing applications is becoming common in the biopharmaceutical industry but is not sufficiently well studied. Orientation of the impellers off-centered and/or at an angle is necessary to enhance mixing and eliminate swirling that would result without a baffle in a standard tank. This study focuses on characterizing mixing in vessels with the hydrofoil axial flow impellers mounted off-center at 10 degrees to the vertical. Geometrically similar vessels ranging from 100 to 5000 L working volume were used in this study. Mixing performance was successfully correlated to vessel geometric factors.     

Function and evolution of 'green' GSK3/Shaggy-like kinases

Glycogen synthase kinase 3 (GSK3) proteins, also known as SHAGGY-like kinases, have many important cell signalling roles in animals, fungi and amoebae. In particular, GSK3s participate in key developmental signalling pathways and also regulate the cytoskeleton. GSK3-encoding genes are also present in all land plants and in algae and protists, raising questions about possible ancestral functions in eukaryotes. Recent studies have revealed that plant GSK3 proteins are actively implicated in hormonal signalling networks during development as well as in biotic and abiotic stress responses. In this review, we outline the mechanisms of Arabidopsis GSK3 action, summarize GSK3 functions in dicot and monocot flowering plants, and speculate on the possible functions of GSK3s in the earliest-evolving land plants.     

Verification of the final anion exchange chromatography in the r-hGH manufacturing process

In this study, final anion exchange chromatography in the recombinant human growth hormone (r-hGH) manufacturing process was validated using a validation protocol that was consistent with both policy and standard operation procedure (SOP). Two buffer solutions used in chromatography were first validated and were found to satisfy pre-established acceptance criteria as follows: pH: 8.2, endotoxin: < 0.6 EU/mL, bioburden test: negative. Final anion exchange chromatography was conducted using a DEAE Sepharose FF Resin and eluted with a linear gradient of 30 to 110 mM NaCl in 50 mM Tris-HCl buffer at a flow rate of 15 L/h. Three consecutive batches of hGH solutions were generated via anion exchange chromatography, which was performed within pre-established operating parameters determined through in-process control. When all three batches were assessed by the pre-established sampling plan and tested for quality control, this purification process was shown to satisfy pre-established acceptance criteria; endotoxin: ≤ 0.5 EU/mg, ECP: ≤ 1.4 ppm, IEF: same removal distance, hGH content by Native-PAGE: 100%, purity by HPLC: ≥ 99%, yield by UV scanning: 87 to 89%, hGH monomer protein content by HPLC: 99%. Therefore, the final anion chromatography process was successfully validated in this study, and this method consistently yielded hGH solutions that satisfied pre-established criteria for subsequent processing.