Effect of wine yeast monoculture practice on the biodiversity of non-Saccharomyces yeasts

AIMS: The objective of this work was to study the effect of the use of Saccharomyces cerevisiae monocultures over the biodiversity of non-Saccharomyces yeasts in wine-producing areas in Chile. METHODS AND RESULTS: Microvinifications were carried out with grape musts of two areas. In one of them, the fermentation is carried out mainly in a spontaneous manner, whereas in the other the musts are inoculated with commercial yeasts. The isolated yeasts were identified by the internal transcribed (ITS)/restriction fragment length polymorphism technique. In the industrial production area less variability of yeast genera was observed as compared with the traditional area, an observation that is greatest at the end of the fermentation. Furthermore, a study of the production of extracellular enzymes was done. The majority of the yeasts showed at least one of the activities assayed with the exception of beta-glycosidase. CONCLUSION: The results suggest that in the industrialized area the diversity of yeasts is less in the traditional area. Likewise, the potentiality of the non-Saccharomyces yeasts as enzyme producers with industrial interest has been confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the negative effect of the use of monocultures over the biodiversity of yeasts in wine-producing regions.     

Optimization of a rapid method for studying the cellular location of beta-glucosidase activity in wine yeasts

AIMS: To improve a method for determining beta-glucosidase activity and to apply it in yeasts isolated from wine ecosystems from "La Mancha" region and to know its cellular location. METHODS AND RESULTS: A total of 82 wine yeasts were identified (PCR/RFLP) and evaluated for their beta-glucosidase activity. First, they were qualitatively evaluated by growth on YNB cellobiose, the activity was quantified using different culture media, under aerobic and anaerobic conditions and cells after 24-72 h of growth. To study the location activity, five fractions were obtained (supernatant, whole cell, cell wall, cytosol and cell membrane). The enzymatic assays were optimized, being: growth in YP cellobiose for 72 h in aeration conditions and, after cell removing, enzyme analysis with 128 g l(-1) of cellobiose as substrate, for 30 min at 30 degrees C. The genus that displayed the greatest activity were Pichia, Hanseniaspora and Rhodotorula, and the activity was intracellular. CONCLUSIONS: The study showed that beta-glucosidase activity was induced by the carbon source and was aerobic dependent. The non-Saccharomyces species displayed the greatest activity, which was intracellular and strain-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a reliable method for screening beta-glucosidase activity in yeasts isolated from wine ecosystems. This activity is very important in the release of monoterpenols from glycoside precursors for the enhancement of wine aromas.     

Cell-recycle batch fermentation using immobilized cells of flocculent Saccharomyces cerevisiae wine strains

Five, highly flocculeng strains of Saccharomyces cerevisiae, isolated from wine, were immobilized in calcium alginate beads to optimize primary must fermentation. Three cell-recycle batch fermentations (CRBF) of grape musts were performed with the biocatalyst and the results compared with those obtained with free cells. During the CRBF process, the entrapped strains showed some variability in the formation of secondary products of fermentation, particularly acetic acid and acetaldehyde. Recycling beads of immobilized flocculent cells is a good approach in the development and application of the CRBF system in the wine industry.     

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.     

Differential killer sensitivity as a tool for fingerprinting wine-yeast strains ofSaccharomyces cerevisiae

The extreme variability of the killer phenomenon in nature, expressed differently in different strains of the same yeast species, embodies an exceptional potential for the discrimination of yeasts at the strain level. Killer-sensitive relationships between a killer reference panel of 24 yeasts belonging to 13 species of six genera, and different industrial wine-starters ofSaccharomyces cerevisiae can be used profitably for a rapid and simple fingerprinting procedure.     

Breeding strategies for combining fermentative qualities and reducing off-flavor production in a wine yeast model

In agricultural sciences, breeding strategies have historically been used to select new, optimized plant varieties or animal breeds. Similar strategies are possible for genetic improvement of wine yeasts. We optimized 11 relevant enological traits in a single clone using successive hybridization and segregation steps. A hybrid obtained by crossing two parent strains derived from commercial wine yeasts showed that some of the traits were readily optimized. Dominance/recessivity, heterosis and transgression were observed among 51 segregating progeny. On the basis of this information, all the optimal characters from both parents were combined in a single strain following two targeted sexual crosses. This article presents a powerful methodology for obtaining a single wine strain with numerous fermentative qualities that does not produce off-flavors.     

Trifluoroleucine resistance as a dominant molecular marker in transformation of strains of Saccharomyces cerevisiae isolated from wine

The resistance to 5,5,5-trifluoro-DL-leucine, encoded by the dominant allele LEU4-1, was used as a selectable marker to transform laboratory and natural Saccharomyces cerevisiae strains by the lithium acetate procedure. Results of transformation of S. cerevisiae laboratory and wine natural strains showed that trifluoroleucine resistance is a very effective selection marker and can be widely used to transform prototrophic S. cerevisiae strains. The LEU4-1 gene could also be exploited to improve wine flavour, as indicated by the higher isoamyl alcohol content of the transformants compared to the parental strains.     

16S-ARDRA, a tool for identification of lactic acid bacteria isolated from grape must and wine

Lactic acid bacteria (LAB) are found in a great variety of habitats, including grape must and wines. There is a close relationship between the species of LAB which develop during fermentation and the eventual quality of the wine. For these reasons analytical techniques allowing fast and reliable identification of wine LAB are needed. In this work a simple and accurate protocol for identifying species of LAB isolated from grape must and wine is presented. This protocol is based on the amplification, directly from colony, of 16S rDNA and later digestion with one of the following restriction enzymes BfaI, MseI and AluI. A sequential use of the three enzymes is proposed to simplify LAB wine identification, first MseI, then BfaI and finally, if necessary, AluI digestion. The technique was able to discriminate 32 of the 36 LAB reference species tested and allowed the identification of 342 isolates from musts and wines. The isolates belonged to the species: Lactobacillus brevis, L. collinoides, L. coryniformis, L. bilgardii, L. mali, L. paracasei, Leuconostoc mesenteroides, Oenococcus oeni, Pediococcus parvulus and P. pentosaceus.     

Life cycle assessment as a tool for improving process performance: A case study on boron products

This paper explores the use of LCA as a tool for process environmental management, thereby moving the focus from product to process oriented analysis. The emphasis is on Improvement Assessment in which the “hot spots” in the system are targeted for maximum environmental improvements. In this context, it is useful to use multiobjective optimisation which renders Valuation unnecessary. The approach is illustrated by the case study of the system processing boron ores to make five different products. The results of Inventory Analysis and Impact Assessment are presented and discussed. In Improvement Assessment, a number of improvement options are identified and evaluated, using system optimisation. It is shown that the site environmental performance can be improved over current operation by an average of 20% over the whole life cycle. Thus the study demonstrates that the optimisation approach to environmental process management may assist in identifying optimal ways to operate a process or plant from “cradle to grave”. This may help the process industries not only to comply with legislation but also provide a framework for taking a more proactive approach to environmental management leading to more sustainable industrial operations and practices.     

Screening of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine strains for the production of acetic acid

In this study 80 wine strains of Saccharomyces cerevisiae were characterized for the production of acetic acid. A significant variability in the production levels was determined among the strains, which produced from a few mg/l to more than 1 g/l. Fifteen strains, differing in acetic acid production, were tested in fermentation of grape musts of different varieties (Aglianico Basilicata, Aglianico Apulia, Cannonau, Bombino nero, Nero d'Avola, Vermentino, Fiano). The results emphasized a great strain variability, but the best strain behaviour was strictly related to the must composition, which is a determinant factor on the expression of the best strain potentiality. Therefore, this study, confirming the high/low production of acetic acid as a strain characteristic, demonstrated also that the inoculated fermentation becomes more advantageous when the starter culture is chosen in relation to the interaction of yeast strain/vine variety.     

Marine bacterial diversity as a resource for novel microbial products

Marine bacteria are an important and relatively unexplored resource for novel microbial products. In this review, we discuss a number of issues relevant to the industrial potential of marine microorganisms including how marine and terrestrial bacteria differ, both physiologically and taxonomically, and what constitute reasonable expectations of the biosynthetic capabilities of marine bacteria relative to terrestrial bacteria and to marine macroorganisms. Also discussed is the concept that bacterial associations with marine plants and animals, which range from casual encounters to obligate symbioses, provide unique opportunities for bacterial adaptation. It is proposed that some of these adaptations would not be selected for in the absence of environmental parameters associated with the host, and that these adaptations can include the biosynthesis of unique metabolic products.     

Satellite imagery as a tool for monitoring species diversity: an assessment

1. A landscape of 5 × 5·5 km in the Karnataka region of the Western Ghats of India was mapped into seven landscape element types, using field identification of types as well as supervised and unsupervised classification of satellite imagery.
2. Plant communities distributed in these landscape element types were surveyed in the field using 246 quadrats of 10 × 10 m, in order to assess whether these types could be distinguished in terms of species composition. All angiosperms excluding grasses, which could not be identified accurately in the field, were recorded for this purpose.
3. Landscape element types identified in the field harboured significantly distinctive sets of species of flowering plants, and were also by and large distinctive in terms of their species richness.
4. Landscape element types could be identified accurately on the basis of supervised classification: the types thus demarcated harboured distinctive sets of flowering plants.
5. Landscape element types coupled to satellite imagery could then be used to organize a programme of monitoring biodiversity.
6. Unsupervised classification of satellite imagery did not permit classification of landscape element types with a high enough level of accuracy. In consequence, the demarcated landscape element types did not harbour significantly distinctive sets of species of flowering plants. Unsupervised classification is therefore not appropriate in a programme of monitoring biodiversity.     

High power ultrasonics as a novel tool offering new opportunities for managing wine microbiology

Industrial scale food and beverage processes that utilize microorganisms are typically faced with issues related to the exclusion, suppression or elimination of spoilage organisms. Yet the use of traditional anti-microbial treatments such as heat, chemical biocides or sterile filtration may themselves be restricted by regulations or else be undesirable due to their adverse sensory impacts on the product. High power ultrasound (HPU) is a technology whose application has been evaluated if not exploited in several food and beverage processes but has yet to be introduced into the wine industry. This review examines the research findings from related industries and highlights possible applications and likely benefits of the use of HPU in winemaking.     

Characterization and technological properties of Oenococcus oeni strains from wine spontaneous malolactic fermentations: a framework for selection of new starter cultures

Aims:  To characterize the genetic and phenotypic diversity of 135 lactic acid bacteria (LAB) strains isolated from Italian wines that undergone spontaneous malolactic fermentation (MLF) and propose a multiphasic selection of new Oenococcus oeni malolactic starters.
Methods and Results:  One hundred and thirty-five LAB strains were isolated from 12 different wines. On the basis of 16S amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes and 16S rRNA gene sequencing, 120 O. oeni strains were identified. M13-based RAPD analysis was employed to investigate the molecular diversity of O. oeni population. Technological properties of different O. oeni genotypes were evaluated in synthetic medium at increasing selective pressure, such as low pH (3·5, 3·2 and 3·0) and high ethanol values (10, 11 and 13% v/v). Finally, the malolactic activity of one selected strain was assessed in wine by malolactic trial in winery.
Conclusions:  The research explores the genomic diversity of wine bacteria in Italian wines and characterizes their malolactic metabolism, providing an efficient strategy to select O. oeni strains with desirable malolactic performances and able to survive in conditions simulating the harsh wine environment.
Significance and Impact of the Study:  This article contributes to a better understanding of microbial diversity of O. oeni population in Italian wines and reports a framework to select new potentially O. oeni starters from Italian wines during MLF.     

Ribonuclease P RNA gene sequencing as a tool for molecular dereplication of myxobacterial strain collections

AIMS: Efficient strain dereplication is of great value during the generation of bacterial strain collections for industrial screening. We evaluated the utilization of the RNase P RNA gene (rnpB) sequence as a tool for molecular dereplication of myxobacteria. METHODS AND RESULTS: 16S rDNA (approx. 1 x 5 kbp) and rnpB (approx. 0 x 3 kbp) sequences were obtained and aligned. From 50 strains, we obtained 20 different sequences for the 16S rDNA and 24 for rnpB. Intersequence similarity was lower for rnpB than for 16S rDNA. CONCLUSIONS: rnpB allows the rapid discrimination of similar strains, with a higher resolution power as compared with 16S rRNA gene sequencing. It not only gives better discrimination, but is also faster and cheaper than 16S rDNA sequencing. SIGNIFICANCE AND IMPACT OF THE STUDY: Myxobacteria isolation and cultivation require time and experience. The application of rnpB sequencing to early myxobacterial strain dereplication may help in the generation of diverse strain libraries of these bacteria.     

Genetic analysis of polyketide synthase and peptide synthetase genes in cyanobacteria as a mining tool for secondary metabolites

Molecular screening using degenerate PCR to determine the presence of secondary metabolite genes in cyanobacteria was performed. This revealed 18 NRPS and 19 PKS genes in the 21 new cyanobacterial strains examined, representing three families of cyanobacteria (Nostocales, Chroococales and Oscillatoriales). A BLAST analysis shows that these genes have similarities to known cyanobacterial natural products. Analysis of the NRPS adenylation domain indicates the presence of novel features previously ascribed to both proteobacteria and cyanobacteria. Furthermore, binding-pocket predictions reveal diversity in the amino acids used during the biosynthesis of compounds. A similar analysis of the PKS ketosynthase domain shows significant structural diversity and their presence in both mixed modules with NRPS domains and individually as part of a PKS module. We have been able to classify the NRPS genes on the basis of their binding-pockets. Further, we show how this data can be used to begin to link structure to function by an analysis of the compounds Scyptolin A and Hofmannolin from Scytonema sp. PCC 7110.     

Immature pollen-derived doubled haploid formation in barley cv. Golden Promise as a tool for transgene recombination

Barley transformation mediated by Agrobacterium tumefaciens is routinely performed in a number of laboratories. However, elimination of selectable marker genes and formation of plants homozygous for the transgene via conventional segregation is laborious and time-consuming. Here we suggest a concept that includes the production of primary transgenic plants via infection of immature embryos with A. tumefaciens followed by androgenetic generation of a segregating population of entirely homozygous plants. Selectable marker-free, truebreeding plants carrying a single-opy transgene integrant may thus be efficiently and rapidly obtained. However, amenability to Agrobacterium-mediated transformation as well as androgenetic potential is genotype-dependent. Efficient genetic transformation by infection of immature embryos is so far confined to the spring type cultivar ‘Golden Promise’ which, however, turned out to be recalcitrant in pollen embryogenesis. To facilitate androgenetic generation of homozygous segregants from primary transformants, we have established a method for embryogenic pollen culture in cv. Golden Promise that includes conventional cold-treatment and subsequent preculture of immature pollen under starvation conditions prior to transfer to complete nutrient medium. Further we show that conditioning of the pollen culture medium by co-culture of immature wheat pistils as well as addition of pistil-preconditioned medium considerably support androgenetic development. Employment of the established method using immature pollen of primary transgenic plants demonstrates that selectable marker-free, true-breeding transgenic progeny can be rapidly obtained pursuing the concept proposed. The protocol presented will be useful in functional genomics as well as in molecular breeding approaches.