Characterization of the recombinant Brettanomyces anomalus ß-glucosidase and its potential for bioflavouring.

AIM: Plant materials used in the food industry contain up to five times more aromas bound to glucose (glucosides) than free, unbound aromas, making these bound aromas an unused flavouring potential. The aim of this study was to identify and purify a novel ß-glucosidase from Brettanomyces yeasts that are capable of releasing bound aromas present in various food products. METHODS AND RESULTS: We screened 428 different yeast strains for ß-glucosidase activity and are the first to sequence the whole genome of two Brettanomyces yeasts (Brettanomyces anomalus and Brettanomyces bruxellensis) with exceptionally high ß-glucosidase activity. Heterologous expression and purification of the identified B. anomalus ß-glucosidase showed that it has an optimal activity at a higher pH (5·75) and lower temperature (37°C) than commercial ß-glucosidases. Adding this B. anomalus ß-glucosidase to cherry beers and forest fruit milks resulted in increased amounts of benzyl alcohol, eugenol, linalool and methyl salicylate compared to Aspergillus niger and Almond glucosidase. CONCLUSIONS: The newly identified B. anomalus ß-glucosidase offers new possibilities for food bioflavouring. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to sequence the B. anomalus genome and to identify the ß-glucosidase-encoding genes of two Brettanomyces species, and reports a new bioflavouring enzyme.

Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains.

Recent studies have suggested a correlation between genotype groups of Brettanomyces bruxellensis and their source of isolation. To further explore this relationship, the objective of this study was to assess metabolic differences in carbon and nitrogen assimilation between different B. bruxellensis strains from three beverages, including beer, wine, and soft drink, using Biolog Phenotype Microarrays. While some similarities of physiology were noted, many traits were variable among strains. Interestingly, some phenotypes were found that could be linked to strain origin, especially for the assimilation of particular α- and ß-glycosides as well as α- and ß-substituted monosaccharides. Based upon gene presence or absence, an α-glucosidase and ß-glucosidase were found explaining the observed phenotypes. Further, using a PCR screen on a large number of isolates, we have been able to specifically link a genomic deletion to the beer strains, suggesting that this region may have a fitness cost for B. bruxellensis in certain fermentation systems such as brewing. More specifically, none of the beer strains were found to contain a ß-glucosidase, which may have direct impacts on the ability for these strains to compete with other microbes or on flavor production.

Comparative genome sequencing to assess the genetic diversity and virulence attributes of 15 Vibrio anguillarum isolates.

Vibrio anguillarum is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. However, the diversity and virulence mechanisms of this pathogen are still insufficiently known. In this study, we aimed to increase our understanding of V. anguillarum diversity and virulence through comparative genome analysis of 15 V. anguillarum strains, obtained from different hosts or non-host niches and geographical regions, among which 10 and 5 strains were found to be virulent and avirulent, respectively, against sea bass larvae. First, the 15 draft genomes were annotated and screened for putative virulence factors, including genes encoding iron uptake systems, transport systems and non-ribosomal peptide synthetases. Second, comparative genome analysis was performed, focusing on single nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). Five V. anguillarum strains showed a remarkably high nucleotide identity. However, these strains comprise both virulent and avirulent strains towards sea bass larvae, suggesting that differences in virulence may be caused by subtle nucleotide variations. Clearly, the draft genome sequence of these 15 strains represents a starting point for further genetic research of this economically important fish pathogen.

Unstable microsatellite repeats facilitate rapid evolution of coding and regulatory sequences.

Tandem repeats are intrinsically highly variable sequences since repeat units are often lost or gained during replication or following unequal recombination events. Because of their low complexity and their instability, these repeats, which are also called satellite repeats, are often considered to be useless 'junk' DNA. However, recent findings show that tandem repeats are frequently found within promoters of stress-induced genes and within the coding regions of genes encoding cell-surface and regulatory proteins. Interestingly, frequent changes in these repeats often confer phenotypic variability. Examples include variation in the microbial cell surface, rapid tuning of internal molecular clocks in flies, and enhanced morphological plasticity in mammals. This suggests that instead of being useless junk DNA, some variable tandem repeats are useful functional elements that confer 'evolvability', facilitating swift evolution and rapid adaptation to changing environments. Since changes in repeats are frequent and reversible, repeats provide a unique type of mutation that bridges the gap between rare genetic mutations, such as single nucleotide polymorphisms, and highly unstable but reversible epigenetic inheritance.

Comparative immunogenicity of two vaccination schedules of a combined hepatitis A and B vaccine in healthy volunteers.

In 1996, a combined vaccine against both hepatitis A and B was licensed and commercialized and has been recommended for healthcare personnel in Belgium. This study compares the immunogenicity against hepatitis B virus (HBV) and safety of two vaccination schedules (0-1-12 months and 0-1-6 months) with this vaccine. This is a randomized, stratified and controlled study in healthy adult workers, who are not occupationally exposed to HBV. Seroconversion (≥1 IU/L) and seroprotection (≥10 IU/L) rates were compared using Fisher's exact test; geometric mean concentrations (GMCs) of anti-HBs were compared using one-way ANOVA. All statistical analyses were carried out with SPSS 11 on Apple Macintosh. A total of 399 subjects were enrolled in the study, and 356 were analysed according to the protocol. The rate of ≥10 IU/L at 6 months was 70.6% in the group 0-1-12 and 79.9% in the group 0-1-6; this rate decreased to 55.9% at 12 months in the first group. Seroconversion and seroprotective rates against HBV measured at month 13 in group 0-1-12 (98.9% and 95.6%) and measured at month 7 in group 0-1-6 (99.4% and 97.1%) were not statistically significantly different. GMC of anti-HBs after the 0-1-12 schedule was more than two fold higher than after 0-1-6 schedule (P < 0.001). Reported side effects were comparable in both groups with a slight tendency to fewer side effects in the 0-1-12 group after the third dose. The results from our study show that the completed schedule 0-1-12 offers at least equal protective immunogenicity against HBV as the completed 0-1-6 schedule. People not receiving their third dose at 6 months can be given this dose up to 12 months after the first dose. The drawback of this flexibility, however, is the longer time period before the protection becomes effective.

Parameters affecting ethyl ester production by Saccharomyces cerevisiae during fermentation.

Volatile esters are responsible for the fruity character of fermented beverages and thus constitute a vital group of aromatic compounds in beer and wine. Many fermentation parameters are known to affect volatile ester production. In order to obtain insight into the production of ethyl esters during fermentation, we investigated the influence of several fermentation variables. A higher level of unsaturated fatty acids in the fermentation medium resulted in a general decrease in ethyl ester production. On the other hand, a higher fermentation temperature resulted in greater ethyl octanoate and decanoate production, while a higher carbon or nitrogen content of the fermentation medium resulted in only moderate changes in ethyl ester production. Analysis of the expression of the ethyl ester biosynthesis genes EEB1 and EHT1 after addition of medium-chain fatty acid precursors suggested that the expression level is not the limiting factor for ethyl ester production, as opposed to acetate ester production. Together with the previous demonstration that provision of medium-chain fatty acids, which are the substrates for ethyl ester formation, to the fermentation medium causes a strong increase in the formation of the corresponding ethyl esters, this result further supports the hypothesis that precursor availability has an important role in ethyl ester production. We concluded that, at least in our fermentation conditions and with our yeast strain, the fatty acid precursor level rather than the activity of the biosynthetic enzymes is the major limiting factor for ethyl ester production. The expression level and activity of the fatty acid biosynthetic enzymes therefore appear to be prime targets for flavor modification by alteration of process parameters or through strain selection.

Effects of CO2 on the formation of flavour volatiles during fermentation with immobilised brewer's yeast.

Immobilised-cell fermentors offer great benefits compared to traditional free-cell systems. However, a major problem is unbalanced flavour production when these fermentors are used for the production of alcoholic beverages. One of the keys to obtaining better control over flavour formation may be the concentration of dissolved CO2, which has inhibitory effects on yeast growth and metabolism. This article demonstrates that the presence of immobilisation matrices facilitates the removal of CO2 from the liquid medium, which results in a low level of dissolved CO2 during fermentation. Moreover, the formation of volatile higher alcohols and esters was greatly enhanced in the immobilised-cell system when compared to the free cell system. By sparging a CO2 flow (45 ml/min) into the immobilised-cell system, cell growth was reduced by 10-30% during the active fermentation stage, while the fermentation rate was unaffected. The uptake of branched-chain amino acids was reduced by 8-22%, and the formation of higher alcohols and esters was reduced on average by 15% and 18%, respectively. The results of this study suggest that mismatched flavour profiles with immobilised-cell systems can be adjusted by controlling the level of dissolved CO2 during fermentation with immobilised yeast.

Bioflavoring and beer refermentation.

Various techniques are used to adjust the flavors of foods and beverages to new market demands. Although synthetic flavoring chemicals are still widely used, flavors produced by biological methods (bioflavors) are now more and more requested by consumers, increasingly concerned with health and environmental problems caused by synthetic chemicals. Bioflavors can be extracted from plants or produced with plant cell cultures, microorganisms or isolated enzymes. This Mini-Review paper gives an overview of different systems for the microbial production of natural flavors, either de novo, or starting with selected flavor precursor molecules. Emphasis is put on the bioflavoring of beer and the possibilities offered by beer refermentation processes. The use of flavor precursors in combination with non-conventional or genetically modified yeasts for the production of new products is discussed.

Yeast flocculation: what brewers should know.

For many industrial applications in which the yeast Saccharomyces cerevisiae is used, e.g. beer, wine and alcohol production, appropriate flocculation behaviour is certainly one of the most important characteristics of a good production strain. Yeast flocculation is a very complex process that depends on the expression of specific flocculation genes such as FLO1, FLO5, FLO8 and FLO11. The transcriptional activity of the flocculation genes is influenced by the nutritional status of the yeast cells as well as other stress factors. Flocculation is also controlled by factors that affect cell wall composition or morphology. This implies that, during industrial fermentation processes, flocculation is affected by numerous parameters such as nutrient conditions, dissolved oxygen, pH, fermentation temperature, and yeast handling and storage conditions. Theoretically, rational use of these parameters offers the possibility of gaining control over the flocculation process. However, flocculation is a very strain-specific phenomenon, making it difficult to predict specific responses. In addition, certain genes involved in flocculation are extremely variable, causing frequent changes in the flocculation profile of some strains. Therefore, both a profound knowledge of flocculation theory as well as close monitoring and characterisation of the production strain are essential in order to gain maximal control over flocculation. In this review, the various parameters that influence flocculation in real-scale brewing are critically discussed. However, many of the conclusions will also be useful in various other industrial processes where control over yeast flocculation is desirable.