Determination of optimal sample preparation for aldehyde extraction from pale malts and their quantification via headspace solid-phase microextraction followed by gas chromatography and mass spectrometry.

Aldehydes originating from malt play an important role in beer flavour deterioration. In order to better understand the influence of malting process on beer staling, it is necessary to acquire a reliable analytical methodology for determination of beer staling aldehydes in malt. Therefore, the aim of this study was to evaluate extraction parameters, which allow quantification of beer staling aldehydes present in pale malts. The method was validated with respect to linearity (R > 0.9988), limit of detection (0.28 - 0.99 µg/L), limit of quantification (0.92 - 3.31 µg/L), accuracy (± 5%), repeatability (1.3 - 5.3%) and intermediate precision (>20%). The following parameters of sample preparation were evaluated: sample amount, extraction time and temperature, ultrasonication time and oxygen level. Consequently, the best extraction conditions were successfully applied on pale malts. After extraction, the samples were analysed by headspace solid-phase microextraction (HS-SPME) with on fibre carbonyl derivatisation followed by gas chromatography and mass spectrometry (GC-MS). In addition, the salting-out effect during HS-SPME was studied. The method application allowed to identify significant differences (p ≤ 0.05) in the levels of aldehydes among various industrial scale, pale malts. The optimised method could give the information on the aldehyde content introduced into the brewing process and its potential contribution to the overall beer quality.

Flax treatment with strategic enzyme combinations: Effect on chemical fiber composition and ease of fiber extraction.

The effect of treatment of flax with strategic enzyme combinations on the ease of fiber extraction and the chemical fiber composition is reported in this study. To contribute to the increasing demand for bio-based and sustainable materials, it is of great importance to develop optimal enzyme formulations which can replace the yet poorly controlled traditional dew retting process. Regarding the chemical composition of the fiber, enzymatic treatments all resulted in similar improvements, with an enhanced cellulose content of 81 ±â€¯1% after polygalacturonase + xylanase treatment (vs. 64 ±â€¯2% for green fibers). Evaluation of extraction efficiency (EE) showed that several enzyme combinations significantly increased EE in comparison with green fibers. An EE of 23 ±â€¯6% was found for fibers extracted after polygalacturonase + pectinmethylesterase treatment, in comparison with an EE of 11 ±â€¯1% for green fibers. Combinations with three enzymes resulted in a higher reduction of the pectin content of the fibers. The combination of enzymes shows hence promising potential but further evaluation of mechanical performance of fiber reinforced composites is needed.

Enzymatic treatment of flax for use in composites.

Enzymes are highly advantageous compared to dew retting to reach fibers of high and consistent quality. However, no unambiguous insights have been retained from the research, i.e. lacking a clear directive of which enzyme activities are strictly needed. Methods for evaluating enzymatic retting should be standardized, with characterization of chemical, morphological and mechanical properties and analysis of the ease of extraction. Moreover, evaluation should not only be focused on the microscopic level of the fiber but the performance of the resulting composite materials should be assessed as well. The review also covers research challenges for introducing enzymatic treatment in large scale production as well as inherent limitations and economic aspects. Besides their high selectivity and environmentally-friendly processing conditions, applying enzymes may also result in a less severe mechanical post-treatment implying less fiber damage. Moreover, recycling of enzymes and utilization of byproducts may increase the economic feasibility of the process.

Phenotypic landscape of non-conventional yeast species for different stress tolerance traits desirable in bioethanol fermentation.

BACKGROUND: Non-conventional yeasts present a huge, yet barely exploited, resource of yeast biodiversity for industrial applications. This presents a great opportunity to explore alternative ethanol-fermenting yeasts that are more adapted to some of the stress factors present in the harsh environmental conditions in second-generation (2G) bioethanol fermentation. Extremely tolerant yeast species are interesting candidates to investigate the underlying tolerance mechanisms and to identify genes that when transferred to existing industrial strains could help to design more stress-tolerant cell factories. For this purpose, we performed a high-throughput phenotypic evaluation of a large collection of non-conventional yeast species to identify the tolerance limits of the different yeast species for desirable stress tolerance traits in 2G bioethanol production. Next, 12 multi-tolerant strains were selected and used in fermentations under different stressful conditions. Five strains out of which, showing desirable fermentation characteristics, were then evaluated in small-scale, semi-anaerobic fermentations with lignocellulose hydrolysates. RESULTS: Our results revealed the phenotypic landscape of many non-conventional yeast species which have not been previously characterized for tolerance to stress conditions relevant for bioethanol production. This has identified for each stress condition evaluated several extremely tolerant non-Saccharomyces yeasts. It also revealed multi-tolerance in several yeast species, which makes those species good candidates to investigate the molecular basis of a robust general stress tolerance. The results showed that some non-conventional yeast species have similar or even better fermentation efficiency compared to S. cerevisiae in the presence of certain stressful conditions. CONCLUSION: Prior to this study, our knowledge on extreme stress-tolerant phenotypes in non-conventional yeasts was limited to only few species. Our work has now revealed in a systematic way the potential of non-Saccharomyces species to emerge either as alternative host species or as a source of valuable genetic information for construction of more robust industrial S. serevisiae bioethanol production yeasts. Striking examples include yeast species like Pichia kudriavzevii and Wickerhamomyces anomalus that show very high tolerance to diverse stress factors. This large-scale phenotypic analysis has yielded a detailed database useful as a resource for future studies to understand and benefit from the molecular mechanisms underlying the extreme phenotypes of non-conventional yeast species.

Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains.

Brettanomyces (Dekkera) bruxellensis is an ascomycetous yeast of major importance in the food, beverage and biofuel industry. It has been isolated from various man-made ecological niches that are typically characterized by harsh environmental conditions such as wine, beer, soft drink, etc. Recent comparative genomics studies revealed an immense intraspecific diversity, but it is still unclear whether this genetic diversity also leads to systematic differences in fermentation performance and (off-)flavor production, and to what extent strains have evolved to match their ecological niche. Here, we present an evaluation of the fermentation properties of eight genetically diverse B. bruxellensis strains originating from beer, wine and soft drinks. We show that sugar consumption and aroma production during fermentation are determined by both the yeast strain and composition of the medium. Furthermore, our results indicate a strong niche adaptation of B. bruxellensis, most clearly for wine strains. For example, only strains originally isolated from wine were able to thrive well and produce the typical Brettanomyces-related phenolic off-flavors 4-ethylguaiacol and 4-ethylphenol when inoculated in red wine. Sulfite tolerance was found as a key factor explaining the observed differences in fermentation performance and off-flavor production. Sequence analysis of genes related to phenolic off-flavor production, however, revealed only marginal differences between the isolates tested, especially at the amino acid level. Altogether, our study provides novel insights in the Brettanomyces metabolism of flavor production, and is highly relevant for both the wine and beer industry.

A large set of newly created interspecific Saccharomyces hybrids increases aromatic diversity in lager beers.

Lager beer is the most consumed alcoholic beverage in the world. Its production process is marked by a fermentation conducted at low (8 to 15°C) temperatures and by the use of Saccharomyces pastorianus, an interspecific hybrid between Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus. Recent whole-genome-sequencing efforts revealed that the currently available lager yeasts belong to one of only two archetypes, "Saaz" and "Frohberg." This limited genetic variation likely reflects that all lager yeasts descend from only two separate interspecific hybridization events, which may also explain the relatively limited aromatic diversity between the available lager beer yeasts compared to, for example, wine and ale beer yeasts. In this study, 31 novel interspecific yeast hybrids were developed, resulting from large-scale robot-assisted selection and breeding between carefully selected strains of S. cerevisiae (six strains) and S. eubayanus (two strains). Interestingly, many of the resulting hybrids showed a broader temperature tolerance than their parental strains and reference S. pastorianus yeasts. Moreover, they combined a high fermentation capacity with a desirable aroma profile in laboratory-scale lager beer fermentations, thereby successfully enriching the currently available lager yeast biodiversity. Pilot-scale trials further confirmed the industrial potential of these hybrids and identified one strain, hybrid H29, which combines a fast fermentation, high attenuation, and the production of a complex, desirable fruity aroma.

Changes in the hop-derived volatile profile upon lab scale boiling.

Hop terpenes might be oxidized during kettle boiling into more water soluble compounds that could contribute to 'hoppy' aroma of kettle hopped lager beers. Our current research proves that the boiling process induces significant changes in the hop oil volatile profile. The discrimination between volatile profiles of unboiled and boiled hop essential oil was evaluated via principal component and cluster analysis (PCA and CA). HS-SPME-GC-MS analysis revealed quantitative changes (e.g. increases in the levels of oxygenated α-humulene and ß-caryophyllene derivatives) as well as qualitative changes (i.e. detection of compounds, not found in unboiled hop essential oil) in the hop oil volatile profile upon boiling. Many of these compounds were previously found in lager beer and may therefore contribute to beer flavor. Interestingly, the analytical difference between unboiled and boiled hop essential oil proved to be more pronounced as the initial hop essential oil concentration used for boiling was increased. In addition, lager beers spiked with boiled hop oil were described as 'hoppy/spicy' during sensory evaluations. Therefore, the newly formed products and hop oil constituents that are characterized by an increased recovery after boiling, are candidate compounds for 'hoppy' aroma in real brewing practice.

Phenotypic evaluation of natural and industrial Saccharomyces yeasts for different traits desirable in industrial bioethanol production.

Saccharomyces cerevisiae is the organism of choice for many food and beverage fermentations because it thrives in high-sugar and high-ethanol conditions. However, the conditions encountered in bioethanol fermentation pose specific challenges, including extremely high sugar and ethanol concentrations, high temperature, and the presence of specific toxic compounds. It is generally considered that exploring the natural biodiversity of Saccharomyces strains may be an interesting route to find superior bioethanol strains and may also improve our understanding of the challenges faced by yeast cells during bioethanol fermentation. In this study, we phenotypically evaluated a large collection of diverse Saccharomyces strains on six selective traits relevant for bioethanol production with increasing stress intensity. Our results demonstrate a remarkably large phenotypic diversity among different Saccharomyces species and among S. cerevisiae strains from different origins. Currently applied bioethanol strains showed a high tolerance to many of these relevant traits, but several other natural and industrial S. cerevisiae strains outcompeted the bioethanol strains for specific traits. These multitolerant strains performed well in fermentation experiments mimicking industrial bioethanol production. Together, our results illustrate the potential of phenotyping the natural biodiversity of yeasts to find superior industrial strains that may be used in bioethanol production or can be used as a basis for further strain improvement through genetic engineering, experimental evolution, or breeding. Additionally, our study provides a basis for new insights into the relationships between tolerance to different stressors.

Characterization of novel single-variety oxygenated sesquiterpenoid hop oil fractions via headspace solid-phase microextraction and gas chromatography-mass spectrometry/olfactometry.

The volatile composition of novel varietal oxygenated sesquiterpenoid hop oil fractions ("spicy essences") was characterized by headspace solid-phase microextraction in combination with gas chromatography-mass spectrometry. Oxygenated sesquiterpenes represent the major chemical compound class, accounting for at least 65% of the total volatile fraction. In addition to oxygenated sesquiterpenes, spicy hop essences consist of several ketones, sesquiterpene and monoterpene hydrocarbons, and a relatively high number of unidentified compounds. On the basis of their relative composition, spicy hop essences can be fully differentiated according to their varietal origin. Multidimensional gas chromatography in combination with time-of-flight mass spectrometry on spicy hop essence cv. Spalter Select further demonstrated the enormous complexity of this particular hop oil fraction. The aromagram obtained via gas chromatography-olfactometry comprised nine odor-active regions described in terms of "citrus", "green", "haylike", "earthy", "woody", and "spicy". 2-Undecanone, 2-tridecanone, γ-cadinene, α-calacorene, calarene, humuladienone, caryolan-1-ol, caryophyllene oxide enantiomers, and humulene epoxide II are tentatively identified in the odor-active zones.

Assessing the xylanolytic bacterial diversity during the malting process.

The presence of microorganisms producing cell wall hydrolyzing enzymes such as xylanases during malting can improve mash filtration behavior and consequently have potential for more efficient wort production. In this study, the xylanolytic bacterial community during malting was assessed by isolation and cultivation on growth media containing arabinoxylan, and identification by 16S rRNA gene sequencing. A total of 33 species-level operational taxonomic units (OTUs) were found, taking into account a 3% sequence dissimilarity cut-off, belonging to four phyla (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria) and 25 genera. Predominant OTUs represented xylanolytic bacteria identified as Sphingobacterium multivorum, Stenotrophomonas maltophilia, Aeromonas hydrophila and Pseudomonas fulva. DNA fingerprinting of all xylanolytic isolates belonging to S. multivorum obtained in this study revealed shifts in S. multivorum populations during the process. Xylanase activity was determined for a selection of isolates, with Cellulomonas flavigena showing the highest activity. The xylanase of this species was isolated and purified 23.2-fold by ultrafiltration, 40% ammonium sulfate precipitation and DEAE-FF ion-exchange chromatography and appeared relatively thermostable. This study will enhance our understanding of the role of microorganisms in the barley germination process. In addition, this study may provide a basis for microflora management during malting.

On the origin of free and bound staling aldehydes in beer.

The chemistry of beer flavor instability remains shrouded in mystery, despite decades of extensive research. It is, however, certain that aldehydes play a crucial role because their concentration increase coincides with the appearance and intensity of "aged flavors". Several pathways give rise to a variety of key flavor-active aldehydes during beer production, but it remains unclear as to what extent they develop after bottling. There are indications that aldehydes, formed during beer production, are bound to other compounds, obscuring them from instrumental and sensory detection. Because freshly bottled beer is not in chemical equilibrium, these bound aldehydes might be released over time, causing stale flavor. This review discusses beer aging and the role of aldehydes, focusing on both sensory and chemical aspects. Several aldehyde formation pathways are taken into account, as well as aldehyde binding in and release from imine and bisulfite adducts.

Characterization of novel varietal floral hop aromas by headspace solid phase microextraction and gas chromatography-mass spectrometry/olfactometry.

In this study, headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) were optimized and implemented to investigate the volatile composition of novel floral hop essences prepared from four German aroma hop varieties. In total, 91 different constituents were assigned, which were further grouped into monoterpene hydrocarbons, esters, ketones, aldehydes, furans, and oxygenated and nonoxygenated sesquiterpenes. Most volatiles belong to the ester group, whereas the monoterpene hydrocarbon ß-myrcene appears to be the predominant compound in all hop oil preparations investigated. Furthermore, as demonstrated by principal component analysis, varietal floral hop essences are clearly discriminated on the basis of their characteristic volatile composition. Via GC-olfactometry on the floral essence variety Spalter Select, ß-myrcene and 2-undecanone were identified as the most potent odorants. Several hop oil constituents were reported for the first time as impact odorants of hop aroma.

A kinetic study on the isomerization of hop alpha-acids.

In this article, a detailed study on hop alpha-acid isomerization kinetics is presented. Because of the complex wort matrix and interfering interactions occurring during real wort boiling (i.e., trub formation and alpha-acids/iso-alpha-acids complexation), this investigation on alpha-acid isomerization kinetics was performed in aqueous buffer solution as a function of time (0-90 min) and heating temperature (80-100 degrees C). Rate constants and activation energies for the formation of individual iso-alpha-acids were determined. It was found that iso-alpha-acid formation follows first-order kinetics and Arrhenius behavior. Differences in activation energies for the formation of trans- and cis-isomers were noticed, the activation energy for the formation of trans-iso-alpha-acids being approximately 9 kJmol (-1) lower.