Efficient breeding of industrial brewing yeast strains using CRISPR/Cas9-aided mating-type switching.

Yeast breeding is a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. However, breeding of industrial brewing yeast can be challenging, as strains are typically sterile and have large complex genomes. To facilitate breeding, we used the CRISPR/Cas9 system to generate double-stranded breaks in the MAT locus, generating transformants with a single specified mating type. The single mating type remained stable even after loss of the Cas9 plasmid, despite the strains being homothallic, and these strains could be readily mated with other brewing yeast transformants of opposite mating type. As a proof of concept, we applied this technology to generate yeast hybrids with an aim to increase ß-lyase activity for fermentation of beer with enhanced hop flavour. First, a genetic and phenotypic pre-screening of 38 strains was carried out in order to identify potential parent strains with high ß-lyase activity. Mating-competent transformants of eight parent strains were generated, and these were used to generate over 60 hybrids that were screened for ß-lyase activity. Selected phenolic off-flavour positive (POF +) hybrids were further sporulated to generate meiotic segregants with high ß-lyase activity, efficient wort fermentation, and lack of POF, all traits that are desirable in strains for the fermentation of modern hop-forward beers. Our study demonstrates the power of combining the CRISPR/Cas9 system with classic yeast breeding to facilitate development and diversification of brewing yeast. KEY POINTS: • CRISPR/Cas9-based mating-type switching was applied to industrial yeast strains. • Transformed strains could be readily mated to form intraspecific hybrids. • Hybrids exhibited heterosis for a number of brewing-relevant traits.

The influence of storage conditions on the chemistry and flavor of hoppy ales.

Understanding the factors which lead to the (in)stability of the chemistry and sensory of hoppy beer styles such as India Pale Ales (IPAs) has become a major concern for many brewers. Therefore, the evolution of several volatiles under different storage conditions (room temperature, cold storage, forced aging) was investigated in eleven hoppy ales and one lager which were commercially produced in Germany. Compared to the lager, the fresh ales contained higher initial aldehyde levels. Furthermore, the distribution of lipid oxidation and Maillard reaction products differed from those typically found in lager beer. Upon storage, significant increases of some staling aldehydes were observed. Interestingly, the concentrations of some hop aroma volatiles like terpenoids (i.e. linalool and geraniol) were relatively stable throughout storage and counteracted "oxidized" impressions in ales high in these compounds. In comparison, hop aroma in some ales was driven by less stable volatiles such as esters.

How deviations in the elemental profile of Humulus lupulus grown throughout the U.S. and Germany influence hop and beer quality.

Recently studies based on limited sample sizes procured from minor hop growing regions have speculated that the elemental profile of hops can possibly be used to authenticate the origin of a hop because changes in hop elemental profiles were realted to growing region and that these changes might also be related to beer quality. To explore this further, 205 hop samples (i.e. 203 whole cone hops and 2 pelletized samples) compromised of 19 varieties were procured from the major hop growing regions (i.e. the U.S. and Germany). These hops were digested with microwave digestion and analyzed for 25 elements using ICP-MS. Hops from most of the U.S. regions (mainly WA) had vastly different elemental profiles than hops from Germany. German hops had significantly lower concentrations for most of the elements except for Cu and K. Interestingly, high alpha varieties had significantly different elemental profiles than varieties bred for aroma purposes. Dry-hopping trials were then performed in an ale and a lager with the hops that had significantly different elemental profiles. Although heavy metals were extracted from hops into beer, at the 5 g/L dry-hopping load used in this study, beer concentrations of these elements remained below regulated water quality standards set by Germany, the U.S., and Canada. Based on electron paramagnetic resonance, dry-hopping had an antioxidant impact on beer regardless of the original elemental profile of the hops which was correlated to hop polyphenol and α/ ß - acid concentrations. Overall these results highlight that many factors including location have the potential to influence the elemental profile of hops and that changes in the elemental profiles of hops can be related to beer quality.

Evaluation and Optimization of APGC Parameters for the Analysis of Selected Hop Essential Oil Volatiles.

Hop essential oil is a mixture of several hundred volatile metabolites that quantitatively and qualitatively distinguish hop varieties. Given the commercial relevance of hops in the brewing industry and the complexity of hop oil, analytical tools enabling a comprehensive characterization of oil constituents are required. At this, atmospheric pressure chemical ionization interfaced to gas chromatography and high-resolution mass spectrometry (APGC-MS) is a promising option that combines soft ionization, high sensitivity, and high resolution. While high sensitivity is required to detect minor or trace-level volatile metabolites, soft ionization and high resolution enable the reliable identification of unknowns based on exact masses of the molecular ion or the protonated molecule. Twenty-two volatile metabolites typically found in hop oil were studied in respect to their APGC ionization behavior. For 15 compounds, APGC-MS did not yield high molecular ion or protonated molecule intensities and considerable in-source fragmentation was observed. APGC-MS parameter optimization (cone gas flow and cone voltage) was able to yield the maximum absolute intensity for the base peak. However, in-source fragmentation could not be prevented, leading to spectra with either the protonated molecule or a characteristic fragment ion as the base peak. APGC-MS operated under optimized parameters was applied to a hop essential oil sample to verify the effect of optimization. By estimating the limit of quantification for the 22 compounds, it is concluded that APGC-MS is well suited to analyze major, minor, and trace-level volatiles from hops.

Tradition as a Stepping Stone for a Microbial Defined Water Kefir Fermentation Process: Insights in Cell Growth, Bioflavoring, and Sensory Perception.

A process development from a traditional grain-based fermentation to a defined water kefir fermentation using a co-culture of one lactic acid bacterium and one yeast was elaborated as a prerequisite for an industrially scalable, controllable, and reproducible process. Further, to meet a healthy lifestyle, a low ethanol-containing product was aimed for. Five microbial strains-Hanseniaspora valbyensis, Dekkera bruxellensis, Saccharomyces cerevisiae, Liquorilactobacillus nagelii, and Leuconostoc mesenteroides-were used in pairs in order to examine their influence on the fermentation progress and the properties of the resulting water kefir products against grains as a control. Thereby, the combination of H. valbyensis and L. mesenteroides provided the best-rated water kefir beverage in terms of taste and low ethanol concentrations at the same time. As a further contribution to harmonization and reduction of complexity, the usage of dried figs in the medium was replaced by fig syrup, which could have been proven as an adequate substitute. However, nutritional limitations were faced afterward, and thus, an appropriate supplementation strategy for yeast extract was established. Finally, comparative trials in 5-L scale applying grains as well as a defined microbial consortium showed both water kefir beverages characterized by a pH of 3.14, and lactic acid and aromatic sensory properties. The product resulting from co-culturing outperformed the grain-based one, as the ethanol level was considerably lower in favor of an increased amount of lactic acid. The possibility of achieving a water kefir product by using only two species shows high potential for further detailed research of microbial interactions and thus functionality of water kefir.

Evaluation of Variety, Maturity, and Farm on the Concentrations of Monoterpene Diglycosides and Hop Volatile/Nonvolatile Composition in Five Humulus lupulus Cultivars.

Pentose-hexose monoterpene alcohol glycosides were isolated and semiquantitatively measured in dried Humulus lupulus cones using UHPLC-qTOF-MS/MS and HPLC fractionation followed by GC-MS. The samples evaluated included hop cones from five important dual-purpose cultivars (varieties) in the United States, from two locations (farms) per variety and from three distinct harvest time points (maturities) per location, as dictated by dry-matter (% w/w) at the time of harvest. Hop variety accounted for the biggest variation among the concentrations of pentose-hexose monoterpene alcohol glycosides as well as other volatile and nonvolatile chemical factors measured in the samples. This indicates that genetics plays a major role in hop flavor production. Interestingly, "maturity", or ripeness at the time of harvest, was the next most significant factor impacting the concentrations of pentose-hexose monoterpene alcohol glycosides along with most of the other volatile and nonvolatile factors (such as total oil concentration and composition). However, maturity notably had a bigger impact on some cultivars such as Sabro, Mosaic, Simcoe, and Citra. Surprisingly, farm (i.e., location, farming practices, etc.) accounted for the least amount of variation among the concentrations of the different analytical factors. These results highlight the importance of breeding/genetics as well as considering hop maturity/ripeness at the time of harvest on the production and subsequent development of analytical chemical factors associated with driving hoppy beer flavor. It is essential for future studies assessing the impact of different farming practices and locations (i.e., regionality, terroir, etc.) on the constituents in hops important for hoppy beer flavor to consider and account for the impact of hop maturity as well as genetics.

Analysis of Hop-Derived Thiols in Beer Using On-Fiber Derivatization in Combination with HS-SPME and GC-MS/MS.

The quantitation of the hop varietal thiols 4-mercapto-4-methyl-2-pentanone (4MMP), 3-mercapto-1-hexanol (3MH), and 3-mercaptohexylacetate (3MHA) from beer is challenging. This primarily relates to their low concentration (ng/L levels) and their reactivity. Published assays for thiol quantitation from beer include complex and/or time-consuming sample preparation procedures involving manual handling and use reagents that are harmful because they contain mercury. To facilitate thiol analysis from beer, the current article is concerned with the implementation of an automated headspace solid-phase microextraction (HS-SPME) on-fiber derivatization (OFD) approach using 2,3,4,5,6-pentafluorobenzyl bromide followed by gas chromatography-tandem mass spectrometry (GC-MS/MS). Optimization of HS-SPME and MRM conditions was based on a central composite design approach. The final OFD-HS-SPME-GC-MS/MS method yielded limits of quantitation below the sensory thresholds of 4MMP, 3MH, and 3MHA. Method validation and application on beers brewed with German, Australian, and US hops, as well as with added fruits displayed excellent method performance.

Evaluating the Chemical Components and Flavor Characteristics Responsible for Triggering the Perception of "Beer Flavor" in Non-Alcoholic Beer.

Forty-two commercial non-alcoholic beer (NAB) brands were analyzed using sensory and chemical techniques to understand which analytes and/or flavors were most responsible for invoking the perception of "beer flavor" (for Northern Californian consumers). The aroma and taste profiles of the commercial NABs, a commercial soda, and a carbonated seltzer water (n = 44) were characterized using replicated descriptive and CATA analyses performed by a trained sensory panel (i.e., 11 panelists). A number of non-volatile and volatile techniques were then used to chemically deconstruct the products. Consumer analysis (i.e., 129 Northern Californian consumers) was also used to evaluate a selection of these NABs (i.e., 12) and how similar they thought the aroma, taste and mouthfeels of these products were to beer, soda, and water. The results show that certain constituents drive the aroma and taste profiles which are responsible for invoking beer perception for these North American consumers. Further, beer likeness might not be a driver of preference in this diverse beverage class for Northern Californian consumers. These are important insights for brewers planning to create products for similar markets and/or more broadly for companies interested in designing other functional/alternative food and beverage products.

Characterizing Volatile and Nonvolatile Factors Influencing Flavor and American Consumer Preference toward Nonalcoholic Beer.

In this study, the chemical and sensory profiles of 42 different nonalcoholic beer (NAB) brands/styles already on the global market and produced through several different brewing techniques were evaluated. A trained panel (i.e., 11 panelists) performed standard-driven descriptive and check-all-that-apply analyses in triplicate to sensorially characterize the aroma and taste/mouthfeel profiles of 42 commercial NABs, a commercial soda, and a commercial seltzer water (n = 44). These beers were also chemically deconstructed using several different analytical techniques targeting volatile and nonvolatile compounds. Consumer analysis (n = 129) was then performed to evaluate the Northern Californian consumer hedonic liking of a selection (n = 12) of these NAB brands. These results provide direction to brewers and/or beverage producers on which techniques they should explore to develop desirable NAB offerings and suggest chemical targets that are indicators of specific flavor qualities and/or preference for American consumers.