Barley-derived beer brewing by-products contain a high diversity of hydroxycinnamoylagmatines and their dimers.

To aid valorisation of beer brewing by-products, more insight into their composition is essential. We have analysed the phenolic compound composition of four brewing by-products, namely barley rootlets, spent grain, hot trub, and cold trub. The main phenolics detected were hydroxycinnamoylagmatines and dimers thereof. Barley rootlets contained the highest hydroxycinnamoylagmatine content and cold trub the highest dimer content. Additionally, variations in (dimeric) hydroxycinnamoylagmatine composition and content were observed in fourteen barley rootlet samples. The most abundant compound in all rootlets was the glycosylated 4-O-7'/3-8'-linked heterodimer of coumaroylagmatine and feruloylagmatine, i.e. CouAgm-4-O-7'/3-8'-(4'Hex)-DFerAgm. Structures of glycosylated and hydroxylated derivatives of coumaroylagmatine were elucidated by NMR spectroscopy after their purification from a rootlet extract. An MS-based decision tree was developed, which aids in identifying hydroxycinnamoylagmatine dimers in complex mixtures. In conclusion, this study shows that the diversity of phenolamides and (neo)lignanamides in barley-derived by-products is larger than previously reported.

Non-Conventional Yeast: Behavior under Pure Culture, Sequential and Aeration Conditions in Beer Fermentation.

The use of wild yeasts, isolated from different environments, is becoming the most interesting option for the production of new beers. The objective of this study is to evaluate the potential of seven non-conventional yeast strains from five different species (Saccharomyces cerevisiae, Hanseniaspora guilliermondii, Metschnikowia pulcherrima, Torulaspora delbrueckii, and Zygosaccharomyces bailii) isolated from Madrid agriculture to produce type ale beer. Wild yeast strains were evaluated at laboratory and pilot plant scales under different fermentation conditions (pure, aerated, and sequential culture). Strain S. cerevisiae SafAle S-04 was used as a reference. Throughout the fermentation of beer, volatile compounds were determined by GC and residual sugars by HPLC, among other parameters. The yeast strains used for the fermentation in pure culture conditions were unable to ferment maltose and maltotriose (0.73-1.18% v/v of ethanol). The results of the study under aerated conditions showed varying levels of higher alcohol and ester concentrations. It should be noted that the strain CLI 1057 (S. cerevisiae) fermented maltose in the presence of oxygen (Kluyver effect). This strain also showed a high production of 4-vinyl guaiacol, making it suitable for producing beers with a phenolic profile. Finally, three strains (H. guilliermondii, Z. bailii, and T. delbrueckii) were evaluated in sequential culture together with commercial strain and found to improve the organoleptic characteristics of the brewed beer. These approaches offer the opportunity to add new product characteristics to the beers.

Comparative genetic and physiological characterisation of Pectinatus species reveals shared tolerance to beer-associated stressors but halotolerance specific to pickle-associated strains.

Obligate anaerobic bacteria from the genus Pectinatus have been known to cause beer spoilage for over 40 years. Whole genome sequencing was performed on eleven beer spoilage strains (nine Pectinatus frisingensis, one Pectinatus cerevisiiphilus and one Pectinatus haikarae isolate), as well as two pickle spoilage species (Pectinatus brassicae MB591 and Pectinatus sottacetonis MB620) and the tolerance of all species to a range of environmental conditions was tested. Exploration of metabolic pathways for carbohydrates, amino acids and vitamins showed little difference between beer spoilage- and pickle spoilage-associated strains. However, genes for certain carbohydrate- and sulphur-containing amino acid-associated enzymes were only present in the beer spoilage group and genes for specific transporters and regulatory genes were uniquely found in the pickle spoilage group. Transporters for compatible solutes, only present in pickle-associated strains, likely explain their experimentally observed higher halotolerance compared to the beer spoilers. Genes involved in biofilm formation and ATP Binding Cassette (ABC) transporters potentially capable of exporting hop-derived antimicrobial compounds were found in all strains. All species grew in the presence of alcohol up to 5% alcohol by volume (ABV) and hops extract up to 80 ppm of iso-α-acids. Therefore, the species isolated from pickle processes may pose novel hazards in brewing.

A Plasmid-Encoded Putative Glycosyltransferase Is Involved in Hop Tolerance and Beer Spoilage in Lactobacillus brevis.

Lactobacillus brevis beer-spoiling strains harbor plasmids that contain genes such as horA, horC, and hitA which are known to confer hop tolerance. The L. brevis beer-spoiling strain UCCLBBS124, which possesses four plasmids, was treated with novobiocin, resulting in the isolation of UCCLBBS124 derivatives exhibiting hop sensitivity and an inability to grow in beer. One selected derivative was shown to have lost a single plasmid, here designated UCCLBBS124_D, which harbors the UCCLBBS124_pD0015 gene, predicted to encode a glycosyltransferase. Hop tolerance and growth in beer were restored when UCCLBBS124_pD0015 was introduced in one of these hop-sensitive derivatives on a plasmid. We hypothesize that this gene modifies the surface composition of the polysaccharide cell wall, conferring protection against hop compounds. Furthermore, the introduction of this gene in trans in L. brevis UCCLB521, a strain that cannot grow in and spoil beer, was shown to furnish the resulting strain with the ability to grow in beer, while its expression also conferred phage resistance. This study underscores how the acquisition of certain mobile genetic elements plays a role in hop tolerance and beer spoilage for strains of this bacterial species.IMPORTANCELactobacillus brevis is a member of the lactic acid bacteria and is often reported as the causative agent of food or beverage spoilage, in particular, that of beer. Bacterial spoilage of beer may result in product withdrawal or recall, with concomitant economic losses for the brewing industry. A very limited number of genes involved in beer spoilage have been identified and primarily include those involved in hop resistance, such as horA, hitA, and horC However, since none of these genes are universal, it is clear that there are likely (many) other molecular players involved in beer spoilage. Here, we report on the importance of a plasmid-encoded glycosyltransferase associated with beer spoilage by L. brevis that is involved in hop tolerance. The study highlights the complexity of the genetic requirements to facilitate beer spoilage and the role of multiple key players in this process.

Biodiversity and Classification of Phages Infecting Lactobacillus brevis.

Lactobacillus brevis is a lactic acid bacterium that is known as a food and beverage spoilage organism, and more specifically as a beer-spoiler. Phages of L. brevis have been described, but very limited data is available regarding temperate phages of L. brevis. Temperate phages may exert benefits to the host, while they may also be employed to combat beer spoilage. The current study reports on the incidence of prophage sequences present in nineteen distinct L. brevis genomes. Prophage induction was evaluated using mitomycin C exposure followed by genome targeted-PCR, electron microscopy and structural proteome analysis. The morphological and genome sequence analyses revealed significant diversity among L. brevis prophages, which appear to be dominated by members of the Myoviridae phage family. Based on this analysis, we propose a classification of L. brevis phages into five groups.

Isolation and Characterization of Lactobacillus brevis Phages.

Lactobacillus brevis has been widely used in industry for fermentation purposes. However, it is also associated with the spoilage of foods and beverages, in particular, beer. There is an increasing demand for natural food preservation methods, and in this context, bacteriophages possess the potential to control such spoilage bacteria. Just a few studies on phages infecting Lactobacillus brevis have been performed to date and in the present study, we report the isolation and characterization of five virulent phages capable of infecting Lb. brevis strains. The analysis reveals a high diversity among the isolates, with members belonging to both, the Myoviridae and Siphoviridae families. One isolate, designated phage 3-521, possesses a genome of 140.8 kb, thus representing the largest Lb. brevis phage genome sequenced to date. While the isolated phages do not propagate on Lb. brevis beer-spoiling strains, phages showed activity against these strains, impairing the growth of some Lb. brevis strains. The results highlight the potential of bacteriophage-based treatments as an effective approach to prevent bacterial spoilage of beer.

Comparative genome analysis of the Lactobacillus brevis species.

BACKGROUND: Lactobacillus brevis is a member of the lactic acid bacteria (LAB), and strains of L. brevis have been isolated from silage, as well as from fermented cabbage and other fermented foods. However, this bacterium is also commonly associated with bacterial spoilage of beer. RESULTS: In the current study, complete genome sequences of six isolated L. brevis strains were determined. Five of these L. brevis strains were isolated from beer (three isolates) or the brewing environment (two isolates), and were characterized as beer-spoilers or non-beer spoilers, respectively, while the sixth isolate had previously been isolated from silage. The genomic features of 19 L. brevis strains, encompassing the six L. brevis strains described in this study and thirteen L. brevis strains for which complete genome sequences were available in public databases, were analyzed with particular attention to evolutionary aspects and adaptation to beer. CONCLUSIONS: Comparative genomic analysis highlighted evolution of the taxon allowing niche colonization, notably adaptation to the beer environment, with approximately 50 chromosomal genes acquired by L. brevis beer-spoiler strains representing approximately 2% of their total chromosomal genetic content. These genes primarily encode proteins that are putatively involved in oxidation-reduction reactions, transcription regulation or membrane transport, functions that may be crucial to survive the harsh conditions associated with beer. The study emphasized the role of plasmids in beer spoilage with a number of unique genes identified among L. brevis beer-spoiler strains.

Structural and functional analysis of pCI65st, a 6.5 kb plasmid from Streptococcus thermophilus NDI-6.

The 6.5 kb cryptic plasmid pCI65st from Streptococcus thermophilus NDI-6, a strain isolated from the Indian fermented milk dahi, was subcloned and sequenced. Five putative ORFs were identified. ORF1 could encode a 315 aa polypeptide almost identical to the RepA protein of previously sequenced S. thermophilus plasmids, indicating that pCI65st is one of the pC194 group of small gram-positive rolling-circle plasmids. ORFs 2 and 4 were virtually identical and could specify proteins of approximately 150 aa with significant similarity to the small heat-shock proteins described from a variety of gram-positive bacteria. ORF3 could encode a 415 aa protein similar to enolase, an enzyme involved in glycolysis and gluconeogenesis. ORF5 could encode a 412 aa protein which had high similarity to the HsdS (specificity) proteins of type I restriction-modification systems. Variants of strain NDI-6 which lacked pCI65st were readily isolated after subculture of the parent strain at 32 degrees C. The plasmid-bearing parent culture was significantly more resistant to a temperature shift from 42 degrees C to 62 degrees C than its plasmid-free variant and expressed proteins which corresponded with the predicted translation products from ORF2 and ORF4. In addition, plasmid-free mutants were lysed in broth by bacteriophages to which the parent culture was resistant.