Natural Occurrence of Nitrite-Related Compounds in Malt and Beer.

Despite sufficient control of volatile N-nitrosamines in foods and beverages, little attention remained on nonvolatile nitroso compounds, which are mostly unknown and relative to nitrite reactions. In a recent study, new compounds related to reactions of nitrite in beer were pyruvic acid oxime, 4-nitrosophenol, 4-cyanophenol, N-nitrosoproline ethyl ester, nitrosoguaiacol, and 2-methoxy-5-nitrophenol, as well as the already known N-nitrosoproline. The present study is intended to observe their natural occurrence in commercial beers and malts. All 22 nitrite-related products (N-products) were monitored in almost 200 samples of beers and malts. As many as 17 N-products were detected in malts, and all 22 N-products were found in beers. The hierarchical clustering grouped samples based on similarities of detected N-products by frequency of their appearance and level of response. Between N-products and N-nitrosodimethylamine concentrations in malts, only moderate Pearson correlations were found. The same applied to N-product correlations with the apparent total nitroso compound determination in beers.

Sensomic comparison of lager beers fermented by selected Saccharomyces pastorianus yeast strains.

Selection of the appropriate yeast strain is one of the most crucial steps in a brewery. Traditionally, yeast strain's abilities during beer fermentation are described according to brewer's experiences. Hence, these descriptions could be inaccurate and strictly based on sensory experiences. In this study, lager beers fermented by four traditional bottom-fermented yeast strains were characterized in detail by sensomic approach. The obtained results revealed that yeast strains can influence most of the sensory-related components in beer, not only esters and higher alcohols, but also carbonyls, amino acids, saccharides, fatty acids, heterocyclic compounds, hop oils, and other hop-related components. By comparison of chemical and sensory characteristics of each studied beer, the theoretical importance of sensory interactions on beer flavor perception was also revealed as the general conception that the beers with similar flavors have also similar chemical profiles (and vice versa) was seemed as not valid.

Characterization of Nitrite-Related Reaction Products in Beer.

The effect of nitrites in foods and beverages still raises discussion due to the possible formation of harmful nitroso compounds. However, as most of these compounds in beer were not structurally characterized yet, the research about their toxicological relevance for consumers is limited. This study is focused on identification of the products formed by nitrite (or isotopically labeled nitrite 15N) reactions in beer using gas chromatography with tandem mass spectrometry. In total, 19 products were identified, and some of them were structurally characterized and confirmed by comparing retention indices and mass spectra of standard/synthesized compounds. Identified compounds were representatives of nitroso, nitro, oxime, and even cyano compounds. For the peaks which were not structurally identified, primary structural characteristics were also listed. Found products were further screened in 16 authentic beer samples which showed the apparent occurrence of found compounds in non-treated beers.

DeepReI: Deep learning-based gas chromatographic retention index predictor.

Retention index in gas chromatographic analyses is an essential tool for appropriate analyte identification. Currently, many libraries providing retention indices for a huge number of compounds on distinct stationary phase chemistries are available. However, situation could be complicated in the case of unknown unknowns not present in such libraries. The importance of identification of these compounds have risen together with a rapidly expanding interest in non-targeted analyses in the last decade. Therefore, precise in silico computation/prediction of retention indices based on a suggested molecular structure will be highly appreciated in such situations. On this basis, a predictive model based on deep learning was developed and presented in this paper. It is designed for user-friendly and accurate prediction of retention indices of compounds in gas chromatography with the semi-standard non-polar stationary phase. Simplified Molecular Input Entry System (SMILES) is used as the model's input. Architecture of the model consists of 2D-convolutional layers, together with batch normalization, max pooling, dropout, and three residual connections. The model reaches median absolute error of prediction of the retention index for validation and test set at 16.4 and 16.0 units, respectively. Median percentage error is lower than or equal to 0.81% in the case of all mentioned data sets. Finally, the DeepReI model is presented in R package, and is available on https://github.com/TomasVrzal/DeepReI together with a user-friendly graphical user interface.

Pyrolytic profiling nitrosamine specific chemiluminescence detection combined with multivariate chemometric discrimination for non-targeted detection and classification of nitroso compounds in complex samples.

The problems of contamination of many products by nitroso compounds have been discussed since 1970's and have been partially solved, namely, the contamination by carcinogenic volatile N-nitrosamines. However, there is still a gap in knowing non-volatile nitroso compounds in terms of both the determination of these compounds and the description of their toxicity. Therefore, a procedure for their detailed non-targeted study is necessary to be developed. Based on these facts, a new method permitting the detection and the classification of nitroso compound groups, such as N-nitroso, C-nitroso, and interfering substances in the nitrosamine specific chemiluminescence detection after previous gas chromatographic separation, was developed. The method is based on signal profiling of chromatographic peaks recorded by a chemiluminescence detector at different pyrolytic temperatures and subsequent multivariate chemometric classification. The resulting classification function by linear discriminant analysis shows good performance with total accuracy of 96.12% after the method validation. The method was successfully applied and demonstrated on a non-targeted beer sample analysis. Nitroso compounds detected by the method were selected for detailed structural analysis by GC-MS/MS. The combination of the presented method with the MS/MS instrumentation provides a really powerful analytical tool for the identification of unknown nitroso compounds in complex samples. This study represents a valuable contribution to the protocols of identification of organic compounds with the nitrogen functional groups - toxicologically and analytically important nitroso compounds.

The chemical profiling of fatty acids during the brewing process.

BACKGROUND: Although fatty acids have a beneficial effect on yeast growth during fermentation, their effect on foam and sensory stability of beer is negative. In general, long-chain fatty acids originate from raw materials, whereas short-chain acids are produced by yeast during fermentation. If the concentration of short-chain fatty acids, especially isovaleric and butyric acid, overreaches a sensory threshold, then an unpleasant aroma, such as cheesy or sweaty feet, can be formed in beer. RESULTS: The distribution of fatty acids, from the preparation of sweet wort to the final beer, was studied using chemometric evaluation. Differences were observed between the decoction and infusion system using four barley varieties. Attention was paid to the behavior of short-chain fatty acids, namely isovaleric acid. The concentration of isovaleric acid in commercial beers brewed in infusion and decoction systems was approximately 1.4 and 1.0 mg L-1 , respectively. The same trend was observed in experimental samples (1.3 and 0.5 mg L-1 , respectively). This phenomenon was confirmed experimentally; based on the results, this possibly explains why, during the fermentation, isovaleric acid is coupled with the redox state of yeast cell, which is given by the wort composition (i.e. by the mashing process). CONCLUSION: The formation of isovaleric acid is not only caused by microbiology infection or by oxidized hops, but also is influenced by the mashing process. © 2018 Society of Chemical Industry.

Tracking, Behavior and Fate of 58 Pesticides Originated from Hops during Beer Brewing.

The study presents tracking of 58 pesticide residues associated with hops to estimate their carryover into brewed beer. The pesticides were spiked onto organic hops at a concentration of 15 mg/kg, and the wort was boiled with the artificially contaminated hops and fermented on a laboratory scale. Samples were collected during the whole brewing process and pesticide residues were extracted using a method known as QuEChERS (quick, easy, cheap, effective, rugged, and safe). An HPLC-HR-MS/MS method was developed and validated to identify and quantitate pesticide residues in treated hops, spent hops, hopped wort, green beer, and beer samples. Quantitation was achieved using standard addition with isotopically labeled standards. The carryover percentages into hopped wort and the percentages of decay reduction relative to the amount spiked on hops were calculated. The relationship between the partition coefficients n-octanol-water (log P values) and the residual ratios ( RW and RB) of a pesticide were evaluated to predict their behavior during hopping of wort and fermentation. Pesticides with a high log P values (>3.75) tended to remain in spent hops. The pesticides that have a low log P value up to approximately 3 could represent the demarcation lines of appreciable transfer rate of pesticides from hops to beer. Consequently, the pesticides were divided into three categories depending upon their fate during the brewing process. The most potential risk category represents a group involving the thermostable pesticides, such as azoxystrobin, boscalid, dimethomorph, flonicamid, imidacloprid, mandipropamid, myclobutanil, and thiamethoxam, which were transferred at high rates from the pesticide enriched hops into beer during the laboratory brewing trial. These results can be used as a guideline in the application of pesticides on hop plants that would reduce the level of pesticide residues in beer and their exposure in humans.

Inhibitory effect of hop fractions against Gram-positive multi-resistant bacteria. A pilot study.

AIM: Our research focused on the antimicrobial effects of purified hop (Humulus lupulus L.) fractions including α-bitter acids (humulones), ß-bitter acids (lupulones) and xanthohumol, and a commercial CO2 hop extract of bitter acids against reference and multi-resistant strains of Gram-positive and Gram-negative bacteria and against selected yeast strains. METHODS: In vitro testing of antimicrobial activity was performed according to standard testing protocols (EUCAST). The effects of hop extracts on bacterial/yeast strains at concentrations below MICs were also determined and the antimicrobial potential of hop extracts was compared with selected antibiotics using optical density measurement. RESULTS: The fractions were effective not only against reference strains of Gram-positive bacteria but, more importantly, against their methicillin- and vancomycin-resistant variants. No antimicrobial effect was detected against Gram-negative bacterial strains. Among the tested substances, xanthohumol was identified as the hop fraction with the most potent antimicrobial properties. It was also found that hop substances exerted their antimicrobial effects at concentrations considerably lower than the determined MICs, with the strongest effect in case of α-bitter acids in enterococci. CONCLUSION: The search for and research of new compounds with antimicrobial properties represents a possible solution to the current global problem of bacterial resistance. Our data suggest a desirable activity of hop fractions against some multi-resistant bacterial strains. Thus, hops might find use as a source of potential antimicrobial agents applicable in both human and veterinary medicine.

Antibiofilm activity of bioactive hop compounds humulone, lupulone and xanthohumol toward susceptible and resistant staphylococci.

Bacterial biofilms pose a serious medical problem due to their significant resistance to antimicrobials, and staphylococci are recognized as the most frequent cause of biofilm-associated infections. The hop plant (Humulus lupulus L.) contains substances that have been determined to act as anti-infective agents against bacteria, mainly in planktonic form. Therefore, we decided to investigate the antibiofilm properties of H. lupulus L.-derived compounds (humulone, lupulone and xanthohumol) against a selected group of Staphylococcus spp., including methicillin-susceptible and resistant strains. All tested hop compounds were shown to possess antimicrobial properties against all tested staphylococci, both planktonic and biofilm-dwelling, with no significant difference between resistant and susceptible strains. All compounds lowered the number of bacterial cells released from the biofilm, with the strongest effect seen for lupulone, followed by xanthohumol. Moreover, lupulone and xanthohumol were not only able to penetrate the biofilm and reduce the number of bacteria within it, but their higher concentrations (∼60 µg/mL for xanthohumol and ∼125 µg/mL for lupulone) reduced the number of surviving bacterial cells to zero.

Strong antimicrobial activity of xanthohumol and other derivatives from hops (Humulus lupulus L.) on gut anaerobic bacteria.

Anaerobic bacteria, such as Bacteroides fragilis or Clostridium perfringens, are part of indigenous human flora. However, Clostridium difficile represents also an important causative agent of nosocomial infectious antibiotic-associated diarrhoea. Treatment of C. difficile infection is problematic, making it imperative to search for new compounds with antimicrobial properties. Hops (Humulus lupulus L.) contain substances with antibacterial properties. We tested antimicrobial activity of purified hop constituents humulone, lupulone and xanthohumol against anaerobic bacteria. The antimicrobial activity was established against B. fragilis, C. perfringens and C. difficile strains according to standard testing protocols (CLSI, EUCAST), and the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBC) were calculated. All C. difficile strains were toxigenic and clinically relevant, as they were isolated from patients with diarrhoea. Strongest antimicrobial effects were observed with xanthohumol showing MIC and MBC values of 15-107 µg/mL, which are close to those of conventional antibiotics in the strains of bacteria with increased resistance. Slightly higher MIC and MBC values were obtained with lupulone followed by higher values of humulone. Our study, thus, shows a potential of purified hop compounds, especially xanthohumol, as alternatives for treatment of infections caused by select anaerobic bacteria, namely nosocomial diarrhoea caused by resistant strains.

Analysis of 100-Year-Old Beer Originated from the Czech Republic.

Three bottles of different beers were found in 2015 during a reconstruction of the brewery of the Raven Trading s.r.o. company in Záhlinice, Czech Republic. Thanks to good storage conditions, it was possible to analyze their original characteristics. All three bottles contained most probably lager type beer. One beer had sulfuric and fecal off-flavors; it was bright with the original extract of 10.3° Plato. The second beer, with an original extract of 7.6° Plato, was dark and very acidic, resembling Lambic. DNA analysis proved the presence of Dekkera bruxellensis, which corresponded to its chemical profile (total acidity, FAN, ethyl acetate, total esters). The third beer contained traces of carbon dioxide bubbles, was light brown and slightly bitter, with an original extract 10.4° Plato. Because it obviously underwent a natural aging process, sweetness, honey, and fruity off-flavors were detected and transformation products of iso-α-acids were found.

A novel approach for identification of biologically active phenolic compounds in complex matrices using hybrid quadrupole-orbitrap mass spectrometer: A promising tool for testing antimicrobial activity of hops.

The phenolic compounds, secondary metabolites of hops represent a large family of compounds that could be subsequently divided into smaller groups based on the similarities between their chemical structures. The antibacterial, antifungal and antiviral properties of hops are well known, but there is a lack of information about antimicrobial activities of individual hop compounds. This study was carried out with an objective to identify compounds present in hops that have potential antibacterial activity. In the first stage of experiment, the active compounds with potential anti-microbial activity had to be extracted from hop cones. Therefore, minced hop cones were applied on solid growth medium inoculated with Staphylococcus aureus. The active substances that migrated into the medium created an inhibition zone. In the second stage of experiment, the inhibition zones were cut out from Petri dishes, active compounds were extracted from these zones and consequently analyzed using LC-HRMS. These complex assays were developed and optimized. The data were acquired by using a quadrupole-orbitrap hybrid mass spectrometer by targeted-MS2 experiment in both ionization modes. The MS method has been developed as a screening method with a subsequent fragmentation of compound of interest on the base of inclusion mass list. The unknown compounds extracted from inhibition zones have been identified either by searching against a database or their structure has been elucidated on the basis of their fragmentation spectra. On the basis of this experiment the list of active compounds with potential anti-microbial activities was enhanced.

Phylogenetic relatedness determined between antibiotic resistance and 16S rRNA genes in actinobacteria.

BACKGROUND: Distribution and evolutionary history of resistance genes in environmental actinobacteria provide information on intensity of antibiosis and evolution of specific secondary metabolic pathways at a given site. To this day, actinobacteria producing biologically active compounds were isolated mostly from soil but only a limited range of soil environments were commonly sampled. Consequently, soil remains an unexplored environment in search for novel producers and related evolutionary questions. RESULTS: Ninety actinobacteria strains isolated at contrasting soil sites were characterized phylogenetically by 16S rRNA gene, for presence of erm and ABC transporter resistance genes and antibiotic production. An analogous analysis was performed in silico with 246 and 31 strains from Integrated Microbial Genomes (JGI_IMG) database selected by the presence of ABC transporter genes and erm genes, respectively. In the isolates, distances of erm gene sequences were significantly correlated to phylogenetic distances based on 16S rRNA genes, while ABC transporter gene distances were not. The phylogenetic distance of isolates was significantly correlated to soil pH and organic matter content of isolation sites. In the analysis of JGI_IMG datasets the correlation between phylogeny of resistance genes and the strain phylogeny based on 16S rRNA genes or five housekeeping genes was observed for both the erm genes and ABC transporter genes in both actinobacteria and streptomycetes. However, in the analysis of sequences from genomes where both resistance genes occurred together the correlation was observed for both ABC transporter and erm genes in actinobacteria but in streptomycetes only in the erm gene. CONCLUSIONS: The type of erm resistance gene sequences was influenced by linkage to 16S rRNA gene sequences and site characteristics. The phylogeny of ABC transporter gene was correlated to 16S rRNA genes mainly above the genus level. The results support the concept of new specific secondary metabolite scaffolds occurring more likely in taxonomically distant producers but suggest that the antibiotic selection of gene pools is also influenced by site conditions.

Qualitative determination of ß-acids and their transformation products in beer and hop using HR/AM-LC-MS/MS.

Hops represent an important source of ß-acids with antimicrobial and sensory properties. Transformation products of ß-acids formed during their oxidation, mainly hulupones, have been shown to have an interesting kind of bitterness. Their structures were recently elucidated using LC-TOFMS and 1D/2D NMR in solution after thermal treatment of the hop ß-acids. This study demonstrates the advantages of MS detection with high resolution and accurate mass measurements. The structure of transformation products in an experimental solution of oxidized ß-acids was elucidated using a newly developed method by hybrid quadrupole-Orbitrap MS. In addition to already known structures, two new ones were identified and named epoxycohulupone and epoxyhulupone. The method was verified on real samples; the profiles of these products in Sládek hops harvested in 2008 and 2012 and in corresponding beers were compared. For this purpose, a new QuEChERS assay was used for the preparation of beer samples.

Lincomycin biosynthesis involves a tyrosine hydroxylating heme protein of an unusual enzyme family.

The gene lmbB2 of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in Escherichia coli, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis.

Use of chemical indicators of beer aging for ex-post checking of storage conditions and prediction of the sensory stability of beer.

The rate of beer aging is affected by storage conditions including largely time and temperature. Although bottled beer is commonly stored for up to 1 year, sensorial damage of it is quite frequent. Therefore, a method for retrospective determination of temperature of stored beer was developed. The method is based on the determination of selected carbonyl compounds called as "aging indicators", which are formed during beer aging. The aging indicators were determined using GC-MS after precolumn derivatization with O-(2,3,4,5,6-pentaflourobenzyl)hydroxylamine hydrochloride, and their profile was correlated with the development of old flavor evolving under defined conditions (temperature, time) using both a mathematical and statistical apparatus. Three approaches, including calculation from regression graph, multiple linear regression, and neural networks, were employed. The ultimate uncertainty of the method ranged from 3.0 to 11.0 °C depending on the approach used. Furthermore, the assay was extended to include prediction of beer tendency to sensory aging from freshly bottled beer.

Characterization of N-demethyllincosamide methyltransferases LmbJ and CcbJ.

Chemical diversity: Two SAM-dependent N-methyltransferases-LmbJ from the biosynthesis of the antibiotic lincomycin and CcbJ from celesticetin biosynthesis-have been characterized and compared. Both tested enzymes form multimers and are able to utilize N-demethyllincomycin, the natural substrate of LmbJ, with comparable efficiency.

Microbial communities show parallels at sites with distinct litter and soil characteristics.

Plant and microbial community composition in connection with soil chemistry determines soil nutrient cycling. The study aimed at demonstrating links between plant and microbial communities and soil chemistry occurring among and within four sites: two pine forests with contrasting soil pH and two grasslands of dissimilar soil chemistry and vegetation. Soil was characterized by C and N content, particle size, and profiles of low-molecular-weight compounds determined by high-performance liquid chromatography (HPLC) of soil extracts. Bacterial and actinobacterial community composition was assessed by terminal restriction fragment length polymorphism (T-RFLP) and cloning followed by sequencing. Abundances of bacteria, fungi, and actinobacteria were determined by quantitative PCR. In addition, a pool of secondary metabolites was estimated by erm resistance genes coding for rRNA methyltransferases. The sites were characterized by a stable proportion of C/N within each site, while on a larger scale, the grasslands had a significantly lower C/N ratio than the forests. A Spearman's test showed that soil pH was correlated with bacterial community composition not only among sites but also within each site. Bacterial, actinobacterial, and fungal abundances were related to carbon sources while T-RFLP-assessed microbial community composition was correlated with the chemical environment represented by HPLC profiles. Actinobacteria community composition was the only studied microbial characteristic correlated to all measured factors. It was concluded that the microbial communities of our sites were influenced primarily not only by soil abiotic characteristics but also by dominant litter quality, particularly, by percentage of recalcitrant compounds.

Determination of 15 isomers of chlorobenzoic acid in soil samples using accelerated sample extraction followed by liquid chromatography.

A study was conducted to elaborate a fast, simple and efficient method for determination of 15 isomers chlorobenzoic acids (CBAs) in soil using HPLC-UV. Artificially contaminated soil samples were extracted using accelerated solvent extraction (ASE) with 1% acetic acid in a mixture of hexane and acetone (1:1, V/V) under a pressure of 10.34 MPa and temperature of 150°C. The recovery of the ASE method was above 82%. The extracts were concentrated; dimethyl sulfoxide was used to prevent CBA volatilization and the final analysis was performed with a C18 XBridge HPLC column employing a mobile phase consisting of acetonitrile and 0.1% trifluoracetic acid in water. A HPLC procedure with gradient elution and UV detection was developed and validated. The method exhibited a linear range for 2-CBA; 2,6-CBA; 3-CBA; 4-CBA; 2,3-CBA; 2,3,6-CBA; 2,5-CBA; and 2,4-CBA from 5 to 120 µg/mL with a limit of quantification (LOQ) of 5 µg/mL, RSD from 2.42 to 9.42% and accuracy from 82 ± 2 to 103 ± 3%. The linear range of determination of 2,4,6-CBA, 3,4-CBA, 2,3,5,6-CBA, 3,5-CBA, 2,3,5-CBA, 2,3,4,5,6-CBA and 2,3,4,5-CBA was 10-120 µg/mL with LOQ 10 µg/mL, RSD from 0.74 to 5.84% and accuracy from 94 ± 1 to 114 ± 1%. The optimized analytical procedure was finally applied on two historically PCB contaminated soils and 9 CBAs were quantified in the samples.

The UHPLC-DAD fingerprinting method for analysis of extracellular metabolites of fungi of the genus Geosmithia (Acomycota: Hypocreales).

A new simple ultra-high-performance liquid chromatography method with diode array detection (UHPLC-DAD) was developed for chemical fingerprinting analysis of extracellular metabolites in fermentation broth of Geosmithia spp. The SPE method employing Oasis MCX strong cation-exchange mixed-mode polymeric sorbent was chosen for extraction of the metabolites. The analyses were performed on an Acquity UPLC BEH C18 column (100 × 2.1 mm i.d.; particle size, 1.7 µm; Waters) using a gradient elution program with an aqueous solution of trifluoroacetic acid and acetonitrile as the mobile phase. The applicability of the method was proved by analysis of 38 strains produced by different species and isolated from different sources (hosts). The results revealed the correlation of obtained UHPLC-DAD fingerprints with taxonomical identity.