Comparison of odor-active compounds in the spicy fraction of hop (Humulus lupulus L.) essential oil from four different varieties

The "spicy" character of hops is considered to be a desirable attribute in beer, associated with "noble hop aroma". However, the compounds responsible have yet to be adequately identified. Odorants in four samples of the spicy fraction of hop essential oil were characterized using gas chromatography-olfactometry (GC-O) and CharmAnalysis. Four hop varieties were compared, namely, Target, Saaz, Hallertauer Hersbrucker, and Cascade. Odor-active compounds were tentatively identified using comprehensive two-dimensional gas chromatography (GCxGC) combined with time-of-flight mass spectrometry (TOFMS). An intense "woody, cedarwood" odor was determined to be the most potent odorant in three of the four spicy fraction samples. This odor coincided with a complex region where between 8 and 13 compounds were coeluting in each of the four spicy fractions. The peak responsible was determined by (i) correlating peak areas with Charm values in eight hop samples and (ii) heart-cut multidimensional gas chromatography-olfactometry (MDGC-O). The compound responsible was tentatively identified as 14-hydroxy-beta-caryophyllene. Other important odorants identified were geraniol, linalool, beta-ionone, and eugenol.     

Relationships between antioxidant activity, color, and flavor compounds of crystal malt extracts.

Aqueous extracts were prepared from five barley crystal malts (color range 15-440 degrees EBC, European Brewing Convention units). Antioxidant activity was determined by using the 2,2'-azinobis(3-ethylbenothiazoline-6-sulfonic acid) (ABTS(*)(+)) radical cation scavenging method. Antioxidant activity increased with increasing color value although the rate of increase decreased with increasing color value. Color was measured in CIELAB space. Extracts of the 15, 23, and 72 degrees EBC malts followed the same dilution pathway as did the 148 degrees EBC sample at higher dilution levels, indicating that they could each be used to give the same color by appropriate dilution. The 440 degrees EBC sample followed a different dilution pathway, indicating that different compounds were responsible for color in this extract. Fifteen selected volatile compounds were monitored using gas chromatography/mass spectrometry (GC/MS). Levels of methylpropanal, 2-methylbutanal, and 3-methylbutanal were highest for the 72 degrees EBC sample. When odor threshold values of the selected compounds were taken into account, 3-methylbutanal was the most important contributor to flavor. Relationships between levels of the lipid oxidation products, hexanal and (E)-2-nonenal, and antioxidant activity were complex, and increasing antioxidant activity for samples in the range of 15-148 degrees EBC did not result in reduced levels of these lipid-derived compounds. When different colored malt extracts were diluted to give the same a* and b* values, calculated antioxidant activity and amounts of 3-methylbutanal, hexanal, and (E)-2-nonenal decreased with increasing degrees EBC value.     

Analysis of hop acids and their oxidized derivatives and iso-alpha-acids in beer by capillary electrophoresis-electrospray ionization mass spectrometry

This study investigates the applicability of on-line coupling of capillary electrophoresis with electrospray ionization tandem mass spectrometry (CZE-ESI-MS) for the separation and characterization of alpha- and beta-acids and oxidized hop acids from crude extracts of different hop varieties. CZE-ESI-MS with negative-ion electrospray ionization proved to be a suitable technique for the determination of these types of natural compounds and their oxidized derivatives. The CZE parameters (pH, concentration, and buffer type) and ESI-MS parameters (nature and flow rate of the sheath liquid, nebulizer pressure, drying gas flow rate, temperature, and compound stability) were optimized. The optimized method provides the potential for a fast qualitative determination of hop acids and their oxidation compounds. The method was also applied to the determination of iso-alpha-acids in beer.     

Detection and quantification of provitamin D2 and vitamin D2 in hop (Humulus lupulus L.) by liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry

In this work, ergosterol and ergocalciferol were identified for the first time in hop. In addition, in this article, a simple and reliable analytical methodology for analysis of these compounds in different commercial forms of hop is presented. The performance of the method was assessed by the evaluation of parameters such as absolute recovery (higher than 70%), repeatability (lower than 3 %), linearity ( r(2) > 0.9988) and limits of detection (ranging from 0.034 for ergocalciferol to 0.058 mg/L for ergosterol) and quantification (ranging from 0.113 for ergocalciferol to 0.195 mg/L for ergosterol). On the basis of standard additions applied with the optimized procedure and high-performance liquid chromatography with diode array detection, it appears that the Nugget hop plant (crop 2006) contains 1.84 +/- 0.09 microg/g of ergosterol and 1.95 +/- 0.05 microg/g of ergocalciferol. The identity of the compounds was confirmed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry in the positive ion mode. The presence of ergosterol here reported should have great potential for the assessment of hop as related to the fungal contamination proportion and hence the quality of this raw material.     

Use of GC-olfactometry to identify the hop aromatic compounds in beer

This paper describes a sensorial aroma extract dilution analysis (AEDA) approach to the analysis of beer aromas derived from hops. To obtain an extract with an odor representative of the original product, the XAD extraction procedure was applied and the experimental conditions were optimized. The aromagrams of three beers were compared: one brewed without hops, one brewed with Saaz hop pellets, and one brewed with Challenger hop pellets. One spicy/hoppy compound, unmodified from hop to beer, proved responsible for the most intense odor in both hopped beer extracts. Another flavoring compound in hops, linalool, also survives through the process to the final beer. Other compounds such as gamma-nonalactone and humuladienone, although not found in our extracts of hop, significantly modify beer aromagrams after hopping. Sulfur compounds characteristic of Challenger hops proved to be at least partially responsible for the unpleasant flavor found in the corresponding beer.     

Direct thermal desorption-gas chromatography and gas chromatography-mass spectrometry profiling of hop (Humulus lupulus L.) essential oils in support of varietal characterization

The use of direct thermal desorption-gas chromatography-mass spectrometry (DTD-GC-MS) and DTD-GC-flame ionization detection (DTD-GC-FID) for characterization of hop essential oils is described. Four hop varieties (Nugget, Galena, Willamette, and Cluster) from the Yakima valley (Yakima, WA) 1998 harvest were analyzed by DTD-GC-MS and DTD-GC-FID methodology. Approximately 1 g of hops was needed for the analysis. Hop samples were prepared for GC-MS and/or GC-FID profiling in approximately 20 min. More than 100 volatile compounds have been identified and quantified for each hop variety. The results were found to be in good agreement with conventional steam distillation-extraction (SDE) data. A calibration curve for determination of essential oil content in hops by DTD-GC-FID has been generated. Quantitation of hop oil content by DTD-GC-FID was shown to be in good agreement with conventional SDE data. The recovery of key oil components valuable for varietal identification was demonstrated to be highly reproducible and characteristic of each variety analyzed when DTD-GC-FID was used for analysis.     

Relevance of organic farming and effect of climatological conditions on the formation of alpha-acids, beta-acids, desmethylxanthohumol, and xanthohumol in hop (Humulus lupulus L.)

The concentrations of alpha-acids, beta-acids, desmethylxanthohumol, and xanthohumol were monitored in the hop varieties Admiral (A), Wye Challenger (WC), and First Gold (FG) during the harvest seasons of 2003 through 2005. Hops grown under an organic regimen were compared to plants grown conventionally in hop fields in close vicinity. The concentrations of the key compounds depended very much on climatological conditions showing, in general, highest levels in poorest weather conditions (2004). Of the three varieties studied, FG was the only one showing a clear trend for higher concentrations of secondary metabolites under organic growing conditions than under conventional farming conditions. Cultivation of A and WC seems to be very sensitive to climatic conditions and environmental stresses caused by pests and diseases, thereby leading to various results. WC proved to be a rich source of bioactive chalcones, particularly desmethylxanthohumol.     

Occurrence of resveratrol and piceid in American and European hop cones

The recent discovery in hops of trans-resveratrol and its glucoside, trans-piceid, both famous in wine for their potential role in the "French paradox", opens new doors to understanding hop health benefits. In the present work, trans-resveratrol and trans-piceid were quantified by HPLC-APCI-MS/MS in 40 American and European hop cone samples from harvests of 2004, 2005, and 2006. Their content varies greatly, in the range 0.5-12 mg/kg. All German varieties revealed low amounts of stilbenes. The highest concentrations were found in Cascade (2004) and Willamette (2004 and 2006), two American low-alpha-acid varieties. Yet, probably because trans-resveratrol is a phytoalexin, a strong influence of harvest year was also observed.     

Identification and in vitro biological activities of hop proanthocyanidins: inhibition of nNOS activity and scavenging of reactive nitrogen species

Oligomeric proanthocyanidins constitute a group of water-soluble polyphenolic tannins that are present in the female inflorescences (up to 5% dry wt) of the hop plant (Humulus lupulus). Humans are exposed to hop proanthocyanidins through consumption of beer. Proanthocyanidins from hops were characterized for their chemical structure and their in vitro biological activities. Chemically, they consist mainly of oligomeric catechins ranging from dimers to octamers, with minor amounts of catechin oligomers containing one or two gallocatechin units. The chemical structures of four procyanidin dimers (B1, B2, B3, and B4) and one trimer, epicatechin-(4beta-->8)-catechin-(4alpha-->8)-catechin (TR), were elucidated using mass spectrometry, NMR spectroscopy, and chemical degradation. When tested as a mixture, the hop oligomeric proanthocyanidins (PC) were found to be potent inhibitors of neuronal nitric oxide synthase (nNOS) activity. Among the oligomers tested, procyanidin B2 was most inhibitory against nNOS activity. Procyanidin B3, catechin, and epicatechin were noninhibitory against nNOS activity. PC and the individual oligomers were all strong inhibitors of 3-morpholinosydnonimine (SIN-1)-induced oxidation of LDL, with procyanidin B3 showing the highest antioxidant activity at 0.1 microg/mL. The catechin trimer (TR) exhibited antioxidant activity more than 1 order of magnitude greater than that of alpha-tocopherol or ascorbic acid on a molar basis.     

Fate of resveratrol and piceid through different hop processings and storage times

Trans-Piceid and trans-resveratrol contents of hop cones, hop pellets, CO2 extracts, and spent hop from American varieties (harvest 2004) were determined by reverse-phase high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry [RP-HPLC-APCI(+)-MS/MS]. Pelletization induced strong stilbene degradation in some cultivars. Similarly, 1 year of storage at 4 degrees C led to a huge loss of trans-piceid, especially in the case of hop cones (much faster than in model media, although well protected from light and oxygen). Therefore, after 8 months of storage, the overall stilbene content was in the same range whatever the conditioned form. Absent in fresh hop cones or pellets, cis-resveratrol was released from cis-piceid in all stored samples. On the other hand, no delta-viniferin was detected despite it is present in light-protected model media spiked with trans-piceid. Because supercritical carbon dioxide proved inefficient for recovering resveratrol and piceid from pellets, spent hop emerged as the most interesting material for subsequent specific stilbene extraction.     

Determination of stilbenes in hop pellets from different cultivars

About 30% of the polyphenols in wort and beer derive from hop, but little is yet known about their nutritional impact. The recent discovery of trans-resveratrol and piceid isomers in hop opens new doors to understanding beer health benefits. In the present work, resveratrol was quantified by HPLC-APCI-MS/MS in pellets from 9 different cultivars. Concentrations ranging from 4 to 9 mg/kg trans-piceid, from 2 to 6 mg/kg cis-piceid, and up to 1 mg/kg trans-resveratrol were detected. As previously shown for total polyphenols and flavonoids, the lower the alpha-acid content, the higher the total stilbene content.     

Chemical composition of the flower oil of Cinnamomum zeylanicum blume

The steam-distilled oil of cinnamon (Cinnamomum zeylanicum) flowers was analyzed by GC and GC-MS. It consists of 23% hydrocarbons and 74% oxygenated compounds. A total of 26 compounds constituting approximately 97% of the oil were characterized. (E)-Cinnamyl acetate (41.98%), trans-alpha-bergamotene (7.97%), and caryophyllene oxide (7.2%) are found to be major compounds. This is the first report on the chemical composition of the flower oil of Cinnamomum zeylanicum.     

Phytochemical and morphological characterization of hop (Humulus lupulus L.) cones over five developmental stages using high performance liquid chromatography coupled to time-of-flight mass spectrometry, ultrahigh performance liquid chromatography photodiode array detection, and light microscopy techniques

Hop (Humulus lupulus L.) inflorescences, commonly known as "hop cones", are prized for their terpenophenolic contents, used in beer production and, more recently, in biomedical applications. In this study we investigated morphological and phytochemical characteristics of hop cones over five developmental stages, using liquid chromatography coupled to time-of-flight mass spectrometry (LC-TOF-MS), and ultrahigh performance liquid chromatography photodiode array detection (UHPLC-PDA) methods to quantitate 21 polyphenolics and seven terpenophenolics. Additionally, we used light microscopy to correlate phytochemical quantities with changes in the morphology of the cones. Significant increases in terpenophenolics, concomitant with glandular trichome development and associated gross morphological changes, were mapped over development to fluctuations in contents of polyphenolic constituents and their metabolic precursor compounds. The methods reported here can be used for targeted metabolic profiling of flavonoids, phenolic acids, and terpenophenolics in hops, and are applicable to quantitation in other crops.     

Inventory methods for finding historically cultivated hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.) in Sweden

The relationship between the conservation of plant genetic resources and their culture history is intimate. Consequently, biodiversity research must also take into account historical and cultural factors. An inventory of plants collected from all over Sweden was made with the aim of establishing a national Swedish gene bank for once cultivated hop. Only female hop, which could be regarded as cultivated hop on the basis of history, were selected. In this study, two different inventory methods were used: one based on plant habitat and the other involving the use of historical documents, primarily large-scale maps from the first half of the seventeenth century, used to locate their cultivars today. The documented history of the hop, combined with its biology, is the basis for the methods used. Hop is the only Swedish crop that according to a 1442 law had to be cultivated. The law lasted for over 400 years. Since the hop is a perennial, dioecious plant and only female individuals are cultivated, over time very few genetic recombination events are expected. Today, it is possible to connect and identify living plants using historical documents. The degree of connection between today’s living plants and the historical evidence for hop cultivation differ between the two methods.     

AFLP fingerprinting in hop: analysis of the genetic variability of the Tettnang variety

Hops are mostly grown as an ingredient for beer production where quality and aroma properties play an important role. Tett­nanger is one of the famous aroma varieties, that had been considered to be an old landrace. As its genetic origin was unknown, the level of genetic variability was assessed by AFLP fingerprints. A representative sample of 279 individual Tett­nanger plants was analyzed by a set of six primer combinations, which had previously been proven to reveal a high level of polymorphism. Very little polymorphism was detected among the samples from Tettnang. By comparing Tett­nanger to 16 related hop varieties, which could be clearly distinguished from each other by AFLP fingerprints, two individual plants from Tett­nanger were identified to belong to the varieties Hallertauer mfr. or Backa, respectively. One further plant was considered to be a mutant of Tett­nanger. It was concluded that the variety Tett­nanger constitutes one clone. The robustness of the AFLP technique was demonstrated by using hop DNA from leaves, cones, and pellets. Except for pellets all sources of plant material yielded DNA for highly reproducible, informative AFLP-banding patterns. Our results showed, that the AFLP technique was a feasible tool for determining the level of genetic variability within one hop variety and between varieties.     

Identification and genetic structure of wild Italian <Emphasis Type="Italic">Humulus lupulus</Emphasis> L. and comparison with European and American hop cultivars using nuclear microsatellite markers

Nine genic SSR loci were used to evaluate the genetic diversity and identify accessions in wild Italian Humulus lupulus L., in comparison with widely cultivated European and U.S. commercial cultivars. A collection of 80 wild hop samples from Italy and 43 hop cultivars from Europe and U.S., were characterized. Allelic frequency analysis revealed 65 distinct Italian genotypes and differentiated all the commercial cultivars; moreover, specific alleles were observed for wild and cultivated hops. The number of alleles identified in the wild population were 104 and 123 within all the accessions. The maximum polymorphic information content was evidenced for locus HlGA23 in the Italian wild population and in the whole set of accessions (0.905 and 0.902 respectively). The dendrogram constructed from Euclidean distance with the UPGMA method showed two main clusters, one including commercial American and European accessions and one mostly composed by wild Italian accessions. Model-based clustering (Bayesian method) placed the accessions into five germplasm groups, one of which was characterized by Italian genotypes only. The study showed for the first time the great biodiversity present in Italy, and the remarkable differences with European and American hops. It was also found that within the population of north-central Italy a large genetic variability is present, suited to be studied and exploited; this genetic wealth could be used in future breeding programs in order to develop new hop varieties carrying characteristics useful for brewers.     

Quantitative analysis of aroma compounds in carrot (Daucus carota L.) cultivars by capillary gas chromatography using large-volume injection technique

Dynamic headspace sampling was used to collect aroma compounds from raw samples of four carrot (Daucus carota L.) cultivars (Brasilia, Duke, Fancy, and Cortez). The collected volatiles were analyzed by capillary GC-FID and GC-MS using large-volume cool on-column injection (LVI-COC). Of the 36 compounds identified, 6 had not been previously detected in carrots. Significant differences between the carrot cultivars were found for 31 of the identified volatiles as well as for total monoterpenes, sesquiterpenes, and total volatile content. Mono- and sesquiterpenes accounted for about 98% of the total volatile mass in all cultivars. LVI-COC injection was used to determine the loss of carrot volatiles during concentration of headspace samples under a stream of nitrogen. The loss among major monoterpenes in the concentrated samples varied from 16% for p-cymene to >40% for alpha-pinene as compared to nonconcentrated samples. The loss among high-boiling sesquiterpenes varied from not detectable (beta-caryophyllene, alpha-humulene, and caryophyllene oxide) to approximately 7% for (E)- and (Z)-gamma-bisabolene.     

Mechanisms of cancer chemoprevention by hop bitter acids (beer aroma) through induction of apoptosis mediated by Fas and caspase cascades

The bitter acids of hops (Humulus lupulus L.) mainly consist of alpha-acids, beta-acids, and their oxidation products that contribute the unique aroma of the beer beverage. Hop bitter acids displayed a strong growth inhibitory effect against human leukemia HL-60 cells, with an estimated IC(50) value of 8.67 microg/mL, but were less effective against human histolytic lymphoma U937 cells. Induction of apoptosis was confirmed in HL-60 cells by DNA fragmentation and the appearance of a sub-G1 DNA peak, which were preceded by dissipation of mitochondrial membrane potential, cytochrome c release, and subsequent induction of pro-caspase-9 and -3 processing. Cleavages of PARP and DFF-45 were accompanied with activation of caspase-9 and -3 triggered by hop bitter acids in HL-60 cells. The change in the expression of Bcl-2, Bcl-X(L), and Bax in response to hop bitter acids was studied, and the Bcl-2 protein level slightly decreased; however, the Bcl-X(L) protein level was obviously decreased, whereas the Bax protein level was dramatically increased, indicating that the control of Bcl-2 family proteins by hop bitter acids might participate in the disruption of mitochondrial integrity. In addition, the results showed that hop bitter acids promoted the up-regulation of Fas and FasL prior to the processing and activation of pro-caspase-8 and cleavage of Bid, suggesting the involvement of a Fas-mediated pathway in hop bitter acids-induced cells. Taken together, these findings suggest that a certain intimate link might exist between receptor- and mitochondria-mediated death signalings that committed to cell death induced by hop bitter acids. The induction of apoptosis by hop bitter acids may offer a pivotal mechanism for their chemopreventive action.