Capillary electrophoresis in association with chemometrics approach for bitterness hop (Humulus lupulus L.) classification.

The precursor compounds related to the bitterness of beer are called α-acids. These compounds are extracted from the hop, which is an important ingredient in the brewing process. These compounds were analyzed by capillary electrophoresis. The electrophoretic method used 160 mmol/L of ammonium carbonate (pH 9) as BGE (background electrolyte), a voltage of +20 kV in a capillary with 50 µm of internal diameter and with a 62.5 cm of total length (54 cm effective). The samples were injected in hydrodynamic mode applying a pressure of 25 mbar for 5 s and the analytes were detected at 230 nm. A hydromethanolic extraction during 3 h was considered as the optimum condition for the sample preparation using MeOH/H2 O 80:20 v/v as the extract solution. From the optimized conditions the electropherograms were evaluated for their use as input for chemometric modeling. Preprocessing investigation for electrophoretic data taking into account the alignment, denoising and baseline correction, and variable selection were considered before the chemometric modeling using principal component analysis (PCA). The electrophoretic data were systematically evaluated to find the optimum conditions to modeling. A PCA analysis for all tests was carried out using different preprocessing methods and, an explained variance higher than 90% was achieved in all of them. The optimized chemometric method worked with aligned and meancentered data. From this approach, a simple and efficient method to classify hop samples with high and low α-acids content without the use of analytical standards was established from a simple electrophoretic analysis.

Quantitation of 4-Methyl-4-sulfanylpentan-2-one (4MSP) in Hops by a Stable Isotope Dilution Assay in Combination with GC×GC-TOFMS: Method Development and Application To Study the Influence of Variety, Provenance, Harvest Year, and Processing on 4MSP Concentrations.

A stable isotope dilution assay was developed for quantitation of 4-methyl-4-sulfanylpentan-2-one (4MSP) in hops. The approach included the use of 4-(13C)methyl-4-sulfanyl(1,3,5-13C3)pentan-2-one as internal standard, selective isolation of hop thiols by mercurated agarose, and GC×GC-TOFMS analysis. Application of the method to 53 different hop samples revealed 4MSP concentrations between <1 and 114 µg/kg. Notably high concentrations were associated with United States varieties such as Citra, Eureka, Simcoe, and Apollo, whereas 4MSP was absent from traditional German and English varieties. Further experiments showed that besides the variety, also harvest year and storage vitally influenced 4MSP concentrations, whereas the impact of provenance was less pronounced. Hop processing such as drying and pelletizing had only a minor impact on 4MSP concentrations. Like the majority of other hop volatiles, 4MSP is predominantly located in the lupulin glands.

Quantitation of selected terpenoids and mercaptans in the dual-purpose hop varieties Amarillo, Citra, Hallertau Blanc, Mosaic, and Sorachi Ace.

Free terpenoids and both free and bound polyfunctional thiols were investigated in five selected dual-purpose hop cultivars. Surprisingly, the dual-purpose Sorachi Ace variety was found to contain higher amounts of farnesene (2101 mg/kg) than aromatic hops such as Saaz but only traces of 3-methylbutylisobutyrate, a compound that usually distinguishes all bitter varieties. All five cultivars investigated here showed an exceptional citrus-like potential explained by either monoterpenic alcohols or polyfunctional thiols. Among the monoterpenic alcohols, ß-citronellol at concentrations above 7 mg/kg distinguished Amarillo, Citra, Hallertau Blanc, Mosaic, and Sorachi Ace from Nelson Sauvin and Tomahawk, two previously investigated dual-purpose hops, while linalool (312 mg/kg) and geraniol (211 mg/kg) remained good discriminating compounds for Nelson Sauvin and Tomahawk, respectively. Regarding polyfunctional thiols, higher amounts of 3-sulfanylhexyl acetate (27 µg/kg) characterized the Citra variety. Free 4-sulfanyl-4-methylpentan-2-one proved discriminant for Sorachi Ace, while the bound form is predominant in Nelson Sauvin. On the other hand, an S-conjugate of 3-sulfanylhexan-1-ol was found in Sorachi Ace at levels not far from those previously reported for Cascade, although the free form was undetected here. Both free and bound grapefruit-like 3-sulfanyl-4-methylpentan-1-ol (never evidenced before the present work) emerged as discriminating compounds for the Hallertau Blanc variety. The apotryptophanase assay also allowed us to evidence for the first time an S-conjugate of 2-sulfanylethan-1-ol.

Influence of two hop (Humulus lupulus L.) varieties on in vitro dry matter and crude protein degradability and digestibility in ruminants.

BACKGROUND: Hop cones contain several antimicrobial substances. The aim of the study was to determine the effects of two hop varieties, Aurora and Dana, on substrate (diet for a dairy cow, producing 30 kg milk daily) in vitro dry matter (DM) and crude protein (CP) degradability and digestibility. RESULTS: In the in vitro trial freshly ground hops were added to the buffered rumen fluid in concentrations simulating the cow's daily intake of 50, 100 and 200 g of hops. Increasing the concentration of hops decreased (P < 0.05) both the average in vitro DM degradabilities of substrate from 725 to 592, 553 and 481 g kg(-1), respectively, and in vitro CP degradabilities of substrate from 752 to 566, 561 and 478 g kg(-1), respectively. The reduction of in vitro DM and CP degradability is counterbalanced by the (invariable) in vitro DM and CP digestibility. The difference between CP digestibility and degradability represents an estimate of the amount of rumen 'bypass' protein which increased with increasing concentration of hops from 172 to 454 g kg(-1). CONCLUSIONS: Decreased DM and CP degradability and increased amount of rumen 'bypass' protein could lower the amounts of protein required by high-producing ruminant animals. However, this supposition needs a validation with in vivo trials.

Effects of hop varieties on ruminal fermentation and bacterial community in an artificial rumen (rusitec).

BACKGROUND: There is a growing interest in the use of hops (Humulus lupulus) as an alternative to antibiotics to manipulate ruminal fermentation. However, the effects of different hop varieties on ruminal fermentation and bacterial populations have not been studied. Here the effects of three hop varieties, Cascade (CAS), Millennium (MIL) and Teamaker (TM), at a level of 800 µg mL(-1) inoculum on ruminal fermentation and microbial populations in an artificial rumen system (rusitec) fed a barley silage-based total mixed ration were investigated. Bacterial populations were assessed using real-time polymerase chain reaction and expressed as a percentage of total bacterial 16S rRNA gene copies. RESULTS: All hops reduced (P < 0.001) total gas, methane and the acetate:propionate ratio. Liquid-associated Fibrobacter succinogenes, Ruminococcus albus and Streptococcus bovis were reduced (P < 0.05) by MIL and TM. Feed particle-associated S. bovis was reduced (P < 0.01) by MIL and TM, but TM and CAS increased (P < 0.01) Ruminobacter amylophilus and Prevotella bryantii respectively. Methanogens were decreased (P < 0.05) by MIL in both liquid and solid fractions and by CAS in the solid fraction. The total amount of α- and ß-acids in hops affected the ruminal fermentation. CONCLUSION: Hop-induced changes in fermentation and microbial populations may improve energy efficiency use in the rumen. Further research is needed to determine the effects of hops on in vivo ruminal fermentation, microbial populations and animal performance.