Alexandr Mikyška

Ultra-high-performance liquid chromatography profiling method for chemical screening of proanthocyanidins in Czech hops.

Hops represent an important natural source of bioactive polyphenols, particularly proanthocyanidins, which can contribute to prevention of several civilization diseases, owing to their antioxidant and radical scavenging activity. We have developed a high-throughput ultra-high-performance liquid chromatography time-of-flight mass spectrometry profiling method, which can be used for monitoring of bioactive proanthocyanidins in hops. The method was applied for analysis of hops of four Czech varieties (Saaz, Sladek, Preminat and Agnus) from the 2011 crop (9 localities, 11 samples) and the 2012 crop (24 localities, 40 samples). Hop samples were extracted by acetone and the analytes were separated on the Acquity UPLC BEH Shield RP18 column. Partial validation of the method revealed a satisfactory intra-day repeatability of the method for retention times (relative standard deviation within 1.39%) as well as areas under the peaks (within 9.89%). Experimental data were evaluated using principal component analysis and cluster analysis. Significant amounts of di-, tri- and tetramer proanthocyanidins consisting of (epi)catechin and (epi)gallocatechin were found in the hop samples. The dependence of the proantocyanidin composition on both the variety and the growing locality was observed. Specifically, the traditional Saaz variety contained more frequently oligomers formed by (epi)catechin units only, whereas the varieties Premiant and Agnus produced oligomers consisting of (epi)catechin as well as (epi)gallocatechin units. The relative abundance of proanthocyanidins in studied hop varieties from the two crops, 2011 and 2012, did correspond to each other. In the further perspective, the method may also be used for prediction of qualitative marks or authenticity verification of hops.

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.

Inhibitory Effects of Fresh Hops on Helicobacter pylori Strains

Cermak P., Paleckova V., Houska M., Strohalm J., Novotna P., Mikyska A., Jurkova M., Sikorova M. (2015): Inhibitory effects of fresh hops on Helicobacter pylori strains. Czech J. Food Sci., 33: 202–207. The goal of this work was to provide evidence for the inhibitory effects of fresh hops on the growth of Helicobacter pylori. Fresh hops were homogenised after harvest and the homogenate was treated using pascalisation (treatment with high pressure). This homogenate was used for testing the growth inhibitory effects on strains of H. pylori, a pathogenic microorganism, isolated from patients suffering from gastritis or gastric ulcers. The tests demonstrated reliable inhibitory effects and open the possibility of hops being used as a supplement to the treatment of H. pylori infections.

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

Hops represent an important source of β-acids with antimicrobial and sensory properties. Transformation products of β-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 β-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 β-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.

Saaz Late - česká odrůda chmele doporučená pro České pivo.

Ve šlechtění chmele lze v uplynulém desetiletí zaznamenat řadu nových trendů, z významných je možno jmenovat zakrslé odrůdy a odrůdy se specifickým aroma . Šlechtění zakrslých odrůd vhodných k pěstování v nízkých konstrukcích (Neve, 1991) bylo započato v Anglii již v 80 . letech minulého století . Velkou výhodou tohoto pěstitelského systému je podstatné snížení nákladů na výstavbu chmelnicové konstrukce a na pracovní sílu v jarním období . Původní trpasličí odrůdy se zde dnes pěstují na několika stech hektarech . Odrůdy jako např . First Gold, Herold, Pioneer či Pilgrim slouží nyní jako prvotní genetický materiál pro šlechtění tohoto typu chmelů i jinde ve světě (USA, Česká republika, Německo) . Další, v současné době velmi populární skupinou odrůd chmele jsou tzv . „flavour hops“ . Jedná se o odrůdy s atraktivním, senzoricky specifickým aroma, které se v plné míře uplatňuje i v pivu díky tomu, že se používají k tzv . „chmelení za studena“, přídavku chmele do ležáckých tanků ve studené fázi výroby piva . Takto chmelená piva získávají zcela jedinečnou chmelovou vůni díky tomu, že se senzoricky uplatňují látky, které běžně při chmelovaru bez užitku vytěkají z vroucí mladiny . Chmelení za studena je doménou především malých a restauračních pivovarů, ale Saaz Late – česká odrůda chmele doporučená pro České pivo

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.

Systém skladování ječmene a jeho vliv na kvalitu sladu a piva.

Mikyška, A. – Prokeš, J.: Systém skladování ječmene a jeho vliv na kvalitu sladu a piva. Kvasny Prum. 55, 2009, č. 3, s. 73–81. Vliv skladování ječmene na kvalitu sladu byl zkoumán v laboratorním měřítku. Ječmeny odrůd Jersey a Tolar byly po ukončení dormance skladovány v množství po 50 kg při teplotě 10 °C v aerobních a anaerobních podmínkách. V intervalech dva týdny, dva a šest měsíců po zahájení skladovacího pokusu byly provedeny mikrosladovací zkoušky. Připravené slady byly zpracovány ve varních pokusech. Komplexní vyhodnocení všech stanovených analytických parametrů kvality sladu metodou shlukové analýzy potvrdilo významnou závislost kvality sladu na vlastnostech odrůdy ječmene a ukázalo i významnou závislost kvality sladu na době skladování ječmene ve vazbě na způsob skladování ječmene. Způsob skladování ovlivnil klíčivost a klíčivou energii ječmene, příjem vody při namáčce, friabilitu sladu a čirost sladiny. V aerobních podmínkách skladování roste riziko vyšších hodnot zákalu sladu. Po šesti měsících skladování v těchto podmínkách se rovněž výrazně snížila friabilita sladu. Některé parametry kvality sladu byly ovlivněny dobou skladování ječmene bez statisticky průkazného vztahu k podmínkám skladování. Slady vyrobené z ječmenů skladovaných delší dobu měly vyšší dosažitelné prokvašení, rozpustný dusík, lepší předpověď filtrovatelnosti, vyšší antioxidační aktivitu a mírně lepší senzorickou kvalitu a stabilitu piva.

Hodnocení obsahu α- a β-hořkých kyselin ve sklizni českých chmelů v roce 2017

Mikyška, A., Jurková, M., 2018: Evaluation of αand β-bitter acids content in harvest of Czech hops in 2017. Kvasny Prum. 64(3): 122–130 This article presents the results of the harvest forecast of the αand β-acid content of the Czech hop varieties in the 2017 harvest (194 samples in total). Hops were analyzed using EBC 7.7 (HPLC) method. The results are discussed in relation to the long-term averages, structure of bitter acids analogs and stability of varietal character. The α-acid content of the Saaz variety (2.81% in dry matter) was 20% rel. lower than the 25-year average, the content of α-acids in the varieties with a later maturity date, Sládek, Premiant, Agnus, Kazbek and Saaz Late, was only slightly lower or comparable to the multi-annual averages. The ratio of αand β-acids, the relative content of cohumulone and the relative content of colupulone were consistent with long-term averages for the tested varieties. The harvest forecast gives a timely information on the α-acids content of the harvest, the results were in good agreement with the results of the whole harvested hops.

Study on technological properties of the historical barley variety Proskovcuv hanacky

Olšovská, J., Mikyška, A., Čejka, P., Slabý, M., Psota, V., 2018: Study of technological properties of the historical barley variety Proskovcuv hanacky. Kvasny Prum. 64(4): 167–172 Proskovcuv hanacky belongs to the famous, historically significant Czech malting barley varieties. For its excellent qualities, this variety was widely used in breeding and today it is the basis of the pedigree of many modern European varieties. The aim of this work was to study the technological, analytical and sensory properties of malt and beer made of this variety and to evaluate its potential for current beer production. Pilsner-type malt made in a floor malt house was characterized by high protein content (13.9%) and very low cytolytic, proteolytic and amylolytic modification. Beer was made from the malt from two craft breweries using a two-mash decoction process and Saaz hops. The beers were lowly fermented (apparent attenuation 67 and 69%) and had a high content of dextrins, betaglucans, arabinoxylans and polyphenols. Sensory evaluation of the beers was similar to current conventional lager-type beers. Beer brewed from this variety using the traditional malting and brewing technology could contribute to an interesting diversification of the offer of smaller breweries.

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), β-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.