Pavel Dostálek

Transfer of Fusarium mycotoxins and ‘masked’ deoxynivalenol (deoxynivalenol-3-glucoside) from field barley through malt to beer

The fate of five Fusarium toxins — deoxynivalenol (DON), sum of 15- and 3-acetyl-deoxynivalenol (ADONs), HT-2 toxin (HT-2) representing the main trichothecenes and zearalenone (ZON) during the malting and brewing processes — was investigated. In addition to these ‘free’ mycotoxins, the occurrence of deoxynivalenol-3-glucoside (DON-3-Glc) was monitored for the first time in a beer production chain (currently, only DON and ZON are regulated). Two batches of barley, naturally infected and artificially inoculated with Fusarium spp. during the time of flowering, were used as a raw material for processing experiments. A highly sensitive procedure employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was validated for the analysis of ‘free’ Fusarium mycotoxins and DON-conjugate in all types of matrices. The method was also able to detect nivalenol (NIV), fusarenon-X (FUS-X) and T-2 toxin (T-2); nevertheless, none of these toxins was found in any of the samples. While steeping of barley grains (the first step in the malting process) apparently reduced Fusarium mycotoxin levels to below their quantification limits (5–10 µg kg−1), their successive accumulation occurred during germination. In malt, the content of monitored mycotoxins was higher compared with the original barley. The most significant increase was found for DON-3-Glc. During the brewing process, significant further increases in levels occurred. Concentrations of this ‘masked’ DON in final beers exceeded ‘free’ DON, while in malt grists this trichothecene was the most abundant, with the DON/DON-3-Glc ratio being approximately 5:1 in both sample series. When calculating mass balance, no significant changes were observed during brewing for ADONs. The content of DON and ZON slightly decreased by a maximum of 30%. Only traces of HT-2 were detected in some processing intermediates (wort after trub removal and green beer).

Continuous Beer Fermentation Using Immobilized Yeast Cell Bioreactor Systems

Traditional beer fermentation and maturation processes use open fermentation and lager tanks. Although these vessels had previously been considered indispensable, during the past decades they were in many breweries replaced by large production units (cylindroconical tanks). These have proved to be successful, both providing operating advantages and ensuring the quality of the final beer. Another promising contemporary technology, namely, continuous beer fermentation using immobilized brewing yeast, by contrast, has found only a limited number of industrial applications. Continuous fermentation systems based on immobilized cell technology, albeit initially successful, were condemned to failure for several reasons. These include engineering problems (excess biomass and problems with CO2 removal, optimization of operating conditions, clogging and channeling of the reactor), unbalanced beer flavor (altered cell physiology, cell aging), and unrealized cost advantages (carrier price, complex and unstable operation). However, recent development in reactor design and understanding of immobilized cell physiology, together with application of novel carrier materials, could provide a new stimulus to both research and application of this promising technology.

Immunochemical determination of gluten in malts and beers

The gluten content in different varieties of barley and malts, and in different types of beers, was determined by a ‘sandwich’ enzyme immunoassay (RIDASCREEN® Gliadin kit). The gluten levels in barley wheat, rye and spelt malts ranged 18.8–45.0, 44.0–68.0, 41.6 and 21.2 g kg–1, respectively. When various types of beer were compared, the gluten concentration increased as follows: alcohol-free beer (<3.0), lager beers (<3.0–8.7 mg l–1), stouts (9.0–15.2 mg l–1) and wheat beers (10.6–41.2 mg l–1). When 10 Czech lager beers were analysed, using both sandwich and competitive ELISA, the results showed that the latter method provided values several times higher than the former. Gluten balance was carried out during the brewing process, starting from the raw materials and terminating at the final beer. Gluten levels decreased due to precipitation during the mashing process, primary and secondary fermentation and, lastly, as a result of adsorption during beer stabilization. The gluten content in beer is, thus, approximately three orders of magnitude lower than in the raw malt.

Continuous immobilized yeast reactor system for complete beer fermentation using spent grains and corncobs as carrier materials

Despite extensive research carried out in the last few decades, continuous beer fermentation has not yet managed to outperform the traditional batch technology. An industrial breakthrough in favour of continuous brewing using immobilized yeast could be expected only on achievement of the following process characteristics: simple design, low investment costs, flexible operation, effective process control and good product quality. The application of cheap carrier materials of by-product origin could significantly lower the investment costs of continuous fermentation systems. This work deals with a complete continuous beer fermentation system consisting of a main fermentation reactor (gas-lift) and a maturation reactor (packed-bed) containing yeast immobilized on spent grains and corncobs, respectively. The suitability of cheap carrier materials for long-term continuous brewing was proved. It was found that by fine tuning of process parameters (residence time, aeration) it was possible to adjust the flavour profile of the final product. Consumers considered the continuously fermented beer to be of a regular quality. Analytical and sensorial profiles of both continuously and batch fermented beers were compared.

Analysis of aldehydes in beer by gas-diffusion microextraction: Characterization by high-performance liquid chromatography–diode-array detection–atmospheric pressure chemical ionization–mass spectrometry

In this work, a recently developed extraction technique for sample preparation aiming the analysis of volatile and semi-volatile compounds named gas-diffusion microextraction (GDME) is applied in the chromatographic analysis of aldehydes in beer. Aldehydes-namely acetaldehyde (AA), methylpropanal (MA) and furfural (FA)-were simultaneously extracted and derivatized with 2,4-dinitrophenylhydrazine (DNPH), then the derivatives were separated and analyzed by high-performance liquid chromatography with spectrophotometric detection (HPLC-UV). The identity of the eluted compounds was confirmed by high-performance liquid chromatography-atmospheric pressure chemical ionization-mass-spectrometry detection in the negative ion mode (HPLC-APCI-MS). The developed methodology showed good repeatability (ca. 5%) and linearity as well as good limits of detection (AA-12.3, FA-1.5 and MA 5.4microgL(-1)) and quantification (AA-41, FA-4.9 and MA 18microgL(-1)); it also appears to be competitive in terms of speed and cost of analysis.

Biotransformations and biological activities of hop flavonoids

Female hop cones are used extensively in the brewing industry, but there is now increasing interest in possible uses of hops for non-brewing purposes, especially in the pharmaceutical industry. Among pharmaceutically important compounds from hops are flavonoids, having proven anticarcinogenic, antioxidant, antimicrobial, anti-inflammatory and estrogenic effects. In this review we aim to present current knowledge on the biotransformation of flavonoids from hop cones with respect to products, catalysis and conversion. A list of microbial enzymatic reactions associated with gastrointestinal microbiota is presented. A comparative analysis of the biological activities of hop flavonoids and their biotransformation products is described, indicating where further research has potential for applications in the pharmaceutical industry.

Growth Model and Metabolic Activity of Brewing Yeast Biofilm on the Surface of Spent Grains: A Biocatalyst for Continuous Beer Fermentation

In the continuous systems, such as continuous beer fermentation, immobilized cells are kept inside the bioreactor for long periods of time. Thus an important factor in the design and performance of the immobilized yeast reactor is immobilized cell viability and physiology. Both the decreasing specific glucose consumption rate ( qim) and intracellular redox potential of the cells immobilized to spent grains during continuous cultivation in bubble‐column reactor implied alterations in cell physiology. It was hypothesized that the changes of the physiological state of the immobilized brewing yeast were due to the aging process to which the immobilized yeast are exposed in the continuous reactor. The amount of an actively growing fraction (Xactim) of the total immobilized biomass ( Xim) was subsequently estimated at approximately Xactim = 0.12 gIB gC−1 (IB = dry immobilized biomass, C = dry carrier). A mathematical model of the immobilized yeast biofilm growth on the surface of spent grain particles based on cell deposition (cell‐to‐carrier adhesion and cell‐to‐cell attachment), immobilized cell growth, and immobilized biomass detachment (cell outgrowth, biofilm abrasion) was formulated. The concept of the active fraction of immobilized biomass (Xactim) and the maximum attainable biomass load (Xmaxim) was included into the model. Since the average biofilm thickness was estimated at ca. 10 μm, the limitation of the diffusion of substrates inside the yeast biofilm could be neglected. The model successfully predicted the dynamics of the immobilized cell growth, maximum biomass load, free cell growth, and glucose consumption under constant hydrodynamic conditions in a bubble‐column reactor. Good agreement between model simulations and experimental data was achieved.

Metal removal by immobilised and non-immobilised Azolla filiculoides

Milled-sieved and epichlorhydrin-immobilised Azolla biosorbed ca. 363 and 320 μmol Cu2+ g−1 from a 100 mg l−1 solution. Efficiency of Cu2+ removal by columns was in the order epichlorohydrin-immobilised Azolla>milled-sieved Azolla>untreated Azolla. The 2.5 g epichlorohydrin-immobilised Azolla column demonstrated complete metal sequestration from ca. 12 l of influent 5 mg Cu2+ l−1 and was still at less than 75% saturation even after ca. 22 l had passed through the column. EDTA effectively desorbed Cu2+ with a ca. 55-fold decrease in volume.

Development of magnetic biosorbents for metal uptake

Two types of magnetic biosorbent were prepared by novel protocols from epichlorhydrin-cross-linked Saccharomyces cerevisiae cell walls and their biosorption characteristics were compared to those of non-magnetic cell walls. The magnetic biosorbents I and II were capable of binding Cu maximally to 225 and 50 mmol/g, Cd to 90 and 25 mmol/g and Ag + to 80 and 45 mmol/g respectively. These values compare with 400, 125 and 75 mmol/g, respec-tively, for non-magnetic cell walls.

The Application of a Sol-Gel Technique to Preparation of a Heavy Metal Biosorbent from Yeast Cells

In this work we compared biosorbents obtained by encapsulation of polysaccharides, isolated from waste brewing biomass, in sol-gel derived silicates and an organic polymer. Biosorbents were prepared by mixing cross-linking-agents—organic or siliceous—with dried cells envelopes. Siliceous prepolymers were synthezised via transesterification and hydrolysis from tetraethoxysilane (TEOS) and methanol. Sorption of Cd2+, Cu2+ and Ag+ by biosorbent granules (0.25–0.6 mm) was examined in batch and in a packed column. The biosorbent prepared by interesterification of TEOS showed a 2–3 times higher intensity of sorption than the biosorbent cross-linked with epichlorohydrin (the most effective cross-linking organic agent) while the sorption capacity of both biosorbents was equal. The specific surface area of the silica matrix was 597 m2/g but only traces of metals were sequestered from solution with a concentration of Cd2+ of 50 mg/l. The biosorbent with a silica matrix is a heterogeneous material containing microporous matrix inclusions of thin cell walls. Its high sorption intensity and good mechanical strength will be useful in continuous metal uptake of low concentrations of metals.