Unraveling the mechanism of the lightstruck flavor of beer

An objectionable off-flavor is produced on exposure of beer to light. Current understanding regarding formation of the lightstruck flavor (LSF) in beer dates from the original reports by Kuroiwa et al. in the early sixties. Using model systems, they showed that LSF was produced in a light-induced reaction involving riboflavin as a sensitizer (riboflavin is the main flavin in beer), isohumulones (hop-derived, beer-bittering compounds), and a sulfur source (e.g. cysteine or sulfur-rich proteins). The off-flavor was attributed to formation of 3-methylbut-2-ene-1-thiol (MBT). This mechanism has been frequently referred to in articles dealing with light-induced off-flavors in beer and all relevant data collected to date do not contradict the Kuroiwa-premise. Although the key components are known for more than three decades, details on the mechanism of interaction between these components are lacking. This Ph.D. project aimed at obtaining a thorough and detailed understanding of the mechanism of formation of LSF in beer. The issue was approached by an in-depth investigation of the interacting components on a molecular level using model systems, consisting of isohumulones, dihydroisohumulones, or tetrahydroisohumulones in the presence or absence of riboflavin or flavin mononucleotide as a sensitizer. Unraveling a photochemical mechanism implies studies in various time domains. Thus, model systems were subjected to both photostationary irradiation and photodynamic flash-initiated experiments. Information on the nature of intervening photo-excited states and intermediates, along with mechanistic features, were retrieved from time-resolved electron paramagnetic resonance (TREPR) and flash photolysis transient absorption spectroscopy (TAS). On characterization and identification of prevalent photoreaction products by comprehensive product analysis, relevant reaction schemes were elaborated to account for formation of key compounds. In a first section, a detailed study of the photolability of isohumulones, dihydroisohumulones, and tetrahydroisohumulones, is described. On laser-flash photolysis at 308 nm, isohumulones and tetrahydroisohumulones yielded strong emissive TREPR signals (originating from the triplet mechanism of chemically induced dynamic electron polarization), while, under identical conditions, no TREPR signal was observed for dihydroisohumulones. This finding provides the first direct spectroscopic evidence for the resistance of dihydroisohumulones to photolysis and confirms conclusively that radicals produced on photolysis of isohumulones and tetrahydroisohumulones originate from the alpha-hydroxyketone chromophore as present in isohumulones and tetrahydroisohumulones. Using well-substantiated simulations, the most prevalent radicals on photolysis of isohumulones were characterized as a five-membered ring ketyl radical in conjunction with a 3-methylbut-2-enyl radical, while photolysis of tetrahydroisohumulones led to production of a similar five-membered ring ketyl radical and a 4-methylpentanoyl radical. Combined TREPR data allowed to propose a mechanism for photodegradation of isohumulones and tetrahydroisohumulones on direct irradiation. Absorption of UV light by the beta-tricarbonyl chromophore leads to the excited singlet state. After intersystem crossing to the excited triplet state, intramolecular triplet energy transfer occurs to the isolated alpha-hydroxyketone, which in the excited state ultimately undergoes Norrish Type I alpha-cleavage, thereby furnishing the radicals as detailed above. Comprehensive product analyses of complex mixtures of photoreaction products derived from isohumulones and tetrahydroisohumulones showed that recombination of the most prevalent radicals as characterized by TREPR accounts for formation of the majority of photoreaction products, while minor photoreaction products were found to originate from other pathways. The main reaction products on direct irradiation of isohumulones were characterized as decarbonylated dehydrated isohumulones, while the predominant reaction products on direct irradiation of tetrahydroisohumulones were identified as dimeric-type compounds formed on recombination of two five-membered ring ketyl radicals. In a second section, the interaction between isohumulones, dihydroisohumulones, and tetrahydroisohumulones with riboflavin or flavin mononucleotide on sensitized irradiation was investigated in detail. Laser-flash photolysis at 355 nm of mixtures containing either isohumulones, dihydroisohumulones or tetrahydroisohumulones, and riboflavin resulted in transient absorption spectra (350-800 nm) dominated by the absorption of a neutral flavin semiquinone radical, while the absorption of triplet-excited riboflavin decreased very rapidly. These observations are consistent with a one-electron transfer from the ground state of either isohumulones, dihydroisohumulones or tetrahydroisohumulones to the excited triplet state of riboflavin. The feasibility of one-electron transfer derives from consideration of the redox potentials of the interacting species. The redox potential of riboflavin shifts from -0.3 V vs. NHE in the ground state to +1.7 V vs. NHE in the excited triplet state. Furthermore, TREPR signals on laser-flash photolysis of mixtures containing isohumulones or tetrahydroisohumulones, in addition to flavin mononucleotide, showed a superimposed signal with respect to the TREPR signal derived from laser-flash photolysis of a mixture containing dihydroisohumulones and flavin mononucleotide, or of a model system consisting of EDTA and flavin mononucleotide. The superimposed signal is derived from further degradation of the cation radicals resulting from one-electron transfer of isohumulones or tetrahydroisohumulones to triplet-excited flavin mononucleotide. Although dihydroisohumulones underwent one-electron transfer in the presence of a triplet-excited flavin, no photolysis of dihydroisohumulones was apparent, even at prolonged exposure times, suggesting occurrence of back-electron transfer. Important photoreaction products found on sensitized irradiation of isohumulones include riboflavin adducts containing a 3-methylbut-2-enyl side chain, oxidized five-membered ring fragments, and decarbonylated isohumulones. The distribution of photoreaction products for tetrahydroisohumulones is very similar, except for riboflavin adducts which were found to include addition of both 3-methylbutyl and 4-methylpentanoyl side chains. The identified photoreaction products are consistent with a reaction mechanism involving one-electron transfer from isohumulones or tetrahydroisohumulones in the ground state to triplet-excited riboflavin. Further degradation of the resulting cation radical leads to a 3-methylbut-2-enyl radical in conjunction with a five-membered ring fragment for isohumulones, or to a 3-methylbutyl or a 4-methylpentanoyl radical and a five-membered ring fragment for tetrahydroisohumulones

Activation of proestrogens from hops (Humulus lupulus L.) by intestinal microbiota; Conversion of isoxanthohumol into 8-prenylnaringenin

Hop, an essential ingredient in most beers, contains a number of prenylflavonoids, among which 8-prenylnaringenin (8-PN) would be the most potent phytoestrogen currently known. Although a number of health effects are attributed to these compounds, only a few reports are available about the bioavailability of prenylflavonoids and the transformation potency of the intestinal microbial community. To test these transformations, four fecal samples were incubated with xanthohumol, isoxanthohumol (IX), and 8-PN. Upon incubation with IX, present in strong ales up to 4 mg/L, 36% was converted into 8-PN in one fecal sample and the estrogenic properties of the sample drastically increased. In an experiment with 12 fecal cultures, this conversion was observed in one-third of the samples, indicating the importance of interindividual variability in the intestinal microbial community. Eubacterium limosum was identified to be capable of this conversion (O-demethylation) of IX into 8-PN, and after strain selection, a conversion efficiency of 90% was achieved. Finally, strain supplementation to a nonconverting fecal sample led to rapid and high 8-PN production at only 1% (v/v) addition. Up to now, the concentration of 8-PN in beer was considered too low to affect human health. However, these results show that the activity of the intestinal microbial community could more than 10-fold increase the exposure concentration. Because prenylflavonoids are present in many beers with IX being the major constituent, the results raise the question whether moderate beer consumption might contribute to increased in vivo levels of 8-PN and even influence human health

Cellular uptake of soy-derived phytoestrogens in vitro and in human whole blood

Epidemiological studies comparing typical Western and traditional Eastern lifestyles indicate that dietary intake of soyderived phytoestrogens, including genistein, daidzein, and equol, may have significant health protective effects on hormone-dependent cancers, osteoporosis and cardiovascular diseases. Phytoestrogens have been demonstrated to exert varying effects depending on tissue, endogenous hormone concentrations, and receptor types. Thus, a detailed understanding of the biodistribution and bioavailability of specific phytoestrogens is required in order to predict the subsequent biologic activities. In this study we aimed to investigate the cellular uptake of these soy-derived phytoestrogens in different cell types, including the mammary MCF-7/6 and MDAB-MB 231 cell lines, the ovarian Ishikawa Var-I cell lines and in murine adipocyte clusters. Furthermore, the biodistribution between serum and cell fraction was also investigated in human whole blood. Equol generally shows a higher cellular uptake when compared with genistein and daidzein. Therefore, equol may be more potent with respect to its relative bioactivity, which is corroborated by the observations of specific health effects associated with the equol-producer phenotype

Heterologous screening of hybridomas for the development of broad-specific monoclonal antibodies against deoxynivalenol and its analogues

Hapten heterology was introduced into the steps of hybridoma selection for the development of monoclonal antibodies (MAbs) against deoxynivalenol (DON). Firstly, a novel heterologous DON hapten was synthesised and covalently coupled to proteins (i.e. bovine serum albumin (BSA), ovalbumin and horseradish peroxidase) using the linkage of cyanuric chloride (CC). After immunisation, antisera from different DON immunogens were checked for the presence of useful antibodies. Next, both homologous and heterologous enzyme-linked immunosorbent assays were conducted to screen for hybridomas. It was found that heterologous screening could significantly reduce the proportion of false positives and appeared to be an efficient approach for selecting hybridomas of interest. This strategy resulted in two kinds of broad-selective MAbs against DON and its analogues. They were quite distinct from other reported DON-antibodies in their cross-reactivity profiles. A unique MAb 13H1 derived from DON-CC-BSA immunogen could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyl-DON > DON > nivalenol, with IC50 ranging from 1.14 to 7.69 mu g/ml. Another preferable MAb 10H10 generated from DON-BSA immunogen manifested relatively similar affinity to DON, 3-acetyl-DON and 15-acetyl-DON, with IC50 values of 22, 15 and 34 ng/ml, respectively. This is the first broad-specific MAb against DON and its two acetylated forms and thus it can be used for simultaneous detection of the three mycotoxins

Sesquiterpene lactones from the extracts of two Balkan endemic Laserpitium species and their cytotoxic activity

Chloroform extracts of the underground parts of two Balkan endemic Laserpitium species, Laserpitium zernyi Hayek and Laserpitium ochridanum Micevski, were chemically investigated. Five unknown guaianolides from the class of slovanolides, of which four were additionally 2 beta-esterified, as well as two lactones, previously identified in other Laserpitium species, were isolated from the L. ochridanum extract. From the L. zernyi extract one slovanolide derivative was isolated for the first time in the genus Laserpitium. In addition, the phenylpropanoid latifolone and six known sesquiterpene lactones, characterised as derivatives of slovanolide and silerolide, were isolated from the extracts of both species. The cytotoxic activities of the total extracts and the isolated compounds were tested using MTT and SRB assays on the two human breast cancer cell lines, MCF 7/6 and MCF 7/AZ. The extracts exerted cytotoxic activities with the IC50 values ranging 65.21-348.25 mu g/mL. The L ochridanum extract was most potent in the MTT test with IC50 values of 65.21 and 66.09 mu g/mL in the MCF 7/AZ and MCF 7/6 cell lines, respectively. The highest cytotoxic activity exerted 2 beta,8 alpha-di-angeloyloxy-10 beta-hydroxy-6 alpha H-guaian-3,(7-11)-dien-12,6-olide, a slovanolide derivative with an additional double bond in lactone ring, on highly invasive MCF 7/6 cell line, with IC50 value 0.7 mu M in both assays tested. Generally, guaianolides with a higher number of ester moieties at the positions 2 beta, 8 alpha, 10 beta or 11 alpha exhibited IC50 values in the micromolar range, while eudesmanolides and guaianolides with a lower number of esters did not induce significant cytotoxicity

Microbial and dietary factors associated with the 8-prenylnaringenin producer phenotype: a dietary intervention trial with fifty healthy post-menopausal Caucasian women

Hop-derived food supplements and beers contain the prenylflavonoids xanthohumol (X), isoxanthohumol (IX) and the very potent phyto-oestrogen (plant-derived oestrogen mimic) 8-prenylnaringenin (8-PN). The weakly oestrogenic IX can be bioactivated via O-demethylation to 8-PN. Since IX usually predominates over 8-PN, human subjects may be exposed to increased doses of 8-PN. A dietary intervention trial with fifty healthy postmenopausal Caucasian women was under-taken. After a 4 d washout period, participants delivered faeces, blank urine and breath samples. Next, they started a 5 d treatment with hop-based supplements that were administered three times per d and on the last day, a 24 h urine sample was collected. A semi-quantitative FFQ was used to estimate fat, fibre, alcohol, caffeine and theobromine intakes. The recoveries of IX, 8-PN and X in the urine were low and considerable inter-individual variations were observed. A five-fold increase in the dosage of IX without change in 8-PN concentration resulted in a significant lower IX recovery and a higher 8-PN recovery. Classification of the subjects into poor (60 %), moderate (25 %) and strong (15 %) 8-PN producers based on either urinary excretion or microbial bioactivation capacity gave comparable results. Recent antibiotic therapy seemed to affect the 8-PN production negatively. A positive trend between methane excretion and 8-PN production was observed. Strong 8-PN producers consumed less alcohol and had a higher theobromine intake. From this study we conclude that in vivo O-demethylation of IX increases the oestrogenic potency of hop-derived products

Design of Peptide-Based Nanovaccines Targeting Leading Antigens From Gynecological Cancers to Induce HLA-A2.1 Restricted CD8+ T Cell Responses

Gynecological cancers are a leading cause of mortality in women. CD8+ T cell immunity largely correlates with enhanced survival, whereas inflammation is associated with poor prognosis. Previous studies have shown polystyrene nanoparticles (PSNPs) are biocompatible, do not induce inflammation and when used as vaccine carriers for model peptides induce CD8+ T cell responses. Herein we test the immunogenicity of 24 different peptides, from three leading vaccine target proteins in gynecological cancers: the E7 protein of human papilloma virus (HPV); Wilms Tumor antigen 1 (WT1) and survivin (SV), in PSNP conjugate vaccines. Of relevance to vaccine development was the finding that a minimal CD8+ T cell peptide epitope from HPV was not able to induce HLA-A2.1 specific CD8+ T cell responses in transgenic humanized mice using conventional adjuvants such as CpG, but was nevertheless able to generate strong immunity when delivered as part of a specific longer peptide conjugated to PSNPs vaccines. Conversely, in most cases, when the minimal CD8+ T cell epitopes were able to induce immune responses (with WT1 or SV super agonists) in CpG, they also induced responses when conjugated to PSNPs. In this case, extending the sequence around the CD8+ T cell epitope, using the natural protein context, or engineering linker sequences proposed to enhance antigen processing, had minimal effects in enhancing or changing the cross-reactivity pattern induced by the super agonists. Nanoparticle approaches, such as PSNPs, therefore may offer an alternative vaccination strategy when conventional adjuvants are unable to elicit the desired CD8+ T cell specificity. The findings herein also offer sequence specific insights into peptide vaccine design for nanoparticle-based vaccine carriers