Ghent University Academic Bibliography

Advanced

Unraveling the mechanism of the lightstruck flavor of beer

Arne Heyerick UGent (2001)
abstract
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.
Please use this url to cite or link to this publication:
author
promoter
UGent
organization
year
type
dissertation (monograph)
subject
keyword
skunky thiol (3-methylbut-2-ene-1-thiol), lightstruck flavor, beer, isohumulones, hop (Humulus lupulus L.)
pages
144 pages
place of publication
Gent
defense location
Ghent
defense date
2001-07-03 16:00
language
English
UGent publication?
yes
classification
D1
copyright statement
I have retained and own the full copyright for this publication
id
530875
handle
http://hdl.handle.net/1854/LU-530875
alternative location
http://lib.ugent.be/fulltxt/RUG01/001/376/233/RUG01-001376233_2010_0001_AC.pdf
date created
2009-03-25 17:01:06
date last changed
2009-03-31 08:31:05
@phdthesis{530875,
  abstract     = {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.},
  author       = {Heyerick, Arne},
  keyword      = {skunky thiol (3-methylbut-2-ene-1-thiol),lightstruck flavor,beer,isohumulones,hop (Humulus lupulus L.)},
  language     = {eng},
  pages        = {144},
  school       = {Ghent University},
  title        = {Unraveling the mechanism of the lightstruck flavor of beer},
  url          = {http://lib.ugent.be/fulltxt/RUG01/001/376/233/RUG01-001376233\_2010\_0001\_AC.pdf},
  year         = {2001},
}

Chicago
Heyerick, Arne. 2001. “Unraveling the Mechanism of the Lightstruck Flavor of Beer”. Gent.
APA
Heyerick, Arne. (2001). Unraveling the mechanism of the lightstruck flavor of beer. Gent.
Vancouver
1.
Heyerick A. Unraveling the mechanism of the lightstruck flavor of beer. [Gent]; 2001.
MLA
Heyerick, Arne. “Unraveling the Mechanism of the Lightstruck Flavor of Beer.” 2001 : n. pag. Print.