Yeast & fermentation L3 Elucidation of the formation mechanism of -mercapto-3-methyl--butanol (M3MB) in beer Shigekuni Noba, Nana Yako, aku Irie, Minoru Kobayashi, etsuya Watanabe Asahi Breweries, ltd Research Laboratories for Alcohol Beverages
ASAHI SUPER DRY No. beer in the Japanese market Karakuchi taste: refreshingly crisp, clear taste ontinually working to improve the flavor
Our technologies Yeast L6:Oomuro et al. Ingredients P39:Kishimoto et al. Flavor my presentation P9:Irie et al.
Source: Demand Media
M3MB Odor Onion, Sweat hreshold.3 ppb Origin Hops -mercapto-3-methyl--butanol
M3MB formation during fermentation,5 M3MB (ppb),4,3,, Brewery A Brewery B Brewery ime (hours) Kobayashi, ASB Meeting, 6
M3MB precursor Origin Hops Generation factor Hot aeration of wort,3-epoxy-3-methyl-butanal (EMB) he discovery of the precursor in food was first reported in 6. Noba, WB, 6
Behavior of the precursor during fermentation,,6 Precursor (EMB,ppb) 8, 4,,3 M3MB (ppb) Precursor_Brewery A Precursor_Brewery B M3MB_Brewery A M3MB_Brewery B, 5 5, ime (hours)
Research objective o identify the factors necessary for M3MB formation
Hypothetical pathway of M3MB formation Yeast Precursor(EMB) M3MB he role of yeast Reduction of aldehyde Substitution of an SH group
Research design Yeast Precursor(EMB) M3MB Yeast () Enzymes () Metabolites
Research design Yeast Precursor(EMB) M3MB Yeast () Enzymes () Metabolites
() Yeast enzyme Yeast addition test Yeast extract addition test
Yeast addition test ppm precursor +. g yeast in acetate buffer (ph5).e+ 7 - M3MB standard - est sample Abundance.5e+ 7,, 5,,,, 8. 9. 3. 3. ime (min)
Yeast addition test ppm precursor +. g yeast in acetate buffer (ph5).e+ 7 - M3MB standard - est sample Abundance.5e+ 7,, 5,, M3MB,, 8. 9. 3. 3. ime (min)
Yeast addition test ppm precursor +. g yeast in acetate buffer (ph5).e+ 7 - M3MB standard - est sample Abundance.5e+ 7,, 5,, Unknown M3MB,, 8. 9. 3. 3. ime (min)
Yeast addition test 59. 5,, OH 3,, 43.,, 3. 85.,3-epoxy-3-methyl-butanol 4 6 8 4 6 m/z his compound wasidentified by comparison with a synthesized reagent.
Yeast extract addition test ppb precursor + rude protein extracted using a DEAE column Precursor (aldehyde) Reduced form (alcohol) ppb 4 6 ime (hours)
Hypothetical pathway of M3MB formation Enzyme Precursor(EMB) Reduced form Reduction of the aldehyde (by alcohol dehydrogenase?)
Short summary he terminal aldehyde of the precursor was converted to alcohol by crude yeast enzymes. he substitution of the SH group was not observed in the two tests; suggesting that yeast metabolites are necessary.
Research design Yeast Precursor(EMB) M3MB Yeast () Enzymes () Metabolites
() Yeast metabolites omparative test of culture media Screening of medium components Addition test Omission test Metabolome analysis Fermentation test using mutant strain
M3MB (ppb) omparative test of culture media after fermentation 4 ppb of precursor was added to the media. unhopped wort YPD N.D. YNB M3MB was produced only from wort and YPD medium.
omparative test of culture media YPDmedium yeast extract peptone glucose YNB medium ammonium sulfate salts vitamins minerals glucose amino acids?
Screening of medium components : Addition test Amino acids concentration in wort: J. Inst. Brew. (), 5 6, 5 M3MB(ppb) after fermentation,4,, All amino acids hr YNB medium + hrwas needed for the formation of M3MB
Screening of medium components : Omission test 4, M3MB(ppb) after fermentation,, All AA hr-omission
Metabolome analysis Yeast at 6 hours of fermentation was used. ontrol: YNB + all AA est: hr omission 8 intracellular metabolites were detected by E-tof/MS Only sulfur metabolism of yeast was affected during growth in hr-omission medium
Yeast sulfur metabolism : all amino acids : hr omission Asp Homoserine 8 4 OAH (O-Acetylhomoserine) 5 Sulfate H S in liquid Homocysteine hr Met
Yeast sulfur metabolism : all amino acids : hr omission Asp Homoserine 8 4 OAH (O-Acetylhomoserine) 5 Sulfate H S in liquid Homocysteine hr Met
Yeast sulfur metabolism : all amino acids : hr omission Asp Homoserine 8 4 OAH (O-Acetylhomoserine) 5 Sulfate H S in liquid Homocysteine hr Met
Yeast sulfur metabolism : all amino acids : hr omission M3MB(ppb) after fermentation 4,,, All AA Homoserine 8 4 Asp hr hr-omission OAH (O-Acetylhomoserine) 5 Sulfate H S in liquid Homocysteine Met
H S behavior during fermentation in hr-omission test All amino acids / hr-omission in YNB (5 L) P-I H S (ppb) 8 4 P-II P-III P-IV 5 5 ime (hours) here was a large difference in the initial concentration of H S (P-I) during fermentation.
H S non-producing mutant,6,4,, Wild type Mutant M3MB (ppb) after fermentation N.D.
Short summary M3MB was formed only in the YNB medium supplemented with threonine. Metabolome analysis revealed that only sulfur metabolism, particularly H S concentration, was affected by threonine omission. M3MB was not formed by the H S nonproducing yeast.
onclusions : wo factors Reduction by yeast enzyme Precursor(EMB) M3MB Substitution of an SH group by H S
Future work Detailed investigation of the M3MB formation factors, identified in this study, is being conducted at our brewery. Some of the results are shown on the poster P9by Irieet al.