P1: SFK/UKS BLBS102-c33 P2: SFK BLBS102-Simpson March 21, 2012 14:5 Trim: 276mm X 219mm 638 Printer Name: Yet to Come Part 5: Fruits, Vegetables, and Cereals Threonine Threonine α-Acetohydroxybutyrate Carbohydrate metabolism α-Ketobutyrate Pyruvate α-Acetohydroxybutyrate α-Acetolactate ILV5 CO2 2,3-Pentanedione Acetylethylcarbinol Bacterial acetolactate decarboxylase α-Acetolactate CO2 Diacetyl CO2 Acetoin Isoleucine Valine Leucine 2,3-Butanediol 2,3-Pentanediol Figure 33.3 The synthesis and reduction of vicinal diketones in Saccharomyces cerevisiae 1975b), approximately 10 times lower than that of pentanedione (Wainwright 1973) The excreted α-acetohydroxy acids are overflow products of the ILV pathway that are nonenzymatically degraded to the corresponding vicinal diketones (Inoue et al 1968) Tetraploid gene dosage series for various ILV genes have been constructed and the obtained yeast strains were used to study the influence of the copy number of ILV genes on the production of vicinal diketones (Debourg et al 1990, Debourg 2002) It was shown that the ILV5 activity is the rate-limiting step in the ILV pathway and responsible for the overflow (Fig 33.3) The nonenzymatic oxidative decarboxylation step is the rate-limiting step and proceeds faster at a higher temperature and a lower pH (Inoue and Yamamoto 1970, Haukeli and Lie 1978) The produced amount of α-acetolactate is very dependent on the used yeast strain The production increases with increasing yeast growth For a classical fermentation, 0.6 ppm α-acetolactate is formed (Delvaux 1998) At high aeration, this value can be increased to 0.9 ppm and in cylindro-conical fermentations tanks even to 1.2–1.5 ppm It has been shown that valine inhibits the synthesis of α-acetolactate through feedback inhibition (Magee and de Robinson-Szulmajster 1968) This inhibition is directed to the protein Ilv6p, which is the regulatory subunit of acetohydroxy acid synthase (the catalytic subunit encoded by ILV2)(Pang and Duggleby 1999, 2001) Because the uptake of valine is delayed in a normal beer fermentation, the suppressive effect of valine accounts for the postponed onset of total diacetyl (sum of actual diacetyl and α-acetolactate), and this effect persists longer in worts with high levels of FAN content In contrast, low FAN levels give two diacetyl peaks as a result of the requirement for valine biosynthesis Therefore, a minimum FAN level above the critical value of 50 ppm (Nakatani et al 1984) or 140 ppm (Pugh et al 1997) should be maintained during the fermentation to ensure the presence of valine in the fermenting wort Yeast cells posses the necessary enzymes (reductases) to reduce diacetyl to acetoin and further to 2,3-butanediol, and 2,3pentanedione to 2,3-pentanediol (Bamforth and Kanauchi 2004) These reduced compounds have much higher taste thresholds and have no impact on the beer flavor (Van Den Berg et al 1983) The reduction reactions are yeast strain dependent The reduction occurs at the end of the main fermentation and during the maturation Sufficient yeast cells in suspension are necessary to obtain an efficient reduction Yeast strains that flocculate early during the main fermentation needs a long maturation time to reduce the vicinal diketones Diacetyl can be complexed using SO2 These complexes cannot be reduced, but diacetyl can again be liberated at a later stage by aldehydes This situation is especially applicable to yeast strains, which produce a lot of SO2 Worts, which are produced using a high content of adjuncts, can be low in free amino acid content These worts can give rise to a high diacetyl peak at the end of the fermentation There are several strategies, which can be chosen to reduce the vicinal diketones amount during fermentation: Since the temperature has a positive effect on the reduction efficiency of the α-acetohydroxy acids, a warm rest period at the end of the main fermentation and a warm maturation are applied in many breweries In this case, temperature should be well controlled to avoid yeast autolysis Since the rapid removal of vicinal diketones requires yeast cells in an active metabolic condition, the addition of 5–10% Krausen (containing active, growing yeast) is a procedure, which gives enhanced transformation of vicinal diketones (NN 2000) This procedure can lead to P1: SFK/UKS BLBS102-c33 P2: SFK BLBS102-Simpson March 21, 2012 14:5 Trim: 276mm X 219mm Printer Name: Yet to Come 33 Biochemistry of Beer Fermentation overproduction of hydrogen sulfide, depending upon the proportions of threonine and methionine carried forward from primary fermentation Heating up the green beer to a high temperature (90◦ C) and hold it there for a short period (ca 7–10 min) to decarboxylate all excreted α-acetohydroxy acids To avoid cell autolysis, yeast cells are removed by centrifugation prior to heating up The vicinal diketones can be further reduced by immobilized yeast cells in a few hours (typically at 4◦ C) (see further) Adding the enzyme α-acetolactate decarboxylase (Godtfredsen et al 1984, Rostgaard-Jensen et al 1987): This enzyme decarboxylates α-acetolactate directly into acetoin (see Fig 33.3) It is not present in S cerevisiae, but has been isolated from various bacteria such as Enterobacter aerogenes, Aerobacter aerogenes, Streptococcos lactis, Lactobacillus casei, Acetobacter aceti, and Acetobacter pasteurianus It has been shown that the addition of α-acetolactate decarboxylase from L casei can reduce the maturation time to 22 hours (Godtfredsen et al 1983, 1984) An example of a commercial product is Maturex L from Novo Nordisk (Denmark) (Jensen 1993) Maturex L is a purified α-acetolactate decarboxylase produced by a genetically modified strain of Bacillus subtilis, which has received the gene from Bacillus brevis The recommended dosage is a` kg per 1000 hL wort, to be added to the cold wort at the beginning of fermentation Using genetic modified yeast strains: a Introducing the bacterial α-acetolactate decarboxylase gene into yeast chromosomes (Fujii et al 1990, Suihko et al 1990, Blomqvist et al 1991, Enari et al 1992, Linko et al 1993, Yamano et al 1994, Tada et al 1995, Onnela et al 1996) Transformants possessed a very high α-acetolactate decarboxylase activity that reduced the diacetyl concentration considerably during beer fermentations b Modifying the biosynthetic flux through the ILV pathway by partially deactivation of ILV2 Spontaneous mutants resistant to the herbicide sulfometuron methyl have been selected These strains showed a partial inactivation of the α-acetolactate synthase activity and some mutants produced 50% less diacetyl compared to the parental strain (Gjermansen et al 1988) c Increasing the flux of α-acetolactate acid isomerorecuctase activity encoded by the ILV5 gene (Dillemans et al 1987) Since α-acetolactate acid isomerorecuctase activity is responsible for the rate-limiting step, increasing its activity reduces the overflow of αacetolactate A multicopy transformant resulted in a 70% decreased production of vicinal diketones (Villaneuba et al 1990), whereas an integrative transformant gave a 50% reduction (Goossens et al 1993) A tandem integration of multiple ILV5 copies resulted also in elevated transciption in a polyploidy industrial yeast strain (Mithieux and Weiss 1995) Vicinal diketones production could be reduced by targeting the mitochondrial Ilv5p to the cytosol (Omura 2008) 639 SECONDARY FERMENTATION During the secondary fermentation or maturation of beer, several objectives should be realized: r Sedimentation of yeast cells r Improvement of the colloidal stability by sedimentation of the tannin–protein complexes r Beer saturation with carbon dioxide r Removal of unwanted aroma compounds r Excretion of flavor-active compounds from yeast to give body and depth to the beer r Fermentation of the remaining extract r Improvement of the foam stability of the beer r Adjustment of the beer color (if necessary) by adding coloring substances (e.g., caramel) r Adjustment of the bitterness of beer (if necessary) by adding hop products In the presence of yeast, the principal changes that occur are the elimination of undesirable flavor compounds, such as vicinal diketones, hydrogen sulfide, and acetaldehyde, and the excretion of compounds enhancing the flavor fullness (body) of beer Vicinal Diketones In traditional fermentation lagering processes, the elimination of vicinal diketones required several weeks and determined the length of the maturation process Nowadays, the maturation phase is much shorter since strategies are used to accelerate the vicinal diketones removal (see the preceding text) Diacetyl is used as a marker molecule The objective during lagering is to reduce the diacetyl concentration below its taste threshold (