Formation Of Acetoin Research Articles

The biosynthesis of ephedrine

It is shown by 13C nuclear magnetic resonance spectroscopy that the labelled C2 fragment of [2,3-13C2]pyruvic acid is transferred intact into the C-methyl group and the adjacent carbon atom of the Ephedra alkaloids, norephedrine, ephedrine, norpseudoephedrine, and pseudoephedrine, in growing plants of Ephedragerardiana. This finding serves to identify pyruvate as the elusive precursor of the aliphatic C2 terminus of the skeleton of the alkaloids. In earlier experiments with C-labelled substrates, label from [3-14C]pyruvic acid was incorporated mainly, but not exclusively, into the C-methyl group of ephedrine, and label from [2-14C]pyruvate was incorporated similarly into the carbon atom adjacent to the C-methyl group. A C6–C1 unit related to benzaldehyde or benzoic acid has long been known to generate the benzylic fragment of the carbon skeleton of the Ephedra alkaloids. It is likely that the carbon skeleton of ephedrine is generated from pyruvate and either benzaldehyde or benzoic acid, by a reaction analogous to the formation of acetoin or diacetyl from pyruvate and acetaldehyde or acetic acid, respectively. Keywords: biosynthesis of ephedrine, Ephedra alkaloids, 13C NMR spectra, ephedrine, biosynthesis of pyruvic acid, incorporation into ephedrine13C NMR spectra.

Impact of aeration on the metabolic end‐products formed from glucose and galactose by Streptococcus lactis

Compared with cultures grown aerobically in batch culture with glucose, aerated cultures of lactic streptococci had a less homolactic type of metabolism when galactose was the carbohydrate source in batch cultures, or when glucose was limiting in chemostat cultures. Differences in end‐products of sugar metabolism between aerated and unaerated cultures were observed. In addition to lactate, formate, acetate and ethanol were produced in anaerobic cultures, whereas acetate and acetoin were formed in aerated cultures. Acetate production in aerated cultures depended on lipoic acid, an essential cofactor of the pyruvate dehydrogenase complex. In a chemically defined medium with glucose as the energy substrate, lipoic acid (or acetate) was an essential growth factor. Formation of acetoin was inversely related to lipoic acid concentration in the growth medium. Although not observed in unaerated cultures, acetoin (and 2,3‐butanediol) was produced in unaerated buffered suspensions metabolizing pyruvate. Aeration caused a modest increase in the activities of aP‐acetolactate synthetase and phosphate acetyl trans‐ferase, but it is unlikely that the increases were sufficient to account for the changes in end‐products of sugar metabolism observed.

Effects of aeration on 2,3-butanediol production from glucose by Klebsiella oxytoca

Higher cell concentrations and greater 2,3-butanediol production were observed in aerobic cultures of Klebsiella oxytoca than with anaerobic cultures. The concentration of butanediol inhibitors such as ethanol and lactic acid are partially suppressed by adequate aeration-agitation. Excessive aeration-agitation leads to the formation of acetoin and acetic acid at the expense of butanediol. With 94.3 g/ l of glucose in the media, aerobic batch cultures produced 38.1 g/ l butanediol with complete substrate use and a productivity of 0.39 g/ l/h.

Enterotoxigenicity of Staphylococcus intermedius of canine origin

Seventy-three staphylococcal strains isolated from pyrodermatitis in dogs were classified as Staphylococcus intermedius (52 strains) or Staphylococcus aureus (21 strains) on the basis of acetoin formation, anaerobic mannitol fermentation, aerobic maltose fermentation, pigmentation, coagulation of human plasma, and reaction on crystal violet agar. Enterotoxin was produced by 13 of the 52 S. intermedius strains and 6 of the S. aureus strains. The highest percentage of enterotoxigenic strains produced enterotoxins C (6 strains), D(7 strains), and E (6 strains). Four strains produced the toxic shock syndrome toxin-1. There was little difference in the antibiotic susceptibility between the enterotoxigenic and non-enterotoxigenic strains.

無蒸煮穀類を用いた食酢の製造

Alcohol fermentation of uncooked corn and rice grain at a mash temperature between 20°C and 25°C resulted in less dissolution of nitrogeneous substances and a lower production of lactic acid. But the alcohol yield was not affected by this control. Proteinase is usually contained in commercial glucoamylase preparations. In alcohol fermentation of uncooked rice grain, proteinase-free glucoamylase had a depressive effect on the production of lactic acid and dissolution of nitrogeneous substances, and the rate of alcohol fermentation was significantly reduced, but this enzyme showed no effect on alcohol fermentation of uncooked corn grain. The lactic acid produced and nitrogeneous substances dissolved in the alcohol fermented beer of corn or rice seemed to lead to formation of acetoin and several volatile acids in the acetic acid fermentation, because the amounts of these substances were roughly in parallel with those of the former substances.

Formation and utilization of acetoin, an unusual product of pyruvate metabolism by Ehrlich and AS30-D tumor mitochondria.

[14C]Pyruvate was rapidly non-oxidatively decarboxylated by Ehrlich tumor mitochondria at a rate of 40 nmol/min/mg of protein in the presence or absence of ADP. A search for decarboxylation products led to significant amounts of acetoin formed when Ehrlich tumor mitochondria were incubated with 1 mM [14C] pyruvate in the presence of ATP. Added acetoin to aerobic tumor mitochondria was rapidly utilized in the presence of ATP at a rate of 65 nmol/min/mg of protein. Citrate has been found as a product of acetoin utilization and was exported from the tumor mitochondria. Acetoin has been found in the ascitic liquid of Ehrlich and AS30-D tumor-bearing animals. These unusual reactions were not observed in control rat liver mitochondria.

Formation of volatile aroma compounds and carbon dioxide in yogurt starter grown in cows' and goats' milk

SummaryYogurt starter B3 was grown in cows' milk, goats' milk I (obtained 2–3 weeks after parturition) and goats' milk II (8 months after parturition), and a number of biochemical and bacteriological parameters were recorded at intervals during incubation for 8 h at 43°C. No appreciable differences between the milks were observed for total viable count, bacteriological ratio of rods to cocci, or decrease in pH. Production of acetaldehyde, however, was considerably lower in goats' milk I (6·2 ppm) and goats' milk II (8·9 ppm) than in cows' milk (18 ppm). Formation of diacetyl and acetoin showed similar patterns in all three milks. The highest production of α-acetolactic acid was observed in goats' milk II, apparently coincident with the highest production of CO2 in this milk.

Butanediol production from lignocellulosic substrates by Klebsiella pneumoniae grown in sequential co-culture with Clostridium thermocellum

A sequential co-culture approach was investigated for the conversion of lignocellulosic substrates to butanediol and ethanol. Growth of Clostridium thermocellum on solka floc and aspenwood xylan resulted in the release of extracellular endoglucanase and xylanase enzymes into the culture medium. Low levels of fermentation products were formed and unutilized sugars accumulated in the medium. Inoculation of Klebsiella pneumoniae as a sequential culture resulted in the rapid utilization of the accumulated sugars and the formation of additional fermentation products, including butanediol, ethanol, and acetoin. This approach was applicable to the use of mixed cellulose and hemicellulose substrates, including steam-exploded aspenwood. Further improvement in solvent production from steam-exploded substrates could be obtained by using a fed-batch approach to circumvent the problem of inhibitors associated with the natural substrates.

Transformation of Citric Acid to Acetic Acid, Acetoin and Diacetyl by Wine Making Lactic Acid Bacteria

A decrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC 27307. a malo-lactic bacterium, grown on the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malolactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid.Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cell-free extract were optimal at pH 6.0. Divalent cations such as Mn2+, Mg2+, Co2+ and Zn2+ activated the citrate lyase. The citrate lyase was completely inhibited by EDTA, Hg2...

Transformation of citric acid to acetic acid, acetoin and diacetyl by wine making lactic acid bacteria.

A decrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC 27307, a malo-lactic bacterium, grown on the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malolactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid. Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cellfree extract were optimal at pH 6.0. Divalent cations such as Mn2+, Mg2+, Co2+ and Zn2+ activated the citrate lyase. The citrate lyase was completely inhibited by EDTA, Hg2+ and Ag2+. The acetoin formation from pyruvic acid was significantly stimulated by thiamine pyrophosphate and CoCl2, and inhibited by oxaloacetic acid. Specific activities of the citrate lyase and acetoin formation were considerably variable among the six strains of malo-lactic bacteria examined. Some activities of irreversible reduction of diacetyl to acetoin were found in the cell-free extracts of four of the malolactic bacteria strains and the optimal pH was 6.0 for this activity of Leu. mesenteroides.

Carbohydrate metabolism in lactic acid bacteria.

The term "lactic acid bacteria" is discussed. An overview of the following topics is given: main pathways of homo- and heterofermentation of hexoses, i.e. glycolysis, bifidus pathway, 6-phosphogluconate pathway; uptake and dissimilation of lactose (tagatose pathway); fermentation of pentoses and pentitols; alternative fates of pyruvate, i.e. splitting to formate and acetate, CO2 and acetate or formation of acetoin and diacetyl; lactate oxidation; biochemical basis for the formation of different stereoisomers of lactate.

Aerobic metabolism of Brochothrix thermosphacta growing on meat surfaces and in laboratory media.

Acetoin and acetic, isobutyric and isovaleric acids are major end‐products, and important components of the spoilage odours, of Brochothrix thermosphacta growing aerobically on meat surfaces or in tryptone‐based medium containing glucose, ribose or glycerol. Acetoin and acetic acid are probably derived entirely from the carbohydrates and isobutyric and isovaleric acids from valine and leucine respectively. Glucose and pH are both important factors in controlling the relative amounts of end‐products, low glucose and near neutral pH favouring fatty acid formation, high glucose and lower pH values favouring acetoin formation.

Studies on the formation of acetoin from acetaldehyde by the pyruvate dehydrogenase complex and its regulation.

1. Pyruvate dehydrogenase complex was isolated from pigeon breast muscle involving steps of isoelectric precipitation, poly(ethyleneglycol) fractionation and separation on a glycerine gradient in the ultracentrifuge. 2. Arsenite, a potent inhibitor of the dihydrolipoyl transcetylase, did not affect the formation of acetoin from acetaldehyde, indicating that the pyruvate dehydrogenase component was operative in this reaction. 3. Production of acetoin by the pyruvate dehydrogenase complex is subject to regulation by phosphorylation and dephosphorylation, the dephosphorylated form only being active. 4. The inhibition by acetaldehyde of the pyruvate dehydrogenase complex could be partly explained by the formation of acetoin as an alternative reaction.

Decreased riboflavin formation in mutants of Aerobacter (Enterobacter) aerogenes deficient in the butanediol pathway

Riboflavin concentration increased linearly for more than 60 h in wild type cultures, whereas in three mutants deficient in the formation of acetoin and 2,3-butanediol the production ceased at the end of exponential growth.

Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes

Aerobacter (Enterobacter) aerogenes wild type and three mutants deficient in the formation of acetoin and 2,3-butanediol were grown in a glucose minimal medium. Culture densities, pH, and diacetyl, acetoin, and 2,3-butanediol levels were recorded. The pH in wild-type cultures dropped from 7.0 to 5.8, remained constant while acetoin and 2,3-butanediol were formed, and increased to pH 6.5 after exhaustion of the carbon source. More 2,3-butanediol than acetoin was formed initially, but after glucose exhaustion reoxidation to acetoin occurred. The three mutants differed from the wild type in yielding acid cultures (pH below 4.5). The wild type and one of the mutants were grown exponentially under aerobic and anaerobic conditions with the pH fixed at 7.0, 5.8, and 5.0, respectively. Growth rates decreased with decreasing pH values. Aerobically, this effect was weak, and the two strains were affected to the same degree. Under anaerobic conditions, the growth rates were markedly inhibited at a low pH, and the mutant was slightly more affected than the wild type. Levels of alcohol dehydrogenase were low under all conditions, indicating that the enzyme plays no role during exponential growth. The levels of diacetyl (acetoin) reductase, lactate dehydrogenase, and phosphotransacetylase were independent of the pH during aerobic growth of the two strains. Under anaerobic conditions, the formation of diacetyl (acetoin) reductase was pH dependent, with much higher levels of the enzyme at pH 5.0 than at pH 7.0. Lactate dehydrogenase and phosphotransacetylase revealed the same pattern of pH-dependent formation in the mutant, but not in the wild type.

Regulation of heart muscle pyruvate dehydrogenase kinase.

1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [(32)P]phosphate from [gamma-(32)P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100mum) and cyclic 3':5'-nucleotides (at 10mum) had no significant effect on kinase activity. 3. The K(m) for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76mum. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The K(m) for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9-25.4mum. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The K(m) for pyruvate in the pyruvate dehydrogenase reaction was 35.5mum. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25-500mum. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate (endogenous or added at 2 or 10mum) the kinase activity was enhanced by low concentrations of pyruvate (25-100mum) and inhibited by a high concentration (500mum). Activation of the kinase reaction was not seen when sodium pyrophosphate was substituted for thiamin pyrophosphate. 7. Under the conditions of the kinase assay, pig heart pyruvate dehydrogenase forms (14)CO(2) from [1-(14)C]pyruvate in the presence of thiamin pyrophosphate. Previous work suggests that the products may include acetoin. Acetoin activated the kinase reaction in the presence of thiamin pyrophosphate but not with sodium pyrophosphate. It is suggested that acetoin formation may contribute to activation of the kinase reaction by low pyruvate concentrations in the presence of thiamin pyrophosphate. 8. Pyruvate effected the conversion of pyruvate dehydrogenase phosphate into pyruvate dehydrogenase in rat heart mitochondria incubated with 5mm-2-oxoglutarate and 0.5mm-l-malate as respiratory substrates. It is suggested that this effect of pyruvate is due to inhibition of the pyruvate dehydrogenase kinase reaction in the mitochondrion. 9. Pyruvate dehydrogenase kinase activity was inhibited by high concentrations of Mg(2+) (15mm) and by Ca(2+) (10nm-10mum) at low Mg(2+) (0.15mm) but not at high Mg(2+) (15mm).

Pyruvic decarboxylase and acetoin formation in Athiorhodaceae.

Studies with cell-free extracts of Rhodopseudomonas palustris strain Q showed that under anaerobic conditions pyruvate was decarboxylated with the stoichiometric formation of carbon dioxide. Other products of the reaction include acetaldehyde and acetoin (acetyl methyl carbinol), indicating the presence of pyruvic decarboxylase. A survey of purple non-sulfur bacteria demonstrated that pyruvic decarboxylase was also formed by R. gelatinosa and R. capsulata and Rhodospirillum molischianum but not by R. rubrum and R. spheroides and Rhodomicrobium vannielii. The enzyme had an optimum pH of 6.25 and an apparent Km of 1.22 × 10−2 M in potassium phosphate buffer (50 mM) at 30C. Pyruvic decarboxylase in photosynthetic bacteria required only thiamine pyrophosphate as a cofactor and it differed in this respect from the enzyme in yeast and in other bacteria which required divalent metal ions as well. Acetoin formation in the Athiorhodaceae, like that in yeast but unlike that in most bacterial species, involved the formation of acetaldehyde from pyruvate.

Effect of acetate upon the formation of acetoin in Klebsiella and Enterobacter and it possible practical application in a rapid voges-proskauer test.

Acetate stimulates the formation of acetoin during 1-h incubation of Voges-Proskauer-positive strains of Klebsiella and Enterobacter. Of these organisms, 124 of 126 strains were recognized as positive in the presence of acetate, and 106 were recognized as positive in its absence.

Effect of Acetate upon the Formation of Acetoin in Klebsiella and Enterobacter and its Possible Practical Application in a Rapid Voges-Proskauer Test

Acetate stimulates the formation of acetoin during 1-h incubation of Voges-Proskauer-positive strains of Klebsiella and Enterobacter. Of these organisms, 124 of 126 strains were recognized as positive in the presence of acetate, and 106 were recognized as positive in its absence.

Further Studies with Submerged Flor Sherry

Very little acetal is formed in 18% flor sherry, and almost none in 14% sherry. Sunlight does not increase acetal concentration appreciably. The flor sherry which had been fortified and barrelaged was superior in quality to that which hadn9t. Rates of acetaldehyde formation are superior from base wines of 3.20-3.50 pH. Oxygen pressure above atmospheric is required for adequate rates of acetaldehyde formation and yeast cell growths in flor fermentations. Acetoin formation parallels the acetaldehyde formation. No changes occur in 2,3-butanediols during flor fermentations.