Specificity of the Westerfeld adaptation of the Voges-Proskauer test.

Aliphatic chain compounds at least four carbons long with vicinal carbonyl groups in the 2,3 positions were detected by the Westerfeld test. Acetoin, which has one carbonyl group and an adjacent hydroxyl group, gave positive results, but methyl action (3-hydroxy-3-methyl-2-butanone) was negative, and subsequent tests supported the conclusion that acetoin is oxidized to diacetyl by alpha-naphthol during the Westerfeld test in the absence or presence of air. 2,3-Pentanedione and 2,3-heptanedione gave positive results, but equimolar concentrations of these compounds gave maximal absorbancy readings that were only 35% (2,3-pentanedione) and 31% (2,3-heptanedione) of those obtained with diacetyl or acetoin. Negative results were obtained with pyruvic acid, 2,3-butylene glycol, and carbon ring compounds (1,2-cyclohexanedione, alloxan, and 3,4-dihydroxy-3-cyclobutene-1,2-dione). alpha-Naphtho could not be replaced in the test by beta-naphthol, 1,2,3,4,-tetrahydroxy-1-naphthol, or 5,6,7,8-tetrahydroxy-1-naphthol. Creatine could not be replaced by arginine, guanidine . HCl, or guanidinoacetic acid.     

Simple and sensitive determination of diacetyl and acetoin in biological samples and alcoholic drinks by gas chromatography with electron-capture detection

Acetoin was quantitatively oxidized into diacetyl by Fe³⁺ in 1 M perchloric acid. The reaction of diacetyl with 4,5-dichloro-1,2-diaminobenzene afforded 6,7-dichloro-2,3-dimethylquinoxaline (DCDMQ), which was extracted by benzene containing aldrin (25 ng/ml) as an internal standard, and determined by gas chromatography with electron-capture detection. The method is very simple and sensitive. The detection limit of DCDMQ (either diacetyl or acetoin) was 10 fmol/μl of the benzene extract, and the determination limit of DCDMQ (either diacetyl or acetoin) was 50 fmol/μl of the extract. Both acetoin and diacetyl could be determined in 0.1 ml of normal human urine or blood, and both were found in rat liver, kidney and brain. The method was also applied to the determination of acetoin and diacetyl in alcoholic drinks.     

Separation of diacetyl, acetoin, and 2,3-butylene glycol by ion-exchange chromatography

Mixtures of diacetyl, acetoin, and 2,3-butylene glycol were quantitatively separated by ion-exchange chromatography on Dowex 1-X8 resin in the bisulfite form. Initial elution with water removed 2,3-butylene glycol from the column. Further elution with 0.1 m NaCl separated acetoin from diacetyl. Sulfite in the eluates was deactivated with $\text{I}{}_2\text{KI}$ reagent. After oxidation by bromine, 2,3-butylene glycol was measured as acetoin. Excess bromine was neutralized by addition of 40% NaOH and saturated Na₂S₂O₅. After separation and conversion of the glycol to acetoin, the Westerfeld colorimetric method was used to determine the three components quantitatively.     

Microbial Production of 2,3-Butylene Glycol from Cheese Whey

Six microorganisms that produced acetoin or diacetyl or both from glucose were tested for the production of 2,3-butylene glycol from lactose. Bacillus polymyxa and Streptococcus faecalis gave positive results and were tested in unmodified wheys. Cottage cheese whey was unsatisfactory, but B. polymyxa produced large amounts of the glycol in sweet whey, about 60 mmol of glycol per 100 mmol of lactose utilized. Aeration and an increased ratio of surface area to volume of whey enhanced the production of glycol. 2,3-Butylene was separated from the spent whey and from acetoin and diacetyl with a Sephadex G-10 column.     

Isolation and properties of Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 mutants producing diacetyl and acetoin from glucose.

Following treatment with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine, three mutants of Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 that produced diacetyl and acetoin from glucose were isolated. The lactate dehydrogenase activity of these mutants was strongly attenuated, and the mutants produced less lactate than the parental strain. The kinetic properties of lactate dehydrogenase of strain CNRZ 483 and the mutants revealed differences in the affinity of the enzyme for pyruvate, NADH, and fructose-1,6-diphosphate. When cultured aerobically, strain CNRZ 483 transformed 2.3% of glucose to acetoin and produced no diacetyl or 2,3-butanediol. Under the same conditions, mutants 483L1, 483L2, and 483L3 transformed 42.0, 78.9, and 75.8%, respectively, of glucose to C4 compounds (diacetyl, acetoin, and 2,3-butanediol). Anaerobically, strain CNRZ 483 produced no C4 compounds, while mutants 483L1, 483L2, and 483L3 transformed 2.0, 37.0, and 25.8% of glucose to acetoin and 2,3-butanediol. In contrast to the parental strain, the NADH balance showed that the mutants regenerated most of the NAD via NADH oxidase under aerobic conditions and by ethanol production under anaerobic conditions.     

Desmutagenic effect of alpha-dicarbonyl and alpha-hydroxycarbonyl compounds against mutagenic heterocyclic amines

The desmutagenic effects of alpha-hydroxycarbonyl compounds, such as glyceraldehyde, glycolaldehyde, dihydroxyacetone, furfural, 5-hydroxymethylfurfural, maltol, acetol and acetoin and alpha-dicarbonyl compounds, such as diacetyl, glyoxal, methyl glyoxal and 2,3-pentanedione were investigated against the mutagenic heterocyclic amines, such as Trp-P-1, Trp-P-2, Glu-P-1, Glu-P-2 and IQ. Most of the carbonyl compounds suppressed the mutagenicity of heterocyclic amines for S. typhimurium TA98, alpha-dicarbonyl compounds showing a higher desmutagenic effect than alpha-hydroxycarbonyl compounds. Among the alpha-hydroxycarbonyl compounds, glyceraldehyde, glycolaldehyde and dihydroxyacetone showed more effective desmutagenicity, and diacetyl among the alpha-dicarbonyl compounds had the highest desmutagenic effect. These carbonyl compounds alone also showed mutagenicity to S. typhimurium TA100 without S9 mix. The reaction of carbonyl compounds with mutagenic heterocyclic amines also eliminated the mutagenicity of the former for S. typhimurium TA100.     

Fermentation of primary alcohols and diols and pure culture of syntrophically alcohol-oxidizing anaerobes

Ethanol, propanol, ethylene glycol, 1,2-propanediol, 1,2-butanediol, acetoin, diacetyl, and 2,3-pentanedione were used as substrates for enrichment and isolation of alcohol-oxidizing fermentative bacteria. Diacetyl and 2,3-pentanedione proved to be highly toxic. With the other substrates, various kinds of bacteria could be isolated which were assigned to three different metabolic groups: (i) homoacetogenic bacteria, and (ii) bacteria forming propionate as reduced end product were isolated from freshwater sources; (iii) bacteria disproportionating acetoin and 1,2-diols to acids and primary alcohols were isolated from marine sediments. The latter oxidized primary alcohols to fatty acids in the presence of hydrogen-oxidizing partners. Syntrophically ethanol-oxidizing cocultures enriched with primary alcohols could be separated with 1,2-diols as substrates into an alcohol-oxidizing organism and a hydrogen-oxidizing homoacetogen. The pathways of alcohol conversion in the disproportionating isolates were studied in detail. Growth experiments as well as enzymological studies demonstrated that acetoin and 1,2-diols were degraded via acetaldehyde which was also an intermediate in syntrophic oxidation of primary alcohols. The environmental importance of the various metabolic types isolated was assessed by most-probable-number enumerations.     

Detection and Identification of Bacteria by Gas Chromatography

Ether extracts of cultures of 29 strains representing 6 species of Bacillus, and of individual strains of Escherichia coli, Aerobacter aerogenes, and Pseudomonas aeruginosa were examined in a gas chromatograph by use of flame ionization and electron capture detectors. Among the products detected were compounds with the chromatographic characteristics of acetic, propionic, and butyric acids, ethyl alcohol, diacetyl, acetoin, and 2,3-butanediol. The differences in peak areas of the various products formed by the bacteria were determined statistically for the chromatograms obtained with the two detectors, and the peaks were arranged in order of decreasing areas to yield a signature for each bacterial strain. Different signatures were obtained for the various genera and species and for strains of the same species. B. licheniformis, B. subtilis, and A. aerogenes formed significant quantities of a number of volatile compounds, and qualitative and quantitative differences between strains were noted. The electron capture detector was particularly sensitive to diacetyl and acetoin as well as to unknown compounds. By use of this detector, the presence of 5 pg of diacetyl and 20 pg of acetoin could be demonstrated. The quantity of acetoin detected in B. subtilis and B. licheniformis cultures was present in as little as 6.3 x 10(-3) muliters of medium.     

Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation

Two 2,3-butanediol dehydrogenases (enzymes 1 and 2; molecular weight of each, 170,000) have been partially purified from Lactococcus lactis subsp. lactis (Streptococcus diacetylactis) D10 and shown to have reductase activity with either diacetyl or acetoin as the substrate. However, the reductase activity with 10 mM diacetyl was far greater for both enzymes (7.0- and 4.7-fold for enzymes 1 and 2, respectively) than with 10 mM acetoin as the substrate. In contrast, when acetoin and diacetyl were present together, acetoin was the preferred substrate for both enzymes, with enzyme 1 showing the more marked preference for acetoin. meso-2,3-Butanediol was the only isomeric product, with enzyme 1 independent of the substrate combinations. For enzyme 2, both the meso and optical isomers of 2,3-butanediol were formed with acetoin as the substrate, but only the optical isomers were produced with diacetyl as the substrate. With batch cultures of strain D10 at or near the point of citrate exhaustion, the main isomers of 2,3-butanediol present were the optical forms. If the pH was sufficiently high (>pH 5), acetoin reduction occurred over time and was followed by diacetyl reduction, and meso-2,3-butanediol became the predominant isomer. Interconversion of the optical isomers into the meso isomer did occur. The properties of 2,3-butanediol dehydrogenases are consistent with diacetyl and acetoin removal and the appearance of the isomers of 2,3-butanediol.     

The D(-)lactic acid and acetoin/diacetyl as potential indicators of the microbial quality of vacuum-packed pork and meat products

The suitability of D(-)lactic acid and/or acetoin/diacetyl as indicators of spoilage of vacuum-packed meat and meat products has been studied. When pork was vacuum-packed, these substances reached measurable amounts after storage for only about 10 days. Although microbial counts stabilized from the 20th day of storage, the D(-)lactic acid and acetoin/diacetyl concentrations increased progressively. These substances could therefore be potential indicators of the storage time of vacuum-packed pork. From a survey carried out with several vacuum-packed meat products from the market, it was concluded that the D(-)lactic acid content could be used as an indicator of the storage time of these products. No consistent results were obtained with acetoin/diacetyl.     

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.     

Effect of lactic acid on diacetyl and acetoin production byLactobacillus casei subsp.rhamnosus ATCC 7469

Lactic acid or its acidity apparently play an important role in the regulation of the biosynthesis of flavor compounds inLactobacillus casei subsp.rhamnosus ATCC 7469. In pyruvate-containing media,L. casei produces lactic acid, acetoin, and diacetyl. A specific pH-dependent system is necessary for both the use of pyruvate and the induction of acetoin and diacetyl production. In cell extracts ofL. casei, lactic acid inhibits the enzymatic activity of acetolactate decarboxylase (ALD) and acetolactate synthetase (ALS); this effect does not occur in whole cells under standard physiological conditions. Lactic acid prevents the use of pyruvate, and the induction of acetoin and diacetyl production. When pyruvate-containing media are used, the pH must be kept close to 6.0 in order to obtain the best production of acetoin and diacetyl.     

Spot Test of Some Carbonyl Compounds Using Their Fluorigenic Reactions with o-Aminodiphenyl

A new spot test on silica gel thin-layers of some carbonyl compounds was described, which was based on their fluorigenic reactions with o-aminodiphenyl dissolved in diluted sulfuric acid. Pyridoxal, higher fatty aldehydes, glycolaldehyde, glyoxylic acid and 2,3-pentanedione gave brilliantly fluorescent spots in UV light by heating with the reagent sprayed. Some other non- or sparingly volatile carbonyls also gave positive results.

The reaction of glyoxal with the reagent was carried out in aqueous solution. A linear relationship between the fluorescence intensity and glyoxal concentration was observed.     

A descriptive model for citrate utilization by Lactococcus lactis ssp lactis bv diacetylactis

A model for the use of citrate by Lactococcus lactis ssp lactis bv diacetylactis CNRZ 125 is proposed. Citrate metabolism by this strain leads to the production of acetate, CO₂ and C4 compounds (diacetyl, acetoin, 2,3-butylene glycol). The model furnishes correct simulations, consistent with published results on the pathways used and on lactose-citrate co-metabolism. Citric acid is incorporated independently of growth. The production of flavoring compounds is a complex process, depending on the rate of citrate utilization, on the proportion of pyruvate arising from citrate and which condenses to form -acetolactate and CO₂, on the rate of transformation of -acetolactate to diacetyl and acetoin, as well as on the rate of reduction of these compounds to 2,3-butylene glycol.     

Impact of Lactobacillus paracasei IMC502 in coculture with traditional starters on volatile and non-volatile metabolite profiles in yogurt

The impact of Lactobacillus paracasei IMC502 co-fermented with traditional starters on metabolites in yogurt was evaluated using metabolomic analysis. Forty-four volatile metabolites were determined using headspace solid phase microextraction and gas chromatography-mass spectrometry, including alcohols, esters, organic acids, carbonyl and sulfur compounds. Acetaldehyde, diacetyl, acetoin, acetone, butanoic and acetic acid were present in yogurts in higher intensity, which are the major volatile metabolites related to yogurt flavor. L. paracasei IMC502 did not affect the amounts of acetaldehyde, diacetyl and acetoin while promoted the formation of acetone and butanoic acid. A total of 196 non-volatile metabolites including nucleosides, amino acids, carbohydrates, lipids were analyzed using UPLC-Q-TOF-MS. Non-volatile metabolite profiles of these two types of yogurts were distinguished and 94 differential metabolites were screened using multivariate statistical analysis, which were mainly associated with the biosynthesis of secondary metabolites, amino acid metabolism and nucleotide metabolism. The impact of storage on metabolites was also investigated. The amounts of the majority of carbonyl compounds, organic acids and free amino acids increased, while those of acetaldehyde, diacetyl and lactose decreased during storage. This study provides insights into the metabolic mechanism of L. paracasei and represents a real advance in the study of the metabolites in yogurt.     

Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu

CitrLactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu, and the activity of NAD-dependent diacetyl reductase was very high. Hemin did not affect the activities of NADH oxidase and lactate dehydrogenase. These results indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that the diacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH when the cells were grown aerobically with hemin or Cu. On the other hand, the Y(Glu) value for the hemincontaining culture was lower than for the culture without hemin, because acetate production was repressed when an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of the dihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu enhanced acetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase was lowered by the addition of lipoic acid. These results indicate that hemin or Cu stimulates acetyl coenzyme A (acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA with hydroxyethylthiamine PP(i). In addition, it appears that acetyl-CoA not used for diacetyl synthesis is converted to acetate.     

Preparation of radioactive diacetyl,acetoin, and 2,3-butylene glycol

Toluene-treated cells of Streptococcus diacetilactis produced large amounts of diacetyl and acetoin without 2,3-butylene glycol. With Na-[3-¹⁴C]pyruvate added to reaction mixtures in place of unlabeled pyruvate, diacetyl with specific activity of 6.1 × 10⁴ cpm/μmol and acetoin with specific activity of 6.8 × 10⁴ cpm/μmol were harvested. Growing cells of Enterobacter aerogens incubated 48 h at 30°C in a complex medium produced large amounts of 2,3-butylene glycol without acetoin or diacetyl. With uniformly labeled [¹⁴C]glucose added to the medium in place of unlabeled glucose, 2,3-butylene glycol with specific activity of 10.8 × 10⁴ cpm/μmol was harvested. The radioactive chemicals were tested and found to be chromatographically homogeneous. Storage frozen in capped containers was especially important for diacetyl, which was found to evaporate rapidly from capped containers at room temperature.     

Mechanism for the formation of 2,3-butanediol stereoisomers in Bacillus polymyxa

The mechanism of the formation of 2,3-butanediol isomers in Bacillus polymyxa was studied. We proposed a new model with NADPH-linked diacetyl reductase (S-acetoin forming) and R(−)-2,3-butanediol dehydrogenase. The two enzymes were separated by Blue Sepharose CL-6B and their stereospecificities were identified using all of the pure isomers of 2,3-butanediol (R(−), S(+)m, and meso), acetoin (R(−) and S(+)) and the separation and measurement of these isomers. The presence of acetoin or butanediol racemase was not confirmed in our experiments.     

Selection of an endogenous 2,3-butanediol pathway in Escherichia coli by fermentative redox balance

Fermentative redox balance has long been utilized as a metabolic evolution platform to improve efficiency of NADH-dependent pathways. However, such system relies on the complete recycling of NADH and may become limited when the target pathway results in excess NADH stoichiometrically. In this study, endogenous capability of Escherichia coli for 2,3-butanediol (2,3-BD) synthesis was explored using the anaerobic selection platform based on redox balance. To address the issue of NADH excess associated with the 2,3-BD pathway, we devised a substrate-decoupled system where a pathway intermediate is externally supplied in addition to the carbon source to decouple NADH recycling ratio from the intrinsic pathway stoichiometry. In this case, feeding of the 2,3-BD precursor acetoin effectively restored anaerobic growth of the mixed-acid fermentation mutant that remained otherwise inhibited even in the presence of a functional 2,3-BD pathway. Using established 2,3-BD dehydrogenases as model enzyme, we verified that the redox-based selection system is responsive to NADPH-dependent reactions but with lower sensitivity. Based on this substrate-decoupled selection scheme, we successfully identified the glycerol/1,2-propanediol dehydrogenase (Ec-GldA) as the major enzyme responsible for the acetoin reducing activity (k_cat/K_m≈0.4 mM⁻¹ s⁻¹) observed in E. coli. Significant shift of 2,3-BD configuration upon withdrawal of the heterologous acetolactate decarboxylase revealed that the endogenous synthesis of acetoin occurs via diacetyl. Among the predicted diacetyl reductase in E. coli, Ec-UcpA displayed the most significant activity towards diacetyl reduction into acetoin (V_max≈6 U/mg). The final strain demonstrated a meso-2,3-BD production titer of 3 g/L without introduction of foreign genes. The substrate-decoupled selection system allows redox balance regardless of the pathway stoichiometry thus enables segmented optimization of different reductive pathways through enzyme bioprospecting and metabolic evolution.     

Exocellular products from Bifidobacterium adolescentis as immunomodifiers in the lymphoproliferative responses of mouse splenocytes

Abstract Pelobacter carbinolicus strain GraBd1 fermented methylacetoin, which is a good carbon source for growth ( μ = 0.16 h⁻¹) of this strict anaerobic bacterium, to acetone, acetate and ethanol (main products), acetoin, 2,3-butanediol and methylbutanediol (minor products). During growth on 2,3-butanediol, acetoin and methyl-acetoin the formation of a protein exhibiting acetoin: DCPIP oxidoreductase activity is induced. This enzyme amounts to a substantial portion of the soluble proteins. In vitro, it cleaves acetoin into acetate and acetaldehyde but reacts also with diacetyl or methylacetoin. We discussed four different models for the degradation of acetoin in the cells and came to the conclusion that in vivo the oxidative-thiolytic acetoin cleavage model is most probably realized in P. carbinolicus .