Assessment of Chemosensory Response to Volatile Compounds in Healthy, Aged, and Neurodegenerative Caenorhabditis elegans Models.

A basic function of the nervous system is to confer the ability to detect external stimuli and generate appropriate behavioral and physiological responses. These can be modulated when parallel streams of information are provided to the nervous system and neural activity is appropriately altered. The nematode Caenorhabditis elegans utilizes a simple and well characterized neural circuit to mediate avoidance or attraction responses to stimuli, such as the volatile odorant octanol or diacetyl (DA), respectively. Aging and neurodegeneration constitute two important factors altering the ability to detect external signals and, therefore, changing behavior. Here, we present a modified protocol to assess avoidance or attraction responses to diverse stimuli in healthy and worm models associated with neurodegenerative diseases.

A New Essential Oil from the Leaves of Gynoxys rugulosa Muschl. (Asteraceae) Growing in Southern Ecuador: Chemical and Enantioselective Analyses.

An essential oil, distilled from the leaves of the Andean species Gynoxys rugulosa Muschl., is described in the present study for the first time. The chemical composition was qualitatively and quantitatively determined by GC-MS and GC-FID, respectively. On the one hand, the qualitative composition was obtained by comparing the mass spectrum and the linear retention index of each component with data from literature. On the other hand, the quantitative composition was determined by calculating the relative response factor of each constituent, according to its combustion enthalpy. Both analyses were carried out with two orthogonal columns of nonpolar and polar stationary phases. A total of 112 compounds were detected and quantified with at least one column, corresponding to 87.3-93.0% of the whole oil mass. Among the 112 detected components, 103 were identified. The main constituents were α-pinene (5.3-6.0%), (E)-ß-caryophyllene (2.4-2.8%), α-humulene (3.0-3.2%), germacrene D (4.9-6.5%), δ-cadinene (2.2-2.3%), caryophyllene oxide (1.6-2.2%), α-cadinol (3.8-4.4%), 1-nonadecanol (1.7-1.9%), 1-eicosanol (0.9-1.2%), n-tricosane (3.3-3.4%), 1-heneicosanol (4.5-5.8%), n-pentacosane (5.8-7.1%), 1-tricosanol (4.0-4.5%), and n-heptacosane (3.0-3.5%). Furthermore, an enantioselective analysis was carried out on the essential oil, by means of two cyclodextrin-based capillary columns. The enantiomers of α-pinene, ß-pinene, sabinene, α-phellandrene, ß-phellandrene, linalool, α-copaene, terpinen-4-ol, α-terpineol, and germacrene D were detected, and the respective enantiomeric excess was calculated.

Enzymes and pathways in microbial production of 2,3-butanediol and 3-acetoin isomers.

2,3-Butanediol (BD) and acetoin (AC) are products of the non-oxidative metabolism of microorganisms, presenting industrial importance due to their wide range of applications and high market value. Their optical isomers have particular applications, justifying the efforts on the selective bioproduction. Each microorganism produces different isomer mixtures, as a consequence of having different butanediol dehydrogenase (BDH) enzymes. However, the whole scene of the isomer bioproduction, considering the several enzymes and conditions, has not been completely elucidated. Here we show the BDH classification as R, S or meso by bioinformatics analysis uncovering the details of the isomers production. The BDH was compared to diacetyl reductases (DAR) and the new enoyl reductases (ER). We observed that R-BDH is the most singular BDH, while meso and S-BDHs are similar and may be better distinguished through their stereo-selective triad. DAR and ER showed distinct stereo-triads from those described for BDHs, agreeing with kinetic data from the literature and our phylogenetic analysis. The ER family probably has meso-BDH like activity as already demonstrated for a single sequence from this group. These results are of great relevance, as they organize BD producing enzymes, to our known, never shown before in the literature. This review also brings attention to nontraditional enzymes/pathways that can be involved with BD/AC synthesis, as well as oxygen conditions that may lead to the differential production of their isomers. Together, this information can provide helpful orientation for future studies in the field of BD/AC biological production, thus contributing to achieve their production on an industrial scale.

Technological properties of Lactococcus lactis subsp. lactis bv. diacetylactis obtained from dairy and non-dairy niches.

Lactococcus lactis subsp. lactis bv. diacetylactis strains are often used as starter cultures by the dairy industry due to their production of acetoin and diacetyl, important substances that add buttery flavor notes in dairy products. Twenty-three L. lactis subsp. lactis isolates were obtained from dairy products (milk and cheese) and dairy farms (silage), identified at a biovar level, fingerprinted by rep-PCR and characterized for some technological features. Fifteen isolates presented molecular and phenotypical (diacetyl and citrate) characteristics coherent with L. lactis subsp. lactis bv. diacetylactis and rep-PCR allowed the identification of 12 distinct profiles (minimum similarity of 90%). Based on technological features, only two isolates were not able to coagulate skim milk and 10 were able to produce proteases. All isolates were able to acidify skim milk: two isolates, in special, presented high acidifying ability due to their ability in reducing more than two pH units after 24 h. All isolates were also able to grow at different NaCl concentrations (0 to 10%, w/v), and isolates obtained from peanut and grass silages presented the highest NaCl tolerance (10%, w/v). These results indicate that the L. lactis subsp. lactis bv. diacetylactis isolates presented interesting technological features for potential application in fermented foods production. Despite presenting promising technological features, the isolates must be assessed according to their safety before being considered as starter cultures.

Fat reduction and whey protein concentrate addition alter the concentration of volatile compounds during Prato cheese ripening.

The impact of fat reduction and the addition of whey protein concentrate (WPC) on the volatile compounds profile of Prato cheese was evaluated for 210 days of storage at 12 °C. Full fatPrato cheese, Prato cheese with fat reduction and reduced fatPrato cheese with 0.5% (w/v) WPC were made, and replicated on a different day. Cheese volatile compounds were extracted by solid phase microextraction (SPME) and separated, identified and quantified by gas chromatography coupled to a mass spectrometry detector (GC-MS). Fat reduction and the WPC addition resulted in lower concentrations of compounds derived from lipolysis and free fatty acid catabolism. Fat reduction generated higher levels of diacetyl and acetoin, both from the citrate metabolism, at days 5 and 30. A similar pattern was observed for sulfur compounds derived from the catabolism of free amino acids, at day 120. Higher levels of diacetyl (day15), dimethyl disulfide (days 150-180) and dimethyl trisulphide (days 150-210) were found for cheese with WPC. These differences might have occurred due to alterations in the structure and polarity of the protein matrix caused by fat reduction and the WPC addition.

Increased chemical acetylation of peptides and proteins in rats after daily ingestion of diacetyl analyzed by Nano-LC-MS/MS.

BACKGROUND: Acetylation alters several protein properties including molecular weight, stability, enzymatic activity, protein-protein interactions, and other biological functions. Our previous findings demonstrating that diacetyl/peroxynitrite can acetylate L-lysine, L-histidine, and albumin in vitro led us to investigate whether diacetyl-treated rats suffer protein acetylation as well. METHODS: Wistar rats were administered diacetyl daily for four weeks, after which they were sacrificed, and their lung proteins were extracted to be analysed by Nano-LC-MS/MS (Q-TOF). A C18 reversed-phase column and gradient elution with formic acid/acetonitrile solutions from 2 to 50% over 150 min were used to separate the proteins. Protein detection was performed using a microTOF-Q II (QTOF) equipped with captive source and an electrospray-ionization source. The data from mass spectrometry were processed using a Compass 1.7 and analyzed using Protein Scape, software that uses Mascot algorithms to perform protein searches. RESULTS: A set of 3,162 acetylated peptides derived from 351 acetylated proteins in the diacetyl-treated group was identified. Among them, 23 targeted proteins were significantly more acetylated in the diacetyl-treated group than in the PBS control. Protein acetylation of the group treated with 540 mg/kg/day of diacetyl was corroborated by Western blotting analysis. CONCLUSIONS: These data support our hypothesis that diacetyl exposure in animals may lead to the generation of acetyl radicals, compounds that attach to proteins, affecting their functions and triggering adverse health problems.

An original method for producing acetaldehyde and diacetyl by yeast fermentation

Abstract In this study a natural culture medium that mimics the synthetic yeast peptone glucose medium used for yeast fermentations was designed to screen and select yeasts capable of producing high levels of diacetyl and acetaldehyde. The presence of whey powder and sodium citrate in the medium along with manganese and magnesium sulfate enhanced both biomass and aroma development. A total of 52 yeasts strains were cultivated in two different culture media, namely, yeast peptone glucose medium and yeast acetaldehyde-diacetyl medium. The initial screening of the strains was based on the qualitative reaction of the acetaldehyde with Schiff's reagent (violet color) and diacetyl with Brady's reagent (yellow precipitate). The fermented culture media of 10 yeast strains were subsequently analyzed by gas chromatography to quantify the concentration of acetaldehyde and diacetyl synthesized. Total titratable acidity values indicated that a total titratable acidity of 5.5 °SH, implying culture medium at basic pH, was more favorable for the acetaldehyde biosynthesis using strain D15 (Candida lipolytica; 96.05 mg L-1 acetaldehyde) while a total titratable acidity value of 7 °SH facilitated diacetyl flavor synthesis by strain D38 (Candida globosa; 3.58 mg L-1 diacetyl). Importantly, the results presented here suggest that this can be potentially used in the baking industry.

An original method for producing acetaldehyde and diacetyl by yeast fermentation.

In this study a natural culture medium that mimics the synthetic yeast peptone glucose medium used for yeast fermentations was designed to screen and select yeasts capable of producing high levels of diacetyl and acetaldehyde. The presence of whey powder and sodium citrate in the medium along with manganese and magnesium sulfate enhanced both biomass and aroma development. A total of 52 yeasts strains were cultivated in two different culture media, namely, yeast peptone glucose medium and yeast acetaldehyde-diacetyl medium. The initial screening of the strains was based on the qualitative reaction of the acetaldehyde with Schiff's reagent (violet color) and diacetyl with Brady's reagent (yellow precipitate). The fermented culture media of 10 yeast strains were subsequently analyzed by gas chromatography to quantify the concentration of acetaldehyde and diacetyl synthesized. Total titratable acidity values indicated that a total titratable acidity of 5.5°SH, implying culture medium at basic pH, was more favorable for the acetaldehyde biosynthesis using strain D15 (Candida lipolytica; 96.05mgL-1 acetaldehyde) while a total titratable acidity value of 7°SH facilitated diacetyl flavor synthesis by strain D38 (Candida globosa; 3.58mgL-1 diacetyl). Importantly, the results presented here suggest that this can be potentially used in the baking industry.

Aroma compounds generation in citrate metabolism of Enterococcus faecium: Genetic characterization of type I citrate gene cluster.

Enterococcus is one of the most controversial genera belonging to Lactic Acid Bacteria. Research involving this microorganism reflects its dual behavior as regards its safety. Although it has also been associated to nosocomial infections, natural occurrence of Enterococcus faecium in food contributes to the final quality of cheese. This bacterium is capable of fermenting citrate, which is metabolized to pyruvate and finally derives in the production of the aroma compounds diacetyl, acetoin and 2,3 butanediol. Citrate metabolism was studied in E. faecium but no data about genes related to these pathways have been described. A bioinformatic approach allowed us to differentiate cit(-) (no citrate metabolism genes) from cit(+) strains in E. faecium. Furthermore, we could classify them according to genes encoding for the transcriptional regulator, the oxaloacetate decarboxylase and the citrate transporter. Thus we defined type I organization having CitI regulator (DeoR family), CitM cytoplasmic soluble oxaloacetate decarboxylase (Malic Enzyme family) and CitP citrate transporter (2-hydroxy-carboxylate transporter family) and type II organization with CitO regulator (GntR family), OAD membrane oxaloacetate decarboxylase complex (Na(+)-transport decarboxylase enzyme family) and CitH citrate transporter (CitMHS family). We isolated and identified 17 E. faecium strains from regional cheeses. PCR analyses allowed us to classify them as cit(-) or cit(+). Within the latter classification we could differentiate type I but no type II organization. Remarkably, we came upon E. faecium GM75 strain which carries the insertion sequence IS256, involved in adaptative and evolution processes of bacteria related to Staphylococcus and Enterococcus genera. In this work we describe the differential behavior in citrate transport, metabolism and aroma generation of three strains and we present results that link citrate metabolism and genetic organizations in E. faecium for the first time.

An original method for producing acetaldehyde and diacetyl by yeast fermentation

Abstract In this study a natural culture medium that mimics the synthetic yeast peptone glucose medium used for yeast fermentations was designed to screen and select yeasts capable of producing high levels of diacetyl and acetaldehyde. The presence of whey powder and sodium citrate in the medium along with manganese and magnesium sulfate enhanced both biomass and aroma development. A total of 52 yeasts strains were cultivated in two different culture media, namely, yeast peptone glucose medium and yeast acetaldehyde-diacetyl medium. The initial screening of the strains was based on the qualitative reaction of the acetaldehyde with Schiff's reagent (violet color) and diacetyl with Brady's reagent (yellow precipitate). The fermented culture media of 10 yeast strains were subsequently analyzed by gas chromatography to quantify the concentration of acetaldehyde and diacetyl synthesized. Total titratable acidity values indicated that a total titratable acidity of 5.5 °SH, implying culture medium at basic pH, was more favorable for the acetaldehyde biosynthesis using strain D15 (Candida lipolytica; 96.05 mg L-1 acetaldehyde) while a total titratable acidity value of 7 °SH facilitated diacetyl flavor synthesis by strain D38 (Candida globosa; 3.58 mg L-1 diacetyl). Importantly, the results presented here suggest that this can be potentially used in the baking industry.

Condensation of Diacetyl with Alkyl Amines: Synthesis and Reactivity of p-Iminobenzoquinones and p-Diiminobenzoquinones.

Condensation reactions between diacetyl and α-branched primary alkylamines under mild and neutral conditions provided a mixture of 2,5-dimethylbenzoquinone(alkylimines), 2,5-dimethylbenzoquinone(bis-alkyldiimines), and N,N'-dialkyl-2,5-dimethylbenzene-1,4-diamines, which were efficiently separated as pure products by column chromatography. Both 2,5-dimethylbenzoquinone(alkylimines) and 2,5-dimethylbenzoquinone(bis-alkyldiimines) underwent an interchange of the alkylimino group when treated with anilines, followed by reductive aromatization, to provide diarylamines and 1,4-dianilinobenzenes, respectively. Evaluation was also made of the reactivity and selectivity of these compounds in the presence of anilines, thiophenols and alkylhalides.

Methylglyoxal is associated with bacteriostatic activity of high fructose agave syrups.

Three α-ketoaldehydes, potentially present in high fructose agave syrups (HFASs) as intermediates of the Maillard reaction, were determined. A previously reported HPLC-FLD procedure based on pre-column derivatisation with 4-methoxy-o-phenylenediamine was adopted, yielding the method quantification limits 0.11 mg/kg, 0.10mg/kg, 0.09 mg/kg for glyoxal, methylglyoxal (MGo) and diacetyl, respectively. The obtained results revealed high concentrations of methylglyoxal in HFASs (average 102 ± 91 mg/kg, range 15.6-315 mg/kg) as compared to commercial Mexican bee honeys or corn syrups. Hydrogen peroxide was generated in all HFASs upon dilution, yet to less extent than in bee honeys. HFASs presented bacteriostatic activity against Bacillus subtilis and Escherichia coli; catalase addition had minimum effect on the assay results in syrups with elevated MGo. Principal component analysis revealed direct association between growth inhibition and MGo. It is concluded that elevated concentration of MGo in HFASs is at least in part responsible for their non-peroxide bacteriostatic activity.

High-performance liquid chromatography determination of glyoxal, methylglyoxal, and diacetyl in urine using 4-methoxy-o-phenylenediamine as derivatizing reagent.

Bioanalytical relevance of glyoxal (Go) and methylglyoxal (MGo) arises from their role as biomarkers of glycation processes and oxidative stress. The third compound of interest in this work is diacetyl (DMGo), a component of different food products and alcoholic beverages and one of the small α-ketoaldehydes previously reported in urine. The original idea for the determination of the above compounds by reversed phase high-performance liquid chromatography (HPLC) with fluorimetric detection was to use 4-methoxy-o-phenylenediamine (4MPD) as a derivatizing reagent and diethylglyoxal (DEGo) as internal standard. Acetonitrile was added to urine for matrix precipitation, and derivatization reaction was carried out in the diluted supernatant at neutral pH (40 °C, 4 h); after acidification, salt-induced phase separation enabled recovery of the obtained quinoxalines in the acetonitrile layer. The separation was achieved within 12 min using a C18 Kinetex column and gradient elution. The calibration detection limits for Go, MGo, and DMGo were 0.46, 0.39, and 0.28 µg/L, respectively. Within-day precision for real-world samples did not exceed 6%. Several urine samples from healthy volunteers, diabetic subjects, and juvenile swimmers were analyzed. The sensitivity of the procedure proposed here enabled detection of differences between analyte concentrations in urine from patients at different clinical or exposure-related conditions.

Bronquiolite associada à exposição a aroma artificial de manteiga em trabalhadores de uma fábrica de biscoitos no Brasil / Bronchiolitis associated with exposure to artificial butter flavoring in workers at a cookie factory in Brazil

OBJETIVO: Relatar quatro casos de bronquiolite decorrente de exposição a aroma artificial de manteiga em uma fábrica de biscoitos no Brasil. MÉTODOS: Descrevemos os achados clínicos, espirométricos e tomográficos nos quatro pacientes, assim como achados de biópsia pulmonar em um dos pacientes. RESULTADOS: Os quatro pacientes eram homens jovens, não fumantes, e desenvolveram obstrução persistente ao fluxo aéreo (relação VEF1/CVF reduzida e VEF1 de 25-44% do previsto) após 1-3 anos de exposição a diacetil, sem a utilização de equipamentos de proteção individual, em uma fábrica de biscoitos. A TCAR mostrou achados indicativos de bronquiolite. Em um paciente, a biópsia pulmonar cirúrgica mostrou bronquiolite obliterante associada a células gigantes. CONCLUSÕES: A bronquiolite decorrente de exposição a flavorizantes artificiais deve ser considerada em trabalhadores com obstrução ao fluxo aéreo no Brasil.

OBJECTIVE: To report the cases of four patients with bronchiolitis caused by exposure to artificial butter flavoring at a cookie factory in Brazil. METHODS: We described the clinical, tomographic, and spirometric findings in the four patients, as well as the lung biopsy findings in one of the patients. RESULTS: All four patients were young male nonsmokers and developed persistent airflow obstruction (reduced FEV1/FVC ratio and FEV1 at 25-44% of predicted) after 1-3 years of exposure to diacetyl, without the use of personal protective equipment, at a cookie factory. The HRCT findings were indicative of bronchiolitis. In one patient, the surgical lung biopsy revealed bronchiolitis obliterans accompanied by giant cells. CONCLUSIONS: Bronchiolitis resulting from exposure to artificial flavoring agents should be included in the differential diagnosis of airflow obstruction in workers in Brazil.

Mecanismo de oxidação aeróbica de acetoacetato e 2-metilacetoacetato catalisada por mioglobina: implicações em desordens cetogênicas / Mechanism of the aerobic oxidation of acetoacetate and 2- methylacetoacetate catalyzed by Mb: implications for ketogenic disorders

Acetoacetato (AA) e 2-metilacetoacetato (MAA) são compostos β-cetoácidos acumulados em diversas desordens metabólicas como no diabetes e na isoleucinemia, respectivamente. Examinamos o mecanismo de oxidação aeróbica de AA e MAA iniciada por intermediários reativos de mioglobina de coração de cavalo (Mb) gerados pela adição de H2O2. Uma rota quimioluminescente que envolve um intermediário dioxetânico cuja termólise gera espécies α-dicarbonílicas (metilglioxal e biacetilo) foi proposta e estudada. Emissão de luz ultra fraca acompanha a reação, e sua intensidade aumenta linearmente pelo aumento da concentração tanto de Mb (10-500 µM) quando AA (10-100 mM). Estudos de consumo de oxigênio mostraram que MAA é, como esperado, quase uma ordem de grandeza mais reativo que AA. Estudos de EPR com captação de spin, utilizando MNP, possibilitaram detectar adutos de MAA atribuíveis a um radical centrado no Cα (aN = 1.55 mT) e ao radical acetila (aN = 0.83 mT). O sinal do radical acetila é totalmente suprimido por sorbato, um conhecido e eficiente supressor de espécies tripletes, o que é consistente com uma rota reacional envolvendo um intermediário dioxetânico. Clivagem-α da ligação carbonila-carbonila do produto biacetilo triplete produziria, de fato, radicais acetila. Além disso, utilizando AA como substrato para Mb/H2O2, um sinal de EPR atribuível ao aduto MNP-AA• (aN = 1.46 mT e aH = 0.34 mT) foi observado e confirmado por efeito isotópico. O consumo de oxigênio e o rendimento de compostos α-dicarbonílicos foram dose-dependentes à concentração de AA ou MAA (1-50 mM) bem como à concentração de H2O2 adicionado às misturas de reação contendo Mb (até 1:10 quando medido o consumo de oxigênio, e até 1:25 quando medido o rendimento de compostos α-dicarbonílicos) e tert-butilhidroperóxido (até 1:200). Os perfis de pH (5,8-7,8) para consumo de oxigênio e rendimento de compostos α-dicarbonílicos mostraram maiores rendimentos para baixos valores de pH, indicativo de ferrilMb...

Acetoacetate (AA) and 2-methylacetoacetate (MAA) are β-ketoacids accumulated in several metabolic disorders such as diabetes and isoleucinemia, respectively. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by the reactive enzyme intermediates formed by the reaction of muscle horse myoglobin (Mb) with H2O2. A chemiluminescent route involving a dioxetane intermediate whose thermolysis yields triplet α-dicarbonyl species (methylglyoxal and diacetyl) is envisaged. Accordingly, the ultraweak light emission that accompanies the reaction increases linearly by raising the concentration of both Mb (10-500 µM) and AA (10- 100 mM). Oxygen uptake studies revealed that MAA is, expectedly, almost one order of magnitude more reactive than AA. EPR spin-trapping studies with MNP detected spin adducts from MAA attributable to an α-carbon-centered radical (aN = 1.55 mT) and to an acetyl radical (aN = 0.83 mT). As the acetyl radical signal is totally suppressed by sorbate, a well-known efficient triplet species quencher, the dioxetane hypothesis seems to be reliable. The α-cleavage of the carbonyl-carbonyl bond of a putative excited triplet diacetyl product would, in fact, leads to an acetyl radical. Furthermore, using AA as substrate for Mb/H2O2, an EPR signal assignable to a MNP-AA• adduct (aN = 1.46 mT and aH = 0.34 mT) was observed and confirmed by isotope effect. Oxygen consumption and α-dicarbonyl yield were also dependent on AA or MAA concentrations (1-50 mM) as well as on the concentration of peroxide added to the Mb-containing reaction mixtures: H2O2 (up to 1:10 when measuring oxygen uptake and up to 1:25 when measuring the α-dicarbonyl yield) and t-butOOH (up to 1:200). The pH profiles (5.8-7.8) of oxygen consumption and α-dicarbonyl yield show higher reaction rates at lower pHs, indicative of a ferrylMb intermediate. Evaluating Mb lesion, both β-ketoacids reduced disorganization of the secondary and tertiary protein structure elicited by H2O2...

A new derivative of dihydroochnaflavone isolated from Luxemburgia species (Ochnaceae) and the complete ¹H and 13C NMR chemical shifts assignments / Um novo derivado da dihidroochnaflavona isolada de Luxemburgia species (Ochnaceae) e o completo assinalamento dos dados de RMN de ¹H e 13C

The biflavonoid 2",3"-dihydroochnaflavone, isolated from the Luxemburgia species (Ochanaceae), was treated with diazomethane to obtain the trimethyl-ether that was treated with pyridine/acetic anhydride to yield a new derivative, 5-acetil-7,4'-dimethyl-flavone-(3'→O-4'")-5"-acetil-7"-methyl-flavanone. The complete ¹H and 13C NMR data assignments of the new derivative were made by the one- and two-dimensional spectral analysis.

O biflavonóide 2",3"-diidroochnaflavona, isolada de espécies de Luxemburgia (Ochanaceae), foi tratada com diazometano e forneceu o éter trimetílico que, em seguida, foi tratado com anidrido acético/piridina obtendo-se um novo derivado, 5-acetil-7,4'-dimetil-flavona-(3'→O-4'")-5"-acetil-7"-metil-flavanona. Fez-se o completo assinalamento dos dados de RMN ¹H e 13C através da análise dos espectros uni- e bidimensionais do derivado.