Fungal-Bacterial Mutualism: Species and Strain-Dependent Simultaneous Modulation of Branched-Chain Esters and Indole Derivatives in Fermented Sausages through Metabolite Cross-Feeding.

The precise impact of species and strain diversity on fungal-bacterial interactions and the overall community functioning has remained unclear. First, our study revealed how Debaryomyces hansenii influences diverse bacteria to accumulate key metabolites in a simulated fermented food system. For flavor, D. hansenii promoted the accumulation of branched-chain esters in Staphylococcus xylosus by promoting growth and facilitating the precursor branched-chain acids transformations but hindered the accumulation of Staphylococcus equorum. Furthermore, fungal-bacterial interactions displayed diversity among S. equorum strains. For bioactive compounds, species and strain diversity of lactic acid bacteria (LAB) also influences the production of indole derivatives. Then, we investigated specific metabolic exchanges under reciprocal interaction. Amino acids, rather than vitamins, were identified as the primary drivers of the bacterial growth promotion. Moreover, precursor transformations by D. hansenii played a significant role in branched-chain esters production. Finally, a synthetic community capable of producing high concentrations of branched-chain esters and indole derivatives was successfully constructed. These results provide valuable insights into understanding and designing synthetic communities for fermented sausages.

Experimental and theoretical estimation of the Hansen solubility parameters of paramylon esters based on the degrees of substitution and chain lengths of their acyl groups.

Paramylon is a natural hydrophilic polysaccharide produced in the pyrenoids of euglenoids, and esterification may render paramylon hydrophobic. Esterification imparts not only thermoplasticity, but also potential compatibilities with other polymer resins and fillers. However, the dependence of the compatibility on the structure of the polymer ester has not yet been systematically studied. To estimate the affinities between paramylon esters and hydrophobic organic solvents/resins, the dependences of their Hansen solubility parameters, which are association indices, on the degrees of substitution and chain lengths of the ester groups were investigated. Experimental and theoretical investigations were conducted using the dissolution and Fedors methods, respectively. Esterification decreased the solubility parameter from 49 (paramylon) to approximately 18 MPa1/2 (paramylon esters), indicating that the potential affinities of paramylon esters for hydrophobic organic solvents/polymers increased. A multiple regression analysis was also performed to investigate the effects of acyl chain length and degree of substitution with acyl groups on the solubility parameter. The solubility parameters of the paramylon derivatives were continuously variable from hydrophilic to -phobic. Hence, esterification with various acyl groups may control the hydrophobicities of paramylon esters, enhancing their miscibilities with various hydrophobic organic solvents and resins.

Insights into the transcriptional regulation of poorly characterized alcohol acetyltransferase-encoding genes (HgAATs) shed light into the production of acetate esters in the wine yeast Hanseniaspora guilliermondii.

Hanseniaspora guilliermondii is a well-recognized producer of acetate esters associated with fruity and floral aromas. The molecular mechanisms underneath this production or the environmental factors modulating it remain unknown. Herein, we found that, unlike Saccharomyces cerevisiae, H. guilliermondii over-produces acetate esters and higher alcohols at low carbon-to-assimilable nitrogen (C:N) ratios, with the highest titers being obtained in the amino acid-enriched medium YPD. The evidences gathered support a model in which the strict preference of H. guilliermondii for amino acids as nitrogen sources results in a channeling of keto-acids obtained after transamination to higher alcohols and acetate esters. This higher production was accompanied by higher expression of the four HgAATs, genes, recently proposed to encode alcohol acetyl transferases. In silico analyses of these HgAat's reveal that they harbor conserved AATs motifs, albeit radical substitutions were identified that might result in different kinetic properties. Close homologues of HgAat2, HgAat3, and HgAat4 were only found in members of Hanseniaspora genus and phylogenetic reconstruction shows that these constitute a distinct family of Aat's. These results advance the exploration of H. guilliermondii as a bio-flavoring agent providing important insights to guide future strategies for strain engineering and media manipulation that can enhance production of aromatic volatiles.

Alice Augusta Ball: The African-American chemist who pioneered the first viable treatment for Hansen's Disease.

Alice Augusta Ball (1892-1916) was a young and dynamic chemist who achieved extraordinary milestones in her short lifespan of 24 years. As an African American and as a woman in the early 20th century, her work and achievements set a benchmark. Her breakthrough work on the extraction of the water-soluble ethyl esters of chaulmoogra oil changed the lives of millions of people affected by Hansen's disease who were socially boycotted and segregated to leprosy colonies and asylums. The chaulmoogra oil esters, which continued to be used for almost two decades, were the first viable treatment of leprosy. Unfortunately, Ball did not receive recognition and honor for her work, and she was forgotten soon after her untimely death.

Chemical composition, sensorial properties, and aroma-active compounds of ciders fermented with Hanseniaspora osmophila and Torulaspora quercuum in co- and sequential fermentations.

In this study, apple juice was fermented using Hanseniaspora osmophila X25-5 in pure culture as well as mixed culture with Torulaspora quercuum X24-4, which was inoculated simultaneously or sequentially. H. osmophila inhibited the growth of T. quercuum, while T. quercuum had little effect on the growth of H. osmophila. The simultaneous fermentation consumed relatively more sugar and resulted in the highest ethanol content. The production of organic acids varied depending on the yeast species employed and inoculation modality. Esters and alcohols were the main volatile families produced during fermentation, while ethyl esters and terpenes contributed most to the temperate fruity aroma. Gas chromatography-olfactometry (GC-O) showed that 3-methyl-1-butanol, ethyl 2-methylbutanoate, phenylethyl alcohol, ß-phenethyl acetate, and ß-damascenone were the most potent odorants in all samples. This study suggested that simultaneous fermentation with H. osmophila and T. quercuum might represent a novel strategy for the future production of cider.

Enhancing wine ester biosynthesis in mixed Hanseniaspora uvarum/Saccharomyces cerevisiae fermentation by nitrogen nutrient addition.

The dynamic changes of wine ester production during mixed fermentation with Hanseniaspora uvarum Yun268 and Saccharomyces cerevisiae F5 was investigated at different levels and timings of nitrogen nutrient addition. Nitrogen additions were performed by supplementing yeast assimilable nitrogen (YAN) into a synthetic grape must with defined composition. Ester precursors and extracellular metabolites involved in ester synthesis were analyzed throughout the fermentation. Results showed that nitrogen additions covering 50-200 mg/L YAN at the point of yeast inoculation slightly affected yeast competition and ester profiles. Interestingly, when YAN was supplemented in the mid-stage, the survival of H. uvarum Yun268 was enhanced, resulting in more than a 2-fold increase in the levels of higher alcohol acetates compared to that at the initial stage. Furthermore, carbon fluxes may be redistributed in the central pathway, which contributed to the production of medium-chain fatty acids and eventually triggered a 1.2-fold elevation in corresponding ethyl ester levels.

Yeast strains as potential aroma enhancers in dry fermented sausages.

Actual healthy trends produce changes in the sensory characteristics of dry fermented sausages therefore, new strategies are needed to enhance their aroma. In particular, a reduction in the aroma characteristics was observed in reduced fat and salt dry sausages. In terms of aroma enhancing, generally coagulase-negative cocci were selected as the most important group from the endogenous microbiota in the production of flavour compounds. Among the volatile compounds analysed in dry sausages, ester compounds contribute to fruity aroma notes associated with high acceptance of traditional dry sausages. However, the origin of ester compounds in traditional dry sausages can be due to other microorganisms as lactic acid bacteria, yeast and moulds. Yeast contribution in dry fermented sausages was investigated with opposite results attributed to low yeast survival or low activity during processing. Generally, they affect sausage colour and flavour by their oxygen-scavenging and lipolytic activities in addition to, their ability to catabolize fermentation products such as lactate increasing the pH and contributing to less tangy and more aromatic sausages. Recently, the isolation and characterization of yeast from traditional dry fermented sausages made possible the selection of those with ability to produce aroma active compounds. Molecular methods were used for genetic typing of the isolated yeasts whereas their ability to produce aroma compounds was tested in different systems such as in culture media, in model systems and finally on dry fermented sausages. The results revealed that the appropriate selection of yeast strains with aroma potential may be used to improve the sensory characteristics of reformulated fermented sausages.

Impact of Debaryomyces hansenii strains inoculation on the quality of slow dry-cured fermented sausages.

Debaryomyces hansenii strains, M4 and P2, isolated from natural fermented sausages were inoculated in slow fermented sausages to study their effect on processing parameters, microbial population, volatile compound and sensory characteristics. The inoculation of D. hansenii strains, M4 and P2, did not affect the ripening process as no differences in pH and Aw were detected. The dominance of the inoculated yeast strains along the process was followed by RAPDs of M13 minisatellite. The inoculated yeasts, P2 and M4, were recovered at the end of the ripening process although P2 appeared in higher counts than M4. The sausages inoculated with P2 resulted in a decrease in lipid oxidation values (TBARS) and a reduction of lipid-oxidation derived aldehydes in addition to a highest acid compound abundance. M4 inoculated sausages resulted in highest sulphur containing compound abundance. However, no differences in consumer acceptance were detected. Moreover, both yeast strains were responsible for the generation of ethyl methyl-branched ester compounds in the dry-cured sausages.

Development of an environmentally acceptable volatile organic compounds recovery and reuse system based on vacuum spray flash with dibasic acid esters as absorbents.

We used dibasic acid esters as the absorbent for a volatile organic compounds (VOC) recovery and reuse system geared mainly toward VOCs from painting and presswork. The system consists of three elements: absorption using dibasic acid esters, separation by vacuum spray flash method, and recovery by condensation. The concentration of both ethyl acetate (EtAc) and isopropyl alcohol (IPA) decreased around 90% at room temperature. Vacuum spray flash was found as a useful process for cost-effective and energy-effective VOC removal system. The difference in the dissolubility parameters (Hansen constant) between dibasic acid esters and VOCs and the boiling point of VOCs was the main contributor to the separation behavior.

Efficient chemical synthesis of a dodecasaccharidyl lipomannan component of mycobacterial lipoarabinomannan.

Lipomannan (LM) is one of the domains of lipoarabinomannan (LAM) glycolipids, the latter being one of several cell surface organic molecules that fortify mycobacterial species against external attack. Some members of mycobacterial families are pathogenic, most notably Mycobacterium tuberculosis and Mycobacterium leprae, while others are nonpathogenic, and used in the clinic, such as Mycobacterium smegmatis. Additional biological significance arises from the fact that LM has been implicated in several health disorders outside of those associated with mycobacterial pathogens, notably for treatment of bladder cancer. LM is comprised of a heavily lipidated phosphoinositide dimannoside headgroup, from which a mannan array, of varied complexity, extends. The latter consists of a 1,6-alpha-linked backbone flanked at position O2, not necessarily regularly, with alpha-linked mannosides. This paper gives an example of lipomannan synthesis in which all of the sugar components, whether functioning as donors or acceptors, are obtained from n-pentenyl orthoesters, themselves in turn prepared in three easy steps from D-mannose. Assembly of the mannan array is facilitated by the exquisite regioselectivity occasioned by the use of ytterbium triflate/N-iodosuccinimide as the trigger for reaction of n-pentenyl orthoesters.

Heavy sulphur compounds, higher alcohols and esters production profile of Hanseniaspora uvarum and Hanseniaspora guilliermondii grown as pure and mixed cultures in grape must.

Hanseniaspora guilliermondii and Hanseniaspora uvarum were tested in grape must fermentations as pure and mixed starter cultures with Saccharomyces cerevisiae. In pure cultures, the specific growth rates found were 0.29 h(-1) for H. uvarum, 0.23 h(-1) for H. guilliermondii and 0.18 h(-1) for S. cerevisiae. No significant differences were observed between these values and those obtained in mixed cultures. Results presented in this work show that growth of apiculate yeasts during the first days of fermentation enhances the production of desirable compounds, such as esters, and may not have a negative influence on the production of higher alcohols and undesirable heavy sulphur compounds. Growth of apiculate yeasts reduced the total content of higher alcohols in wines, when compared to those produced by a pure culture of S. cerevisiae. Furthermore, the highest levels of 2-phenylethyl acetate were obtained when H. guilliermondii was inoculated in grape musts, whereas H. uvarum increased the isoamyl acetate content of wines. Apiculate yeasts produced high amounts of ethyl acetate; however, the level of this compound decreased in mixed cultures of apiculate yeasts and S. cerevisiae. When S. cerevisiae was used as a starter culture, wines showed higher concentrations of glycerol, 2-phenylethanol and ethyl hexanoate. In mixed cultures of apiculate yeasts and S. cerevisiae, wines presented amounts of methionol, acetic acid-3-(methylthio)propyl ester, 4-(methylthio)-1-butanol, 2-mercaptoethanol and cis-2-methyltetrahydro-thiophen-3-ol similar to those produced by a pure culture of S. cerevisiae. An increase in the amounts of 3-(ethylthio)-1-propanol, trans-2-methyltetrahydro-thiophen-3-ol and 3-mercapto-1-propanol was obtained in wines produced from mixed cultures with H. guilliermondii.

Investigating the function of the putative mycolic acid methyltransferase UmaA: divergence between the Mycobacterium smegmatis and Mycobacterium tuberculosis proteins.

Mycolic acids are major and specific lipid components of the cell envelope of mycobacteria that include the causative agents of tuberculosis and leprosy, Mycobacterium tuberculosis and Mycobacterium leprae, respectively. Subtle structural variations that are known to be crucial for both their virulence and the permeability of their cell envelope occur in mycolic acids. Among these are the introduction of cyclopropyl groups and methyl branches by mycolic acid S-adenosylmethionine-dependent methyltransferases (MA-MTs). While the functions of seven of the M. tuberculosis MA-MTs have been either established or strongly presumed nothing is known of the roles of the remaining umaA gene product and those of M. smegmatis MA-MTs. Mutants of the M. tuberculosis umaA gene and its putative M. smegmatis orthologue, MSMEG0913, were created. The lipid extracts of the resulting mutants were analyzed in detail using a combination of analytical techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proton nuclear magnetic resonance spectroscopy, and chemical degradation methods. The M. smegmatis mutants no longer synthesized subtypes of mycolates containing a methyl branch adjacent to either trans cyclopropyl group or trans double bond at the "proximal" position of both alpha- and epoxy-mycolates. Complementation with MSMEG0913, but not with umaA, fully restored the wild-type phenotype in M. smegmatis. Consistently, no modification was observed in the structures of mycolic acids produced by the M. tuberculosis umaA mutant. These data proved that despite their synteny and high similarity umaA and MSMEG0913 are not functionally orthologous.

Identification of trehalose dimycolate (cord factor) in Mycobacterium leprae.

Glycolipids of Mycobacterium leprae obtained from armadillo tissue nodules infected with the bacteria were analyzed. Mass spectrometric analysis of the glycolipids indicated the presence of trehalose 6,6'-dimycolate (TDM) together with trehalose 6-monomycolate (TMM) and phenolic glycolipid-I (PGL-I). The analysis showed that M. leprae-derived TDM and TMM possessed both alpha- and keto-mycolates centering at C78 in the former and at C81 or 83 in the latter subclasses, respectively. For the first time, MALDI-TOF mass analyses showed the presence of TDM in M. leprae.

Use of fumaric acid esters in psoriasis.

Fumaric acid esters (FAE) are chemical compounds derived from the unsaturated dicarbonic acid fumaric acid. The usage of FAEs in treatment of psoriasis was introduced in the late 1950's. In the 1980s more standardized oral preparations of FAEs were developed containing dimethylfumarate (DMF) and salts of monoethylfumarate (MEF) as main compounds. In 1994, Fumaderm an enteric-coated tablet containing DMF and calcium, magnesium and zinc salts of MEF was approved for the treatment of psoriasis in Germany and since then has become the most commonly used systemic therapy in this country. Fumaric acids have been proven to be an effective therapy in patients with psoriasis even though the mechanisms of action are not completely understood. About 50-70% of the patients achieve PASI 75 improvement within four months of treatment and without any long-term toxicity, immunosuppressive effects or increased risk of infection or malignancy. Tolerance is limited by gastrointestinal side effects and flushing of the skin. This article reviews pharmacokinetics, uses, contraindications, dosages and side effects of treatment with FAEs.

The dimycocerosate ester polyketide virulence factors of mycobacteria.

Recent advances in the study of mycobacterial lipids indicate that the class of outer membrane lipids known as dimycocerosate esters (DIMs) are major virulence factors of clinically relevant mycobacteria including Mycobacterium tuberculosis and Mycobacterium leprae. DIMs are a structurally intriguing class of polyketide synthase-derived wax esters discovered over seventy years ago, yet, little was known until recently about their biosynthesis. Availability of several mycobacterial genomes has accelerated progress toward clarifying steps in the DIM biosynthetic pathway and it is our belief that reviewing the bases of our current knowledge will clarify outstanding issues and help direct future endeavors.

Alcohols, esters and heavy sulphur compounds production by pure and mixed cultures of apiculate wine yeasts.

Strains of Hanseniaspora uvarum, Hanseniaspora guilliermondii and Saccharomyces cerevisiae were used as pure or mixed starter cultures in commercial medium, in order to compare their kinetic parameters and fermentation patterns. In pure and mixed cultures, yeasts presented similar ethanol yield and productivity. Pure cultures of H. uvarum and S. cerevisiae showed a specific growth rate of 0.38 h(-1); however, this value decreased when these yeasts were grown in mixed cultures with H. guilliermondii. The specific growth rate of pure cultures of H. guilliermondii was 0.41 h(-1) and was not affected by growth of other yeasts. H. guilliermondii was found to be the best producer of 2-phenylethyl acetate and 2-phenylethanol in both pure and mixed cultures. In pure cultures, H. uvarum led to the highest contents of heavy sulphur compounds, but H. guilliermondii and S. cerevisiae produced similar levels of methionol and 2-methyltetrahydrothiophen-3-one. Growth of apiculate yeasts in mixed cultures with S. cerevisiae led to amounts of 3-methylthiopropionic acid, acetic acid-3-(methylthio)propyl ester and 2-methyltetrahydrothiophen-3-one similar to those obtained in a pure culture of S. cerevisiae; however, growth of apiculate yeasts increased methionol contents of fermented media.

Release mechanism of insulin encapsulated in trehalose ester derivative microparticles delivered via inhalation.

The aim of this study was to evaluate properties of amorphous oligosaccharide ester derivative (OED) microparticles in order to determine drug release mechanisms in the lung. Trehalose OEDs with a wide range of properties were synthesised using conventional methods. The interaction of spray dried amorphous microparticles (2-3 microm) with water was investigated using attenuated total reflectance Fourier transform infra-red spectroscopy (ATR-FTIR) and dynamic vapour sorption (DVS). The in vivo performance of insulin/OED microparticles was assessed using a modified Higuchi kinetic model. A modified Hansen solvent parameter approach was used to analyse the interactions with water and in vivo trends. In water or high humidity, OED powders absorb water, lose relaxation energy and crystallise. The delay of the onset of crystallisation depends on the OED and the amount of water present. Crystallisation follows first order Arrhenius kinetics and release of insulin from OED microparticles closely matches the degree of crystallisation. The induction period depends on dispersive interactions between the OED and water while crystallisation is governed by polarity and hydrogen bonding. Drug release from OED microparticles is, therefore, controlled by crystallisation of the matrix on contact with water. The pulmonary environment was found to resemble one of high humidity rather than a liquid medium.

Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex. Evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene.

Diesters of phthiocerol and phenolphthiocerol are important virulence factors of Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans. They are both long-chain beta-diols, and their biosynthetic pathway is beginning to be elucidated. Although the two classes of molecules share a common lipid core, phthiocerol diesters have been found in all the strains of the M. tuberculosis complex examined although phenolphthiocerol diesters are produced by only a few groups of strains. To address the question of the origin of this diversity 8 reference strains and 10 clinical isolates of M. tuberculosis were analyzed. We report the presence of glycosylated p-hydroxybenzoic acid methyl esters, structurally related to the type-specific phenolphthiocerol glycolipids, in the culture media of all reference strains of M. tuberculosis, suggesting that the strains devoid of phenolphthiocerol derivatives are unable to elongate the putative p-hydroxybenzoic acid precursor. We also show that all the strains of M. tuberculosis examined and deficient in the production of phenolphthiocerol derivatives are natural mutants with a frameshift mutation in pks15/1 whereas a single open reading frame for pks15/1 is found in Mycobacterium bovis BCG, M. leprae, and strains of M. tuberculosis that produce phenolphthiocerol derivatives. Complementation of the H37Rv strain of M. tuberculosis, which is devoid of phenolphthiocerol derivatives, with the fused pks15/1 gene from M. bovis BCG restored phenolphthiocerol glycolipids production. Conversely, disruption of the pks15/1 gene in M. bovis BCG led to the abolition of the synthesis of type-specific phenolphthiocerol glycolipid. These data indicate that Pks15/1 is involved in the elongation of p-hydroxybenzoic acid to give p-hydroxyphenylalkanoates, which in turn are converted, presumably by the PpsA-E synthase, to phenolphthiocerol derivatives.

A robust structure-activity relationship (SAR) model for esters that cause skin irritation in humans.

A structure-activity relationship (SAR) model has been developed to discriminate skin irritant from nonirritant esters. The model is based on the physicochemical properties of 42 esters that were tested in humans for skin irritation. Nineteen physicochemical parameters that represent transport, electronic, and steric properties were calculated for each chemical. Best subsets regression analysis indicated candidate models for further analysis. Regression analyses identified significant models (p < 0.05) that had variables that were also significant (p < 0.05). These candidate models were evaluated using linear discriminant analysis to determine if the irritant esters could be discriminated from nonirritant esters. The stability of the model was evident from the consistency of parameters among ten submodels generated using multiple random sampling of the database. The sensitivity of the ten models, evaluated by "leave-one-out" cross-validation, ranged from 0. 846 to 0.923, with a mean of 0.885 +/- 0.025 (95% CI). The specificity ranged from 0.615 to 0.923, with a mean of 0.738 +/- 0.06 (CI). Compared with nonirritant esters, irritant esters had lower density, lower water solubility, lower sum of partial positive charges, higher Hansen hydrogen bonding parameter, and higher Hansen dispersion parameter. The results indicate that physicochemical features of esters contribute to their ability to cause skin irritation in humans, and that chemical partitioning into the epidermis and intermolecular reactions are likely important components of the response. This model is applicable for prediction of human irritation of esters yet untested.

Location of the mycolyl ester substituents in the cell walls of mycobacteria.

The question of the precise location of mycolic acids, the single most distinctive cell wall entity of members of the Mycobacterium genus, has now been addressed. The free hydroxyl functions of the arabinogalactan component of the mycobacterial cell wall were O-methylated under conditions in which the mycolyl esters were not cleaved. Subsequent replacement of the mycolyl functions with O-ethyl groups resulted in an acid- and base-stable differentially O-alkylated surrogate polysaccharide, more amenable to analysis. Complete hydrolysis, reduction, acetylation, and gas chromatography/mass spectrometry revealed the unexpected finding that the mycolyl substituents were selectively and equally distributed on the 5-hydroxyl functions of terminal- and 2-linked arabinofuranosyl (Araf) residues. Further analysis of the O-alkylated cell wall through partial acid hydrolysis, NaB[2H]4 reduction, pentadeuterioethylation, and gas chromatography/mass spectrometry demonstrated that the mycolyl units are clustered in groups of four on the previously recognized nonreducing terminal pentaarabinosyl unit [beta-Araf-(1----2)-alpha-Araf)2-3, 5-alpha-Araf. However, only about two-thirds of the available pentasaccharide units are so substituted. Thus, the antigenicity of the arabinan component of mycobacterial cell walls may be explained by the fact that about one-third of the pentaarabinosyl units are not mycolyated and are available for interaction with the immune system. On the other hand, the extreme hydrophobicity and impenetrability of the mycobacterial cell may be explained by the same motif also acting as the fulerum for massive esterified paraffin residues. New fundamental information on the structure of mycobacterial cell walls will aid in our comprehension of its impenetrability to antibiotics and role in immunopathogenesis and persistence of disease.