Cédric Paris

Formulation, characterization and pharmacokinetic studies of coenzyme Q10 PUFA’s nanoemulsions

Coenzyme Q(10) (CoQ(10)) is an insoluble antioxidant molecule with great biological value but exhibit poor bioavailability. To improve the bioavailability of CoQ(10), we have proposed to formulate a nanoemulsion consisting of salmon oil, salmon lecithin, CoQ(10) and water. A commercial oily mixture, based on soybean oil and CoQ(10), was used for comparison, as well as a second oily mixture, composed of salmon lecithin, salmon oil and CoQ(10). Salmon oil and salmon lecithin were used as sources of polyunsaturated fatty acids (PUFA). The maximum solubility of CoQ(10) in salmon oil was 81.30 ± 0.08 mg/mL at 37 °C. Mean droplets size of the control and CoQ(10) nanoemulsions was 164 and 167 nm, respectively. The nanoemulsion was stable during 30 days at 25 °C. Bioavailability was evaluated as the area under the curve of CoQ(10) plasma concentration in male Wistar rats following oral administration of the three formulations of CoQ(10). The nanoemulsion increases at twice the bioavailability of CoQ(10) than conventional oily formulations regardless the nature of used fatty acids (soybean and salmon oils). Prepared nanoemulsion represents a vectorization of both LC-PUFAs and CoQ(10). That could be an interesting way to increase the absorption of these two bioactive molecules with natural low availability.

Antioxidant capacity and light-aging study of HPMC films functionalized with natural plant extract

The aims of this work were to functionalize edible hydroxypropyl methylcellulose (HPMC) films with natural coloring biomolecules having antioxidant capacity and to study their photo-aging stability in the films. HPMC films containing a natural red color compound (NRC) at the level of 1, 2, 3 or 4% (v/v) were prepared by a casting method. A slight degradation of films color was observed after 20 days of continuous light exposure. The antioxidant activity of NRC incorporated films was stable during different steps of film formation and 20 days of dark storage. On the other hand, antioxidant activity of samples stored under light was significantly affected after 20 days. FTIR (Fourier Transformed Infrared) spectroscopy was used to characterize the new phenolic polymeric structures and to study the photo-degradation of films. The results showed a good polymerization phenomenon between NRC and HPMC in polymer matrix giving a natural color to the films. NRC showed an ability to protect pure HPMC films against photo-degradation. This phenomenon was directly proportional to the concentration of NRC.

Laccase-catalysed oxidation of ferulic acid and ethyl ferulate in aqueous medium: a green procedure for the synthesis of new compounds.

The enzymatic oxidation of ferulic acid (FA) and ethyl ferulate (EF) with Myceliophthora thermophila laccase, as biocatalyst, was performed in aqueous medium using an eco-friendly procedure to synthesize new active molecules. First, the commercial laccase was ultrafiltrated allowing for the elimination of phenolic contaminants and increasing the specific activity by a factor of 2. Then, kinetic parameters of this laccase were determined for both substrates (FA, EF), indicating a higher substrate affinity for ethyl ferulate. Additionally, enzymatic oxidation led to the synthesis of a FA-major product, exhibiting a molecular mass of 386 g/mol and a EF-major product with a molecular mass of 442 g/mol. Structural analyses by mass spectrometry allowed the identification of dimeric derivatives. The optical properties of the oxidation products showed the increase of red and yellow colours, with FA-products compared to EF-products. Additionally, enzymatic oxidation led to a decrease of antioxidant and cytotoxic activities compared to initial substrates. Consequently, this enzymatic procedure in aqueous medium could provide new compounds presenting optical, antioxidant and cytotoxic interest.

Highly sensitive, quick and simple quantification method for mono and disaccharides in aqueous media using liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS).

A highly sensitive, rapid LC-APCI-MS method for identification and quantification of mono and disaccharides in simple or complex aqueous phase has been developed. This original method is easy to use, no derivation and no post-column injection are needed. The separation is performed with a hydrophilic amino interaction (HILIC) column allowing high-throughput analysis with analysis times of 15 min for monosaccharides to 22 min for disaccharides. The development of the method carried out with 9 standard saccharides allowed to point out a dynamic range from 0.1-25.6 to 1-256 μg mL(-1) depending on the considered sugar. Next, the method was validated on saccharides at known concentrations in water and on 2 real samples: orange juice and aqueous phase obtained after enzymatic hydrolysis of sunflower seeds.

Phenolic characterization and variability in leaves, stems and roots of Micro-Tom and patio tomatoes, in response to nitrogen limitation

Phenolics are implicated in the defence strategies of many plant species rendering their concentration increase of putative practical interest in the field of crop protection. Little attention has been given to the nature, concentration and distribution of phenolics within vegetative organs of tomato (Solanum lycopersicum. L) as compared to fruits. In this study, we extensively characterized the phenolics in leaves, stems and roots of nine tomato cultivars using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-MS(n)) and assessed the impact of low nitrogen (LN) availability on their accumulation. Thirty-one phenolics from the four sub-classes, hydroxycinnamoyl esters, flavonoids, anthocyanins and phenolamides were identified, five of which had not previously been reported in these tomato organs. A higher diversity and concentration of phenolics was found in leaves than in stems and roots. The qualitative distribution of these compounds between plant organs was similar for the nine cultivars with the exception of Micro-Tom because of its significantly higher phenolic concentrations in leaves and stems as compared to roots. With few exceptions, the influence of the LN treatment on the three organs of all cultivars was to increase the concentrations of hydroxycinnamoyl esters, flavonoids and anthocyanins and to decrease those of phenolamides. This impact of LN was greater in roots than in leaves and stems. Nitrogen nutrition thus appears as a means of modulating the concentration and composition of organ phenolics and their distribution within the whole plant.

A single Sfp-type Phosphopantetheinyl transferase plays a major role in the biosynthesis of PKS and NRPS derived metabolites in Streptomyces ambofaciens ATCC23877

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones.

Evaluating Ketoreductase Exchanges as a Means of Rationally Altering Polyketide Stereochemistry

Modular polyketide synthases (PKSs) are multidomain multienzymes responsible for the biosynthesis in bacteria of a wide range of polyketide secondary metabolites of clinical value. The stereochemistry of these molecules is an attractive target for genetic engineering in attempts to produce analogues exhibiting novel therapeutic properties. The exchange of ketoreductase (KR) domains in model PKSs has been shown in several cases to predictably alter the configuration of the β‐hydroxy functionalities but not of the α‐methyl groups. By systematic screening of a broad panel of KR domains, we have identified two donor KRs that afford modification of α‐methyl group stereochemistry. To the best of our knowledge, this provides the first direct in vivo evidence of KR‐catalyzed epimerization. However, none of the introduced KRs afforded simultaneous alteration of methyl and hydroxy configurations in high yield. Therefore, swapping of whole modules might be necessary to achieve such changes in stereochemistry.

Concentration and selective fractionation of an antihypertensive peptide from an alfalfa white proteins hydrolysate by mixed ion-exchange centrifugal partition chromatography

This article reports a promising use of the mixed ion-exchange centrifugal partition chromatography (MIXCPC) technique in the field of downstream processes. A complex alfalfa white protein concentrate hydrolysate (AWPC hydrolysate) showing anti-hypertensive properties was successfully fractionated by MIXCPC to yield a L-valyl-L-tryptophan (VW) enriched fraction in one run. This dipeptide shows an interesting anti-angiotensin converting enzyme (anti-ACE) activity. An analytical method based on RP-LC/MS-MS was developed to quantify the target VW peptide in both the starting material and the enriched fractions. The best results for the MIXCPC fractionation were obtained by the combined use of the quaternary biphasic solvent system, methyl-tert-butylether/acetonitrile/n-butanol/water (2:1:2:5, v/v) in the descending mode, of the lipophilic di(2-ethylhexyl)phosphoric acid (DEHPA) cation-exchanger with an exchanger (DEHPA)/peptides ratio of 15, and of two displacers: calcium chloride and hydrochloric acid. The complexity of the starting material involved the selectivity optimization by splitting the stationary phase into two sections that differed by their triethylamine concentration. From 1g of AWPC hydrolysate containing 0.26% of VW, 30.7 mg of a VW enriched fraction were recovered with a purity of 10.9%, corresponding to a purification factor of 41 and a recovery of 97%.

Identification of metabolic pathways involved in the biosynthesis of flavor compound 3-methylbutanal from leucine catabolism by Carnobacterium maltaromaticum LMA 28

Carnobacterium maltaromaticum strains are widely found in food including fish, meat and some dairy products. Producing a malty/chocolate like aroma due to 3-methylbutanal from the catabolism of leucine is a general characteristic of this species. In this study, we investigated metabolic routes responsible for the biosynthesis of this flavor compound from the catabolism of leucine in C. maltaromaticum LMA 28, a strain isolated from mold ripened soft cheese. Depending on the lactic acid bacterium, leucine can be converted into 3-methylbutanal following two possible metabolic pathways: either directly by α-ketoacid decarboxylase (KADC) pathway or indirectly by α-ketoacid dehydrogenase (KADH) pathway. Both KADC (41.0±3.0 nmol/mg protein/min) and KADH (1.43±0.62 nmol/mg protein/min) activities were detected and determined in vitro in C. maltaromaticum LMA 28. C. maltaromaticum LMA 28 slightly reduced the production of 3-methylbutanal from leucine in the presence of a specific inhibitor of KADH enzyme complex, i.e. sodium meta-arsenite, suggesting that both pathways were involved in vivo in leucine catabolism. Moreover the presence of genes encoding aminotransferase, glutamate dehydrogenase, α-ketoacid decarboxylase, α-ketoacid dehydrogenase and aldehyde dehydrogenase was confirmed. C. maltaromaticum is then the first lactic acid bacterium in which presence of both metabolic routes responsible for the biosynthesis of 3-methylbutanal from leucine catabolism was confirmed in vitro and in vivo as well.

Fluorescent labeling of nisin Z and assessment of anti-listerial action

Biomolecule labeling by fluorescent markers has emerged as an innovative methodology for bio-analytical purposes in food microbiology, medicine and pharmaceutics due to the great advantages of this method such as precision, wide detection limits, and in vivo recognition. Fluorescent nisin Z was synthesized by linking the carboxyl group and amino group of nisin Z and 5-aminoacetamido fluorescein (AAA-flu). This new structure was fully characterized by mass spectrometry with a molecular weight of 3717.3 Da. Intracellular K(+) leakage and transmembrane electrical potential (Δψ) were used to evaluate the antibacterial action of the labeled molecule against three listerial strains and demonstrated that nisin Z endured the labeling process without any activity loss. In vivo activity of labeled nisin was observed by confocal laser microscope which revealed its localization at the septum of listerial cell division site where the membrane-bound cell wall precursor lipid II is maximal. Fluorescent nisin Z showed its great potential as a tool to study antibacterial mechanism of action of nisin in biological systems.