Abel Hiol

Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit

We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.

Production, purification and characterization of an extracellular lipase from Mucor hiemalis f. hiemalis

Abstract Mucor hiemalis f. hiemalis is a major contaminant of cameroonian palm fruit and produces an inducible extracellular lipase in batch fermentation. Rape oil was the best inducer for enzyme production, with the highest activity being achieved after 6 days of incubation. The enzyme was purified 2200-fold by ultrafiltration, ammonium sulfate fractionation, Sephadex G75 chromatography, Q-Sepharose chromatography, and Sephacryl S-200 chromatography. The purified enzyme showed a prominent polypeptide band in polyacrylamide gel electrophoresis, associated with esterase activity according to activity staining. Molecular weight of the lipase was estimated to be 49 kDa using gel filtration on Sephadex G75, and 49 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was identified as a glycoprotein with pI of 4.6. The N-terminal amino acid sequence data (19 residues) and the amino acid composition were determined. The optimum pH and temperature for activity of the enzyme were 7.0 and 40°C, respectively. The lipase was stable in the pH range of 4–9 and at 45°C for 15 min. It hydrolyzed both synthetic and natural triglycerides with optimal activities recorded on tricaprylin and rape oil, respectively. Ca 2+ , Mg 2+ , Co 2+ , Mn 2+ , and Na + -enhanced lipase activity, whereas Fe 2+ , Cu 2+ , Ba 2+ , and surfactants—such as taurocholic acid, triton X-100, and Tween 20—strongly reduced lipase activity. The enzyme activity was not affected by EDTA (ethylenediaminetetraacetic acid disodium dihydrate), PMSF (phenylmethylsulfonylfluoride), (p-chloromercuribenzoic), and Benzamidine.

Metabolomic and Lipidomic Analysis of Serum Samples following Curcuma longa Extract Supplementation in High-Fructose and Saturated Fat Fed Rats.

We explored, using nuclear magnetic resonance (NMR) metabolomics and fatty acids profiling, the effects of a common nutritional complement, Curcuma longa, at a nutritionally relevant dose with human use, administered in conjunction with an unbalanced diet. Indeed, traditional food supplements have been long used to counter metabolic impairments induced by unbalanced diets. Here, rats were fed either a standard diet, a high level of fructose and saturated fatty acid (HFS) diet, a diet common to western countries and that certainly contributes to the epidemic of insulin resistance (IR) syndrome, or a HFS diet with a Curcuma longa extract (1% of curcuminoids in the extract) for ten weeks. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) on the serum NMR profiles and fatty acid composition (determined by GC/MS) showed a clear discrimination between HFS groups and controls. This discrimination involved metabolites such as glucose, amino acids, pyruvate, creatine, phosphocholine/glycerophosphocholine, ketone bodies and glycoproteins as well as an increase of monounsaturated fatty acids (MUFAs) and a decrease of n-6 and n-3 polyunsaturated fatty acids (PUFAs). Although the administration of Curcuma longa did not prevent the observed increase of glucose, triglycerides, cholesterol and insulin levels, discriminating metabolites were observed between groups fed HFS alone or with addition of a Curcuma longa extract, namely some MUFA and n-3 PUFA, glycoproteins, glutamine, and methanol, suggesting that curcuminoids may act respectively on the fatty acid metabolism, the hexosamine biosynthesis pathway and alcohol oxidation. Curcuma longa extract supplementation appears to be beneficial in these metabolic pathways in rats. This metabolomic approach highlights important serum metabolites that could help in understanding further the metabolic mechanisms leading to IR.

Application of Immobilized Lipase from Candida rugosa to Synthesis of Cholesterol Oleate

Candida rugosa lipase (CRL) has been immobilized on two kinds of ion-exchange resins, Duolite A 568 and Amberlite IRC 50. These preparations were investigated as a tool for the production of cholesterol oleate in organic media. An increase in temperature up to 40°C increased the rate of reaction and improved the final ester yield. Under optimal conditions, the reaction yield was followed as a time function, for both lipase preparations with an initial water content of 20%. Then, it was observed that about 78% of the oleic acid was esterified after 10 h using CRL immobilized on Duolite, whereas 73% synthesis of cholesterol oleate was reached with CRL immobilized on Amberlite, for the same incubation time. Also, a difference in reaction yield was noticed for the preparations containing sorbitol. In fact, sorbitol treatment might improve the activity of immobilized lipase by preserving the watershell around the catalyst and by increasing the accessibility of the active site to the substrates. In this way, the reaction yield was enhanced, and an increase of 10% synthesis of cholesterol oleate was obtained in both cases.

Neuronal Conduction of Excitation without Action Potentials Based on Ceramide Production

Background Action potentials are the classic mechanism by which neurons convey a state of excitation throughout their length, leading, after synaptic transmission, to the activation of other neurons and consequently to network functioning. Using an in vitro integrated model, we found previously that peripheral networks in the autonomic nervous system can organise an unconventional regulatory reflex of the digestive tract motility without action potentials. Methodology/Principal Findings In this report, we used combined neuropharmacological and biochemical approaches to elucidate some steps of the mechanism that conveys excitation along the nerves fibres without action potentials. This mechanism requires the production of ceramide in membrane lipid rafts, which triggers in the cytoplasm an increase in intracellular calcium concentration, followed by activation of a neuronal nitric oxide synthase leading to local production of nitric oxide, and then to guanosine cyclic monophosphate. This sequence of second messengers is activated in cascade from rafts to rafts to ensure conduction of the excitation along the nerve fibres. Conclusions/Significance Our results indicate that second messengers are involved in neuronal conduction of excitation without action potentials. This mechanism represents the first evidence—to our knowledge—that excitation is carried along nerves independently of electrical signals. This unexpected ceramide-based conduction of excitation without action potentials along the autonomic nerve fibres opens up new prospects in our understanding of neuronal functioning.

Long-term high fructose and saturated fat diet affects plasma fatty acid profile in rats

As the consumption of fructose and saturated fatty acids (FAs) has greatly increased in western diets and is linked with an increased risk of metabolic syndrome, the aim of this study was to investigate the effects of a moderate (10 weeks) and a prolonged (30 weeks) high fructose and saturated fatty acid (HFS) diet on plasma FA composition in rats. The effects of a few weeks of HFS diet had already been described, but in this paper we tried to establish whether these effects persist or if they are modified after 10 or 30 weeks. We hypothesized that the plasma FA profile would be altered between 10 and 30 weeks of the HFS diet. Rats fed with either the HFS or a standard diet were tested after 10 weeks and again after 30 weeks. After 10 weeks of feeding, HFS-fed rats developed the metabolic syndrome, as manifested by an increase in fasting insulinemia, total cholesterol and triglyceride levels, as well as by impaired glucose tolerance. Furthermore, the plasma FA profile of the HFS group showed higher proportions of monounsaturated FAs like palmitoleic acid [16:1(n-7)] and oleic acid [18:1(n-9)], whereas the proportions of some polyunsaturated n-6 FAs, such as linoleic acid [18:2(n-6)] and arachidonic acid [20:4(n-6)], were lower than those in the control group. After 30 weeks of the HFS diet, we observed changes mainly in the levels of 16:1(n-7) (decreased) and 20:4(n-6) (increased). Together, our results suggest that an HFS diet could lead to an adaptive response of the plasma FA profile over time, in association with the development of the metabolic syndrome.

Effect of physiological harvest stages on the composition of bioactive compounds in Cavendish bananas

The combined influence of maturation, ripening, and climate on the profile of bioactive compounds was studied in banana (Musa acuminata, AAA, Cavendish, cv. Grande Naine). Their bioactive compounds were determined by the Folin-Ciocalteu assay and high-performance thin layer chromatographic (HPTLC) method. The polyphenol content of bananas harvested after 400 degree days remained unchanged during ripening, while bananas harvested after 600 and 900 degree days exhibited a significant polyphenol increase. Although dopamine was the polyphenol with the highest concentration in banana peels during the green developmental stage and ripening, its kinetics differed from the total polyphenol profile. Our results showed that this matrix of choice (maturation, ripening, and climate) may allow selection of the banana (M. acuminata, AAA, Cavendish, cv. Grande Naine) status that will produce optimal concentrations of identified compounds with human health relevance.

Hepatic metabolic effects of Curcuma longa extract supplement in high-fructose and saturated fat fed rats

The metabolic effects of an oral supplementation with a Curcuma longa extract, at a dose nutritionally relevant with common human use, on hepatic metabolism in rats fed a high fructose and saturated fatty acid (HFS) diet was evaluated. High-resolution magic-angle spinning NMR and GC/MS in combination with multivariate analysis have been employed to characterize the NMR metabolite profiles and fatty acid composition of liver tissue respectively. The results showed a clear discrimination between HFS groups and controls involving metabolites such as glucose, glycogen, amino acids, acetate, choline, lysophosphatidylcholine, phosphatidylethanolamine, and β-hydroxybutyrate as well as an increase of MUFAs and a decrease of n-6 and n-3 PUFAs. Although the administration of CL did not counteract deleterious effects of the HFS diet, some metabolites, namely some n-6 PUFA and n-3 PUFA, and betaine were found to increase significantly in liver samples from rats having received extract of curcuma compared to those fed the HFS diet alone. This result suggests that curcuminoids may affect the transmethylation pathway and/or osmotic regulation. CL extract supplementation in rats appears to increase some of the natural defences preventing the development of fatty liver by acting on the choline metabolism to increase fat export from the liver.

Processed Z. Mauritiana Lamk in the Formula of High Nutritional Value Cake

The nutritional value of jujube fruits Ziziphus mauritiana Lamk was processed through an optimized traditional cake procedure. The characteristics of jujube fruit polysaccharides from an accession known as P3 were determined for each of the 5 ripening stages. Therefore, the content of the Alcohol Insoluble Materials, Water Soluble Polysaccharide and Galacturonic Acid was determined at each ripening stage. The degree of methylation (DM) of jujube pectins was less than 50% therefore was classified as low methoxylated pectin (LM). Using the 3rd and the 5th ripening stage, the impact of the drying and cooking was evaluated on selected nutritional characteristics, including vitamin C, total phenolics content and antioxidant capacity. Remarkably, using the fruits from the 3rd stage, the drying process decreased the vitamin C content (74.5%, p<0.05) whereas an increase of 20% (p<0.05) was observed for the cake. Interestingly, the antioxidant activity was unchanged during the drying process. In contrast, after the cooking process the phenolics content and the antioxidant capacity had both increased, by 64% and 30% (p<0.05) respectively. Overall, our results indicated that stage 3 fruits would exhibit higher nutritional qualities than stage 5 fruits. We strongly recommend stage 3 fruits of accession P3 for food applications, including jujube cake processing.