Screening for Volatile α-Unsaturated Ester-Producing Yeasts from the Feces of Wild Animals in South Africa.

α-unsaturated esters are fruity-aromatic compounds which are largely spread in the volatilome of many different fruits, but they are rarely found in the volatilome of yeasts. The yeast S. suaveolens has been recently shown to produce relatively high amounts of α-unsaturated esters and it appears to be an interesting model for the production of these compounds. This study aimed to isolate new α-unsaturated ester-producing yeasts by focusing on strains displaying a similar metabolism to S. suaveolens. While the production of α-unsaturated esters by S. suaveolens is believed to be closely related to its ability to grow on media containing branched-chain amino acids (isoleucine, leucine and valine) as the sole carbon source (ILV+ phenotype), in this study, an original screening method was developed that selects for yeast strains displaying ILV+ phenotypes and is able to produce α-unsaturated esters. Among the 119 yeast strains isolated from the feces of 42 different South African wild animal species, 43 isolates showed the ILV+ phenotype, among which 12 strains were able to produce α-unsaturated esters. Two interesting α-unsaturated esters were detected in two freshly isolated strains, both identified as Galactomyces candidus. These new esters were detected neither in the volatilome of the reference strain S. suaveolens, nor in any other yeast species previously studied for their aroma production. This work demonstrated the efficiency of an original method to rapidly screen for α-unsaturated ester-producing yeasts. In addition, it demonstrated that wild animal feces are interesting resources to isolate novel strains producing compounds with original aromas.

OVAT Analysis and Response Surface Methodology Based on Nutrient Sources for Optimization of Pigment Production in the Marine-Derived Fungus Talaromyces albobiverticillius 30548 Submerged Fermentation.

Pigment production from filamentous fungi is gaining interest due to the diversity of fungal species, the variety of compounds synthesized, and the possibility of controlled massive productions. The Talaromyces species produce a large panel of metabolites, including Monascus-like azaphilone pigments, with potential use as natural colorants in industrial applications. Optimizing pigment production from fungal strains grown on different carbon and nitrogen sources, using statistical methods, is widespread nowadays. The present work is the first in an attempt to optimize pigments production in a culture of the marine-derived T. albobiverticillius 30548, under the influence of several nutrients sources. Nutrient combinations were screened through the one-variable-at-a-time (OVAT) analysis. Sucrose combined with yeast extract provided a maximum yield of orange pigments (OPY) and red pigments (RPY) (respectively, 1.39 g/L quinizarin equivalent and 2.44 g/L Red Yeast pigment equivalent), as well as higher dry biomass (DBW) (6.60 g/L). Significant medium components (yeast extract, K2HPO4 and MgSO4·7H2O) were also identified from one-variable-at-a-time (OVAT) analysis for pigment and biomass production. A five-level central composite design (CCD) and a response surface methodology (RSM) were applied to evaluate the optimal concentrations and interactive effects between selected nutrients. The experimental results were well fitted with the chosen statistical model. The predicted maximum response for OPY (1.43 g/L), RPY (2.59 g/L), and DBW (15.98 g/L) were obtained at 3 g/L yeast extract, 1 g/L K2HPO4, and 0.2 g/L MgSO4·7H2O. Such optimization is of great significance for the selection of key nutrients and their concentrations in order to increase the pigment production at a pilot or industrial scale.

Evaluation of mixed-fermentation of Saccharomyces cerevisiae with Saprochaete suaveolens to produce natural fruity beer from industrial wort.

Fruity beers can be promoted through production of flavoring compounds during fermentation by partial replacement of brewing yeast by non-conventional-yeasts with high aroma production abilities. We evaluated here the use of a wild Saprochaete suaveolens strain, producing atypical aroma compounds, to produce new natural fruity beer, while keeping classical production conditions used in brewing industry. S. suaveolens was inoculated as starter of culture during beer fermentation and the fermentation performance was evaluated through measurement of several physicochemical parameters. The aroma profile of the engineered beers was monitored using HS-SPME GC/MS. The results showed that high fruity aroma and low-ethanol content beers were obtained through single-fermentation using S. suaveolens. We also demonstrated that during mixed-fermentation, S. suaveolens maintained high metabolic activity and allowed production of beer enriched with fruity aroma. Production of high or low ethanol content fruity beer could be achieved by varying the composition of the starter of culture.

Evaluation of Filamentous Fungi and Yeasts for the Biodegradation of Sugarcane Distillery Wastewater.

Sugarcane Distillery Spent Wash (DSW) is among the most pollutant industrial effluents, generally characterized by high Chemical Oxygen Demand (COD), high mineral matters and acidic pH, causing strong environmental impacts. Bioremediation is considered to be a good and cheap alternative to DSW treatment. In this study, 37 strains of yeasts and filamentous fungi were performed to assess their potential to significantly reduce four parameters characterizing the organic load of vinasses (COD, pH, minerals and OD475nm). In all cases, a pH increase (until a final pH higher than 8.5, being an increase superior to 3.5 units, as compared to initial pH) and a COD and minerals removal could be observed, respectively (until 76.53% using Aspergillus terreus var. africanus and 77.57% using Aspergillus niger). Depending on the microorganism, the OD475nm could decrease (generally when filamentous fungi were used) or increase (generally when yeasts were used). Among the strains tested, the species from Aspergillus and Trametes genus offered the best results in the depollution of DSW. Concomitant with the pollutant load removal, fungal biomass, with yields exceeding 20 g·L-1, was produced.

Statistical Optimization of the Physico-Chemical Parameters for Pigment Production in Submerged Fermentation of Talaromyces albobiverticillius 30548.

Talaromyces albobiverticillius 30548 is a marine-derived pigment producing filamentous fungus, isolated from the La Réunion island, in the Indian Ocean. The objective of this study was to examine and optimize the submerged fermentation (SmF) process parameters such as initial pH (4-9), temperature (21-27 °C), agitation speed (100-200 rpm), and fermentation time (0-336 h), for maximum production of pigments (orange and red) and biomass, using the Box-Behnken Experimental Design and Response Surface Modeling (BBED and RSM). This methodology allowed consideration of multifactorial interactions between a set of parameters. Experiments were carried out based on the BBED using 250 mL shake flasks, with a 100 mL working volume of potato dextrose broth (PDB). From the experimental data, mathematical models were developed to predict the pigments and biomass yields. The individual and interactive effects of the process variables on the responses were also investigated (RSM). The optimal conditions for maximum production of pigments and biomass were derived by the numerical optimization method, as follows-initial pH of 6.4, temperature of 24 °C, agitation speed of 164 rpm, and fermentation time of 149 h, respectively.

Production of Aspergillus niger biomass on sugarcane distillery wastewater: physiological aspects and potential for biodiesel production.

BACKGROUND: Sugarcane distillery waste water (SDW) or vinasse is the residual liquid waste generated during sugarcane molasses fermentation and alcohol distillation. Worldwide, this effluent is responsible for serious environmental issues. In Reunion Island, between 100 and 200 thousand tons of SDW are produced each year by the three local distilleries. In this study, the potential of Aspergillus niger to reduce the pollution load of SDW and to produce interesting metabolites has been investigated. RESULTS: The fungal biomass yield was 35 g L-1 corresponding to a yield of 0.47 g of biomass/g of vinasse without nutrient complementation. Analysis of sugar consumption indicated that mono-carbohydrates were initially released from residual polysaccharides and then gradually consumed until complete exhaustion. The high biomass yield likely arises from polysaccharides that are hydrolysed prior to be assimilated as monosaccharides and from organic acids and other complex compounds that provided additional C-sources for growth. Comparison of the size exclusion chromatography profiles of raw and pre-treated vinasse confirmed the conversion of humic- and/or phenolic-like molecules into protein-like metabolites. As a consequence, chemical oxygen demand of vinasse decreased by 53%. Interestingly, analysis of intracellular lipids of the biomass revealed high content in oleic acid and physical properties relevant for biodiesel application. CONCLUSIONS: The soft-rot fungus A. niger demonstrated a great ability to grow on vinasse and to degrade this complex and hostile medium. The high biomass production is accompanied by a utilization of carbon sources like residual carbohydrates, organic acids and more complex molecules such as melanoidins. We also showed that intracellular lipids from fungal biomass can efficiently be exploited into biodiesel.

Flavour production by Saprochaete and Geotrichum yeasts and their close relatives.

In this study, a total of 30 yeast strains belonging to the genera Dipodascus, Galactomyces, Geotrichum, Magnusiomyces and Saprochaete were investigated for volatile organic compound production using HS-SPME-GC/MS analysis. The resulting flavour profiles, including 36 esters and 6 alcohols compounds, were statistically evaluated by cluster and PCA analysis. Two main groups of strains were extracted from this analysis, namely a group with a low ability to produce flavour and a group producing mainly alcohols. Two other minor groups of strains including Saprochaete suaveolens, Geotrichum marinum and Saprochaete gigas were diverging significantly from the main groups precisely because they showed a good ability to produce a large diversity of esters. In particular, we found that the Saprochaete genus (and their closed relatives) was characterized by a high production of unsaturated esters arising from partial catabolism of branched chain amino-acids. These esters were produced by eight phylogenetically related strains of Saprochaete genus.

Contribution of trans-aconitic acid to DPPH scavenging ability in different media.

The antioxidant properties of trans-aconitic acid (TAA) alone or in the presence of usual antioxidants were assessed by DPPH assay. The IC50 value equal to 70mM was very high compared to usual antioxidants (vitamin C and trolox). A joint experimental/theoretical study suggested that hydrogen atom abstraction in TAA by DPPH was located on -CH2- methylene bridge because the corresponding radical was more stabilized than COO(·) and CC(·) radicals. In combination with antioxidants (vitamin C, gallic acid, caffeic acid, trolox), synergy or additivity effects were noticed. The magnitude of the synergistic effect varied between 1.06 and 1.24 depending on the type and concentration of antioxidant for a concentration of TAA equal to 22.3mM. Especially, the addition of TAA at a concentration below 32mM to a solution containing 20µM of vitamin C had a synergy effect. Beyond this concentration, TAA showed an additive effect.

A comparative study on the potential of epiphytic yeasts isolated from tropical fruits to produce flavoring compounds.

In recent years, there has been an increasing interest in identifying and characterizing the yeast flora associated with diverse types of habitat because of the many potential desirable technological properties of these microorganisms, especially in food applications. In this study, a total of 101 yeast strains were isolated from the skins of tropical fruits collected in several locations in the South West Indian Ocean. Sequence analysis of the D1/D2 domains of the large subunit (LSU) ribosomal RNA gene identified 26 different species. Among them, two species isolated from the skins of Cape gooseberry and cocoa beans appeared to represent putative new yeast species, as their LSU D1/D2 sequence was only 97.1% and 97.4% identical to that of the yeasts Rhodotorula mucilaginosa and Candida pararugosa, respectively. A total of 52 Volatile Organic Compounds (VOCs) were detected by Head Space Solid Phase Micro Extraction coupled to Gas Chromatography and Mass Spectroscopy (HS-SPME-GC/MS) from the 26 yeast species cultivated on a glucose rich medium. Among these VOCs, 6 uncommon compounds were identified, namely ethyl but-2-enoate, ethyl 2-methylbut-2-enoate (ethyl tiglate), ethyl 3-methylbut-2-enoate, 2-methylpropyl 2-methylbut-2-enoate, butyl 2-methylbut-2-enoate and 3-methylbutyl 2-methylbut-2-enoate, making them possible yeast species-specific markers. In addition, statistical methods such as Principal Component Analysis allowed to associate each yeast species with a specific flavor profile. Among them, Saprochaete suaveolens (syn: Geotrichum fragrans) turned to be the best producer of flavor compounds, with a total of 32 out of the 52 identified VOCs in its flavor profile.

Physiological and biochemical characteristics of the ethyl tiglate production pathway in the yeast Saprochaete suaveolens.

A yeast identified as Saprochaete suaveolens was investigated for its capacity to produce a large panel of flavouring molecules. With a production of 32 compounds including 28 esters, S. suaveolens seems to be a good producer of fruity flavours and fragrances and especially of unsaturated esters, such as ethyl tiglate. Physiological and biochemical analyses were performed in this study in an attempt to comprehend the metabolic route to the formation of this compound. We show that the accumulation of ethyl tiglate by S. suaveolens is specifically induced by isoleucine. However, and contrary to S. cerevisiae, which harbours a classical Ehrlich pathway leading to the production of 2-methylbutanol from isoleucine, our results provide phenotypic and enzymological evidence of ethyl tiglate biosynthesis in S. suaveolens through the catabolism of this amino acid by the ß-oxidation pathway, which generates tiglyl-CoA as a probable intermediate. A kinetic analysis of this flavour molecule during growth of S. suaveolens on glucose and isoleucine showed a phase of production of ethyl tiglate that culminated concurrently with isoleucine exhaustion, followed by a disappearance of this compound, likely due to reassimilation by the yeast.

Comparison of the concentrations of phenolic constituents in cane sugar manufacturing products with their antioxidant activities.

Polyphenol content and free radical scavenging capacity of seven kinds of sugar manufacturing products (A sugars, clear juices, syrups, massecuite, and A, B, and C molasses) were studied. Seventy-two samples were collected at different stages of the process during two sugar harvests from a local sugar factory (Bois-Rouge, La Réunion). The total phenolic content of sugar products was determined according to the Folin-Ciocalteu assay. Polyphenols of sugar products were extracted with ethyl acetate and quantified by LC-UV-ESI-MS during all of the process. ABTS and DPPH assays were applied to aqueous solution of sugar products, which exhibited interesting free radical scavenging activity. Comparatively, ethyl acetate extracts exhibited higher antioxidant activity. Multivariate analyses (principal component analysis and canonical discriminant analysis) demonstrated a significant correlation between polyphenols and antioxidant activity. Moreover, it was observed that the sugar process results in an increase of the phenolic content and the free radical scavenging capacity of the different products. These products and especially molasses proved to be a rich source of natural antioxidants and may represent an interesting alternative to synthetic food antioxidants.

Assessment of antioxidant activity of cane brown sugars by ABTS and DPPH radical scavenging assays: determination of their polyphenolic and volatile constituents.

Seven cane brown sugars (four from La Réunion, two from Mauritius, and one from France) were investigated for their polyphenol content and volatile composition in relation to their free radical scavenging capacity determined by ABTS and DPPH assays. The thin layer coated on the sugar crystal was extracted by Soxhlet extractor with dichloromethane. The volatile compounds of brown sugars were studied by GC-MS, and 43 compounds were identified. The total phenolic content of brown sugars was determined according to the Folin-Ciocalteu method. Phenolic compounds were quantified in the brown sugar extracts by LC-UV-ESI-MS. Brown sugar aqueous solutions exhibited weak free radical scavenging activity in the DPPH assay and higher antioxidant activity in the ABTS assay at relatively high concentration. The brown sugar extracts showed interesting free radical scavenging properties despite the low concentration of phenolic and volatile compounds. Sugar is a common foodstuff traditionally used for its sweetening properties, which might be accompanied by antioxidant properties arising from molecules (polyphenols, Maillard products) other than sucrose of the cane brown sugars.