Influence of yeasts on bioactive compounds content of traditional sorghum beer (tchapalo) produced in Côte d'Ivoire.

Traditional sorghum beer is reputed for its therapeutic virtues in according the consumers. A number of biological active compounds like phenolic compounds (phenol, tannins, flavonoids, anthocyanins), diets fibers and compounds with clinically demonstrated antimalarial activity (quinine formate, quinine dihydrochloride, chloroquine) and antioxidant activity (2,2-diphenyl-1-picryl-hydrazyl and ferric reducing-antioxidant power methods) were evaluated in sorghum wort and beers fermented by wild yeasts and pure culture of Saccharomyces cerevisiae. The total phenol content in the samples ranged between 1254.69 ± 2.31 and 239.68 ± 11.92 µg/mL GAE. Antioxidant activity with 2,2-diphenyl-1-picryl-hydrazyl analysis method was high in sorghum wort with 73.33 ± 1.15% but with ferric reducing-antioxidant power analysis method, the antioxidant activity was high in beer from pure culture of Saccharomyces cerevisiae. No compounds with clinically demonstrated antimalarial activity were found in the samples. At bioactive compounds (phenolic compounds) content point view, statistical analysis showed similarity between the two beers.

Production of freeze-dried yeast culture for the brewing of traditional sorghum beer, tchapalo.

Freeze-drying is a well-known dehydration method widely used to preserve microorganisms. In order to produce freeze-dried yeast starter culture for the brewing purpose of African sorghum beer, we tested protective agents (sucrose, glucose, glycerol) in combination with support materials (millet, maize, sorghum, and cassava flours) at 1:1 ratio (v/v). The yeast strains Saccharomyces cerevisiae F 12-7 and Candida tropicalis C 0-7 previously isolated from sorghum beer were used in a mixed culture at a ratio of 2:1 (C. tropicalis/S. cerevisiae). After the freeze-drying, the residual water contents were between 0.78 -2.27%, 0.55 -4.09%, and 0.40-2.61%, respectively, with sucrose, glucose and glycerol. The dried yeasts viabilities were between 4.0% and 10.6%. Among the protective agents used, sucrose was found to be the best protectant giving cell viabilities of 8.4-10.6%. Considering the support materials, millet flour was the best support after drying. When the freeze-dried yeast powders were stored at 4°C and room temperature (25-28°C) for up to 3 months, the survival rates were the highest with cassava flour as the support material.

Yarrowia lipolytica lipase Lip2: an efficient enzyme for the production of concentrates of docosahexaenoic acid ethyl ester.

The production of Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) rich in cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) was studied using lipase-catalysed hydrolysis of a mixture of ethyl esters from tuna oil. Lipases from Yarrowia lipolytica (YLL2), Thermomyces lanuginosus (TLL) and Candida rugosa (CRL1, CRL3 and CRL4) were tested. C. rugosa lipases discriminated esters on the basis of their chain length, with less affinity for γ-linolenate, 11-eicosenoate, arachidonate, EPA, DPA and DHA ethyl esters. However, YLL2 and TLL improved discrimination towards DHA, as enzyme selectivity was shown to be mainly based on the position of the double bond closest to the carboxylic group. From the point of view of kinetics, purity and yield, YLL2 was the most effective lipase for DHA purification. Using this enzyme in an open reactor process resulted in the highest concentrations of DHA ethyl ester (77%) and ω-3 esters (81%) with a recovery of 94% and 77% respectively.

Bacterial cell wall macroamphiphiles: pathogen-/microbe-associated molecular patterns detected by mammalian innate immune system.

Innate immune system is the first line of host defense against invading microorganisms. It relies on a limited number of germline-encoded pattern recognition receptors that recognize conserved molecular structures of microbes, referred to as pathogen-/microbe-associated molecular patterns (PAMPs/MAMPs). Bacterial cell wall macroamphiphiles, namely Gram-negative bacteria lipopolysaccharide (LPS), Gram-positive bacteria lipoteichoic acid (LTA), lipoproteins and mycobacterial lipoglycans, are important molecules for the physiology of bacteria and evidently meet PAMP/MAMP criteria. They are well suited to innate immune recognition and constitute non-self signatures detected by the innate immune system to signal the presence of an infective agent. They are notably recognized via their lipid anchor by Toll-like receptors (TLRs) 4 or 2. Here, we review our current knowledge of the molecular bases of macroamphiphile recognition by TLRs, with a special emphasis on mycobacterial lipoglycan detection by TLR2.

Lipoarabinomannan mannose caps do not affect mycobacterial virulence or the induction of protective immunity in experimental animal models of infection and have minimal impact on in vitro inflammatory responses.

Mannose-capped lipoarabinomannan (ManLAM) is considered an important virulence factor of Mycobacterium tuberculosis. However, while mannose caps have been reported to be responsible for various immunosuppressive activities of ManLAM observed in vitro, there is conflicting evidence about their contribution to mycobacterial virulence in vivo. Therefore, we used Mycobacterium bovis BCG and M. tuberculosis mutants that lack the mannose cap of LAM to assess the role of ManLAM in the interaction of mycobacteria with the host cells, to evaluate vaccine-induced protection and to determine its importance in M. tuberculosis virulence. Deletion of the mannose cap did not affect BCG survival and replication in macrophages, although the capless mutant induced a somewhat higher production of TNF. In dendritic cells, the capless mutant was able to induce the upregulation of co-stimulatory molecules and the only difference we detected was the secretion of slightly higher amounts of IL-10 as compared to the wild type strain. In mice, capless BCG survived equally well and induced an immune response similar to the parental strain. Furthermore, the efficacy of vaccination against a M. tuberculosis challenge in low-dose aerosol infection models in mice and guinea pigs was not affected by the absence of the mannose caps in the BCG. Finally, the lack of the mannose cap in M. tuberculosis did not affect its virulence in mice nor its interaction with macrophages in vitro. Thus, these results do not support a major role for the mannose caps of LAM in determining mycobacterial virulence and immunogenicity in vivo in experimental animal models of infection, possibly because of redundancy of function.

Lipoteichoic acid in Streptomyces hygroscopicus: structural model and immunomodulatory activities.

Gram positive bacteria produce cell envelope macroamphiphile glycopolymers, i.e. lipoteichoic acids or lipoglycans, whose functions and biosynthesis are not yet fully understood. We report for the first time a detailed structure of lipoteichoic acid isolated from a Streptomyces species, i.e. Streptomyces hygroscopicus subsp. hygroscopicus NRRL 2387T. Chemical, MS and NMR analyses revealed a polyglycerolphosphate backbone substituted with α-glucosaminyl and α-N-acetyl-glucosaminyl residues but devoid of any amino-acid substituent. This structure is very close, if not identical, to that of the wall teichoic acid of this organism. These data not only contribute to the growing recognition that lipoteichoic acid is a cell envelope component of gram positive Actinobacteria but also strongly support the recently proposed hypothesis of an overlap between the pathways of lipoteichoic acid and wall teichoic acid synthesis in these bacteria. S. hygroscopicus lipoteichoic acid induced signalling by human innate immune receptor TLR2, confirming its role as a microbe-associated molecular pattern. Its activity was partially dependant on TLR1, TLR6 and CD14. Moreover, it stimulated TNF-α and IL-6 production by a human macrophage cell line to an extent similar to that of Staphylococcus aureus lipoteichoic acid. These results provide new clues on lipoteichoic acid structure/function relationships, most particularly on the role of the polyglycerolphosphate backbone substituents.

Identification of mycobacterial alpha-glucan as a novel ligand for DC-SIGN: involvement of mycobacterial capsular polysaccharides in host immune modulation.

Mycobacterium tuberculosis possesses a variety of immunomodulatory factors that influence the host immune response. When the bacillus encounters its target cell, the outermost components of its cell envelope are the first to interact. Mycobacteria, including M. tuberculosis, are surrounded by a loosely attached capsule that is mainly composed of proteins and polysaccharides. Although the chemical composition of the capsule is relatively well studied, its biological function is only poorly understood. The aim of this study was to further assess the functional role of the mycobacterial capsule by identifying host receptors that recognize its constituents. We focused on alpha-glucan, which is the dominant capsular polysaccharide. Here we demonstrate that M. tuberculosis alpha-glucan is a novel ligand for the C-type lectin DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin). By using related glycogen structures, we show that recognition of alpha-glucans by DC-SIGN is a general feature and that the interaction is mediated by internal glucosyl residues. As for mannose-capped lipoarabinomannan, an abundant mycobacterial cell wall-associated glycolipid, binding of alpha-glucan to DC-SIGN stimulated the production of immunosuppressive IL-10 by LPS-activated monocyte-derived dendritic cells. By using specific inhibitors, we show that this IL-10 induction was DC-SIGN-dependent and also required acetylation of NF-kappaB. Finally, we demonstrate that purified M. tuberculosis alpha-glucan, in contrast to what has been reported for fungal alpha-glucan, was unable to activate TLR2.

Isolation of two cell populations from yeast during high-level alcoholic fermentation that resemble quiescent and nonquiescent cells from the stationary phase on glucose.

High-level production of bioethanol (140 g L(-1) in 45 h) in aerated fed-batch cultures of Saccharomyces cerevisiae was shown to be linked to the length of a production phase uncoupled to the growth. The induction of this phase was characterized by metabolic and morphologic changes reminiscent of those occurring in the stationary phase of growth on glucose. Global transcriptomic analysis of ethanol-stressed yeast cells in the uncoupling phase harboured features similar to those from stationary-phase cells on glucose. Two distinct cellular populations were isolated by Percoll density-gradient centrifugation in this uncoupling phase. The lower fraction was enriched by yeast cells that were mostly uniform in size and opalescent, containing a large amount of glycogen and trehalose, and exhibiting high respiratory activity. In contrast, the upper fraction was characterized by cells heterogeneous in size, with one to several small buds, which did not contain storage carbohydrates and which exhibited a poor respiratory competence while retaining a high relative glycolytic activity. These results are discussed in terms of a possible induction of a state similar to the quiescence state previously observed from yeast stationary-phase cultures, in response to ethanol toxicity, whose acquisition may be critical for performing high-level alcoholic fermentation.

Physiological behaviour of Saccharomyces cerevisiae in aerated fed-batch fermentation for high level production of bioethanol.

Saccharomyces cerevisiae was able to produce 20% (v/v) of ethanol in 45 h in a fully aerated fed-batch process recently developed in our laboratory. A notable feature of this process was a production phase uncoupled to growth, the extent of which was critical for high-level ethanol production. As the level of production was found to be highly variable, we investigated on this high variability by means of a detailed physiological analysis of yeast cells in two fed-batch fermentations showing the most extreme behaviour. We found a massive leakage of intracellular metabolites into the growth medium which correlated with the drop of cell viability. The loss of viability was also found to be proportional to the reduction of plasma membrane phospholipids. Finally, the fed-batch processes with the longest uncoupling phase were characterized by induction of storage carbohydrates at the onset of this phase, whereas this metabolic event was not seen in processes with a short uncoupling phase. Taken together, our results suggested that reproducible high-level bioethanol production in aerated fed-batch processes may be linked to the ability of yeast cells to impede ethanol toxicity by triggering a metabolic remodelling reminiscent to that of cells entering a quiescent GO/G1 state.