Evaluation of Fermentative Parameters for the Production of Botryosphaeran (a(1-->3;1-->6)-β-D-glucan) and Mycelial Biomass by Botryosphaeria rhodina MAMB-05

Botryosphaeran is an exopolysaccharide (EPS) of the (1"3;1"6)-β-D-glucan type produced by the fungus Botryosphaeria rhodina MAMB-05. In attempts to enhance its extracellular production, the quantitative effects of different nitrogen sources, fermentation time, inoculum size, C/N ratio, and initial pH were evaluated on the yields of botryosphaeran. Ammonium nitrate was selected as the best nitrogen source in examining these effects using a 2³-factorial central-composite experimental design, and analysis by the response surface method. According to a factorial design, the most important variables influencing both botryosphaeran and mycelium biomass production was inoculum concentration and the time of growth. Under these conditions, optimum botryosphaeran production occurred at 88 h of growth, 0.88 g of mycelium L-1 of nutrient mediumat C/N ratio of 30, and resulted in a 5.2 g L-1 yield of EPS in the fermentation broth. For production of the mycelium biomass, the optimum condition occurred at the same time of growth and inoculum concentration; however the best C/N ratio was 75, and resulted in a yield of 33.8 g L-1 of biomass. The results also indicated that the maximal mycelial growth is not associated to the yields of botryosphaeran production by Botryosphaeria rhodina MAMB-05. DOI: http://dx.doi.org/10.17807/orbital.v7i1.63

Influence of nutrients on enhancing laccase production by Botryosphaeria rhodina MAMB-05

The physiological requirements needed to enhance the production of laccases by the ascomycete Botryosphaeria rhodina MAMB-05 in submerged cultivation were examined under non-induced and induced (veratryl alcohol, VA) conditions. Under non-induced conditions (–VA), the initial pH, C:N ratio, and inorganic N source did not influence laccase production, in contrast to Tween 80, soybean oil, and copper, which significantly increased laccase production, and proline and urea, which suppressed laccase formation. In addition, Tween 60 could serve as the sole carbon source for the production of these enzymes. Under VA-induced conditions of fungal growth, factors such as inoculum type, time-point of addition of inducer, initial pH, C:N ratio, and type of N source, influenced the production of laccases; however, unlike the non-induced conditions, proline and urea did not act as suppressors. Each of these physiological conditions exerted different effects on biomass production. The nutritional conditions examined for B. rhodina MAMB-05 are discussed in relation to their influence on fungal growth and laccase production. [Int Microbiol 2007; 10(3):177-185

Evaluation of the Components Released by Wine Yeast Strains on Protein Haze Formation in White Wine

Cultures of 23 indigenous yeast strains (22 Saccharomyces cerevisiae and a non-Saccharomyces, Torulaspora delbrueckii), isolated from fermentation tanks at wineries in Castilla-La Mancha (Spain), and were performed under winemaking conditions using a synthetic must. Polysaccharide analysis and turbidity assays were conducted so as to observe the capacity of the released mannoproteins against protein haze formation in white wine, and 3 strains (2 Saccharomyces cerevisiae and T. delbrueckii) were chosen for further experiments. The action of a commercial b-glucanolytic enzyme preparation (Lallzyme BETA®), and a β-(1→3)-glucanase preparation from Trichoderma harzianum Rifai were evaluated to release polysaccharides from the different yeast strains’ cell walls. Protection against protein haze formation was strain dependent, and only two strains (Sc2 and Sc4) presented >50% stabilization in comparison to controls. Addition of β-glucanases did not increase the concentrations of polysaccharides in the fermentation musts; however, a significant increase of polymeric mannose (mannoproteins) was detected using an enzymatic assay following total acid hydrolysis of the soluble polysaccharides. Enzymatic treatment presented positive effects and decreased protein haze formation in white wine. DOI http://dx.doi.org/10.17807/orbital.v8i6.86

Evaluation of Fermentative Parameters for the Production of Botryosphaeran (a(1-->3;1-->6)-β-D-glucan) and Mycelial Biomass by Botryosphaeria rhodina MAMB-05

Botryosphaeran is an exopolysaccharide (EPS) of the (1"3;1"6)-β-D-glucan type produced by the fungus Botryosphaeria rhodina MAMB-05. In attempts to enhance its extracellular production, the quantitative effects of different nitrogen sources, fermentation time, inoculum size, C/N ratio, and initial pH were evaluated on the yields of botryosphaeran. Ammonium nitrate was selected as the best nitrogen source in examining these effects using a 2³-factorial central-composite experimental design, and analysis by the response surface method. According to a factorial design, the most important variables influencing both botryosphaeran and mycelium biomass production was inoculum concentration and the time of growth. Under these conditions, optimum botryosphaeran production occurred at 88 h of growth, 0.88 g of mycelium L-1 of nutrient mediumat C/N ratio of 30, and resulted in a 5.2 g L-1 yield of EPS in the fermentation broth. For production of the mycelium biomass, the optimum condition occurred at the same time of growth and inoculum concentration; however the best C/N ratio was 75, and resulted in a yield of 33.8 g L-1 of biomass. The results also indicated that the maximal mycelial growth is not associated to the yields of botryosphaeran production by Botryosphaeria rhodina MAMB-05. DOI: http://dx.doi.org/10.17807/orbital.v7i1.63

Sulfonated (1→6)-β-D-Glucan (Lasiodiplodan): Preparation, Characterization and Bioactive Properties

U radu su proizvedeni sulfonirani derivati β-D-glukana iz gljive Lasiodiplodia theobromae MMPI, nazvani lasiodiplodan (LAS-S), s različitim stupnjevima supstitucije (1,61; 1,42; 1,02 i 0,15), te su okarakterizirani pomoću FTIR spektroskopije i skenirajuće elektronske mikroskopije (SEM). Ispitani su toplinska svojstva, topljivost, te antimikrobni, antioksidacijski i citotoksički učinak derivata. Uspješnost sulfonacije provjerena je FTIR analizom, pri čemu su dobivene specifične vrpce na 1250 cm-1 (S=O, jaki signal za asimetričnu vibraciju rastezanja dvostruke veze) i 810 cm-1 (C-O-S, signal za simetričnu vibraciju povezan s prisustvom C-O-SO3 skupine) u sulfoniranim uzorcima. SEM analizom je potvrđeno da sulfonacija pospješuje morfološke promjene na površini biopolimera, uz pojavu heterogenih fibrilarnih struktura na površini nakon kemijske modifikacije. Sulfonirani je lasiodiplodan imao veliku toplinsku stabilnost, a zbog oksidacije pri gotovo 460 °C opažen je gubitak mase. Sulfonacijom je povećana topljivost lasiodiplodana i njegova antimikrobna aktivnost, osobito spram kvasca Candida albicans i bakterije Salmonella enterica Typhimurium. Prirodni je lasiodiplodan bolje uklanjao ione OH˙, dok je sulfonirani bolje uklanjao ione željeza (FRAP potencijal). Ni prirodni ni sulfonirani lasiodiplodan nisu imali citotoksični učinak na divlje tipove i mutante kvasca Saccharomyces cerevisiae. Uzorci s većim stupnjem supstitucije (1,42 i 1,61) imali su slabije svojstvo indukcije oksidacijskog stresa.Sulfonated derivatives of lasiodiplodan (LAS-S) with different degrees of substitution (1.61, 1.42, 1.02 and 0.15) were obtained and characterized by Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and thermal and solubility analyses. Antimicrobial, antioxidant and cytotoxic potential were also assessed. The sulfonation was confirmed by FTIR analysis with specific bands at 1250 cm–1 (S=O, strong asymmetrical stretching vibration) and at 810 cm–1 (C-O-S, symmetrical vibration associated with the C-O-SO3 group) in the sulfonated samples. SEM demonstrated that sulfonation promoted morphological changes on the surface of the biopolymer with heterogeneous fibrillary structures appearing along the surface following chemical modification. LAS-S showed high thermal stability, with mass loss due to oxidation at temperatures close to 460 °C. Sulfonation increased the solubility of LAS, and in addition, increased the antimicrobial activity, especially against Candida albicans (fungicidal) and Salmonella enterica Typhimurium (bacteriostatic). Native lasiodiplodan (LAS-N) showed higher OH˙ removal capacity, while LAS-S had higher ferric ion reducing antioxidant power (FRAP) potential. LAS-N and LAS-S did not demonstrate lethal cytotoxicity against wild and mutant strains of Saccharomyces cerevisiae. Samples with higher degree of substitution (1.42 and 1.61) showed lower potential to induce oxidative stress

Sulfonation and anticoagulant activity of fungal exocellular β-(1→6)-d-glucan (lasiodiplodan)

AbstractAn exocellular β-(1→6)-d-glucan (lasiodiplodan) produced by a strain of Lasiodiplodia theobromae (MMLR) grown on sucrose was derivatized by sulfonation to promote anticoagulant activity. The structural features of the sulfonated β-(1→6)-d-glucan were investigated by UV–vis, FT-IR and 13C NMR spectroscopy, and the anticoagulant activity was investigated by the classical coagulation assays APTT, PT and TT using heparin as standard. The content of sulfur and degree of substitution of the sulfonated glucan was 11.73% and 0.95, respectively. UV spectroscopy showed a band at 261nm due to the unsaturated bond formed in the sulfonation reaction. Results of FT-IR and 13C NMR indicated that sulfonyl groups were inserted on the polysaccharide. The sulfonated β-(1→6)-d-glucan presented anticoagulant activity as demonstrated by the increase in dose dependence of APTT and TT, and these actions most likely occurred because of the inserted sulfonate groups on the polysaccharide. The lasiodiplodan did not inhibit the coagulation tests

Optimization of Laccase Production, and Characterization of Lignin Degradation Products by Fusarium oxysporum JUMAD-053

The objective of this work was to optimize the culture conditions for laccase production in the presence of Kraft lignin by Fusarium oxysporum JUMAD-053, and to evaluate the biodegradation products of lignin. The fungal isolate that presented highest laccase activity had its production optimized by a statistical factorial design 33 in 15 experimental runs. F. oxysporum presented the highest constitutive laccase titer (5.37 U/mL). Statistical factorial design demonstrated a maximum laccase titer of 9.8 U/mL when assayed against ABTS under the conditions optimized: 1.125% (w/v) yeast extract, 0.5% (w/v) Kraft lignin and 10 days of cultivation. The maximum laccase titer when assayed on DMP was 8.4 U/mL, following the conditions optimized: 1.125% yeast extract, 0.25% Kraft lignin and 7 days of cultivation. The analysis of cultures led to identification of metabolites; two being aromatic: 2,6-dimethoxy benzoic acid and sesamin; also, fumonisin and long-chain fatty acids. As a result of the study, the maximum laccase activities of 9.8 and 8.4 U/mL measured from ABTS and DMP substrates, respectively. The search shows new sources of fungal laccase for obtaining new metabolites of biodegradation from Kraft lignin in culture medium. DOI: http://dx.doi.org/10.17807/orbital.v13i5.162

Optimization of Laccase Production, and Characterization of Lignin Degradation Products by Fusarium oxysporum JUMAD-053

The objective of this work was to optimize the culture conditions for laccase production in the presence of Kraft lignin by Fusarium oxysporum JUMAD-053, and to evaluate the biodegradation products of lignin. The fungal isolate that presented highest laccase activity had its production optimized by a statistical factorial design 33 in 15 experimental runs. F. oxysporum presented the highest constitutive laccase titer (5.37 U/mL). Statistical factorial design demonstrated a maximum laccase titer of 9.8 U/mL when assayed against ABTS under the conditions optimized: 1.125% (w/v) yeast extract, 0.5% (w/v) Kraft lignin and 10 days of cultivation. The maximum laccase titer when assayed on DMP was 8.4 U/mL, following the conditions optimized: 1.125% yeast extract, 0.25% Kraft lignin and 7 days of cultivation. The analysis of cultures led to identification of metabolites; two being aromatic: 2,6-dimethoxy benzoic acid and sesamin; also, fumonisin and long-chain fatty acids. As a result of the study, the maximum laccase activities of 9.8 and 8.4 U/mL measured from ABTS and DMP substrates, respectively. The search shows new sources of fungal laccase for obtaining new metabolites of biodegradation from Kraft lignin in culture medium. DOI: http://dx.doi.org/10.17807/orbital.v13i5.162