Analysis of Aureobasidium pullulans LB83 secretome reveals distinct carbohydrate active enzymes for biomass saccharification.

Aureobasidium pullulans LB83 is a versatile biocatalyst that produces a plethora of bioactive products thriving on a variety of feedstocks under the varying culture conditions. In our last study using this microorganism, we found cellulase activity (FPase, 2.27 U/ml; CMCase, 7.42 U/ml) and other plant cell wall degrading enzyme activities grown on sugarcane bagasse and soybean meal as carbon source and nitrogen, respectively. In the present study, we provide insights on the secretome analysis of this enzymatic cocktail. The secretome analysis of A. pullulans LB83 by Liquid Chromatography coupled to Mass Spectroscopy (LC-MS/MS) revealed 38 classes of Carbohydrate Active enZymes (CAZymes) of a total of 464 identified proteins. These CAZymes consisted of 21 glycoside hydrolases (55.26%), 12 glycoside hydrolases harboring carbohydrate-binding module (31.58%), 4 carbohydrate esterases (10.53%) and one glycosyl transferase (2.63%). To the best of our knowledge, this is the first report on the secretome analysis of A. pullulans LB83.

Production and applications of pullulan from lignocellulosic biomass: Challenges and perspectives.

Pullulan is an exopolysaccharide produced by Aureobasidium pullulans, with interesting characteristics which lead to its application in industries such as pharmaceuticals, cosmetics, food, and others. To reduce production costs for industrial applications, cheaper raw materials such as lignocellulosic biomass can be utilized as a carbon and nutrient source for the microbial process. In this study, a comprehensive and critical review was conducted, encompassing the pullulan production process and the key influential variables. The main properties of the biopolymer were presented, and different applications were discussed. Subsequently, the utilization of lignocellulosics for pullulan production within the framework of a biorefinery concept was explored, considering the main published works that deal with materials such as sugarcane bagasse, rice husk, corn straw, and corn cob. Next, the main challenges and future prospects in this research area were highlighted, indicating the key strategies to favor the industrial production of pullulan from lignocellulosic biomasses.

α-D-mannan from Aureobasidium pullulans (CCMB 324): optimization extraction.

Mannans has been attracted the interest in various sectors due to its promising applications. The low toxicity of mannans allows for their use in cosmetics, pharmaceutical, and biomedical industries. In this study, the α-D-mannan extraction conditions from Aureobasidium pullulans by alkaline extraction were optimized using a Box-Behnken design (BBD). The effect of temperature (°C), pH and extraction time (hours) on the yield of α-mannan was investigated. The conditions that produced the highest yield (26%) were a temperature of 92 °C, extraction time of 3 h and pH 13. In addition, the α-D-mannan structure was confirmed by methylation analysis, 1D and 2D NMR spectroscopic analysis, and GC-MS.

Production of cellulases by Aureobasidium pullulans LB83: optimization, characterization, and hydrolytic potential for the production of cellulosic sugars.

Aureobasidium pullulans LB83 was evaluated for cellulase production under submerged fermentation conditions. Different process variables such as carbon sources (corn cob, sugarcane bagasse, and sugarcane straw), synthetic (urea, ammonium sulfate, and peptone), and non-synthetic (soybean meal, rice, and corn meal) nitrogen sources and inoculum size were evaluated by one parameter at-a-time strategy. Aureobasidium pullulans LB83 showed maximum cellulase activity (FPase, 2.27 U/mL; CMCase, 7.42 U/mL) on sugarcane bagasse. Among the nitrogen sources, soybean meal as a non-synthetic nitrogen sources showed a maximum cellulase activity (FPase 2.45 U/mL; CMCase, 6.86 U/mL) after 60 hr. The inoculum size of 1.6 × 106 CFU/mL had the maximum FPase and CMCase activities of 3.14 and 8.74 U/mL, respectively. For the enzymatic hydrolysis, both the commercial cellulase (10 FPU/g of Cellic CTec 2 (#A) and 10 FPU/g of crude enzyme extract (CEE) (#B), and varying ratio of CTec 2 and CEE in combination #C (5 FPU/g of CTec 2 + 5 FPU/g CEE), combination #D (2.5 FPU/g of CTec 2 + 7.5 FPU/g CEE), and combination #E (7.5 FPU/g of CTec 2 + 2.5 FPU/g CEE) were assessed for enzymatic hydrolysis of delignified sugarcane bagasse. Enzyme combination #C showed maximum hydrolysis yield of 92.40%. The study shows the hydrolytic potential of cellulolytic enzymes from A. pullulans LB83 for lignocellulosic sugars production from delignified sugarcane bagasse.

Improving Nutrition Utilization and Meat Quality of Broiler Chickens Through Solid-State Fermentation of Agricultural By-Products by Aureobasidium Pullulans

A 35 d trial was conducted to evaluate the effects of dietary supplementation of co-fermented agricultural by-products, soybean hulls and Pleurotus eryngii stalk residue (PESR), with Aureobasidium pullulans on nutrients digestibility and meat quality in broilers fed on conventional corn-soybean meal basal diet. A total of 400 1-d-old Ross broilers were allotted to 4 dietary treatments with 4 replicate pens (25 birds per pen). Birds were fed the corn-soybean meal diets supplemented with 0% (CON), 0.5% fermented soybean hulls (0.5% FSBH), 0.5% fermented soybean hulls partially replaced with PESR (0.5% FSHP) and 1.0% FSHP. The broilers fed on the diet that contained fermented products had higher total tract apparent digestibility for hemicellulose than those on CON (p 0.05). Meanwhile, the birds fed on 0.5% FSHP also showed the lightest duodenum and jejunum weight among all groups. Compared with the CON, the inclusion of 1.0% FSHP in the diet significantly decreased ammonia N concentration in cecum followed by 0.5% FSHP and subsequent 0.5% FSBH group. Dietary supplementation of fermented products significantly improved WHC and increased protein content in meat as compared with the CON. Moreover, the fat content in meat decreased in the broilers that received 0.5% FSBH and 1.0% FSHP. In conclusion, dietary supplementation of co-fermented agricultural by-products, PESR and soybean meal, could improve the digestibility of hemicellulose and decrease the cecal ammonia N concentration; moreover, the increased WHC and protein level as well as decreased fat content in meat suggested the promising utilization of these low-cost materials as broiler feed additives.

Improving Nutrition Utilization and Meat Quality of Broiler Chickens Through Solid-State Fermentation of Agricultural By-Products by Aureobasidium Pullulans

A 35 d trial was conducted to evaluate the effects of dietary supplementation of co-fermented agricultural by-products, soybean hulls and Pleurotus eryngii stalk residue (PESR), with Aureobasidium pullulans on nutrients digestibility and meat quality in broilers fed on conventional corn-soybean meal basal diet. A total of 400 1-d-old Ross broilers were allotted to 4 dietary treatments with 4 replicate pens (25 birds per pen). Birds were fed the corn-soybean meal diets supplemented with 0% (CON), 0.5% fermented soybean hulls (0.5% FSBH), 0.5% fermented soybean hulls partially replaced with PESR (0.5% FSHP) and 1.0% FSHP. The broilers fed on the diet that contained fermented products had higher total tract apparent digestibility for hemicellulose than those on CON (p 0.05). Meanwhile, the birds fed on 0.5% FSHP also showed the lightest duodenum and jejunum weight among all groups. Compared with the CON, the inclusion of 1.0% FSHP in the diet significantly decreased ammonia N concentration in cecum followed by 0.5% FSHP and subsequent 0.5% FSBH group. Dietary supplementation of fermented products significantly improved WHC and increased protein content in meat as compared with the CON. Moreover, the fat content in meat decreased in the broilers that received 0.5% FSBH and 1.0% FSHP. In conclusion, dietary supplementation of co-fermented agricultural by-products, PESR and soybean meal, could improve the digestibility of hemicellulose and decrease the cecal ammonia N concentration; moreover, the increased WHC and protein level as well as decreased fat content in meat suggested the promising utilization of these low-cost materials as broiler feed additives.(AU)

Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose

Abstract Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullulan is more costly than other exopolysaccharides, which limits its effective application. The purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullulan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mixtures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose = 80:20) was 0.212 h−1, which was significantly higher than those of potato starch hydrolysate alone (0.146 h−1) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch hydrolysate:glucose:fructose = 80:10:10, 0.166 h−1) with 100 g L−1 total carbon source. The results suggest that mixtures of potato starch hydrolysate and sucrose could promote pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme responsible for pullulan synthesis and promoted effective potato starch hydrolysate conversion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan.

Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose

Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullulan is more costly than other exopolysaccharides, which limits its effective application. The purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullulan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mixtures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose = 80:20) was 0.212 h-¹, which was significantly higher than those of potato starch hydrolysate alone (0.146 h-¹) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch hydrolysate:glucose:fructose = 80:10:10, 0.166 h-¹) with 100 g L-¹ total carbon source. The results suggest that mixtures of potato starch hydrolysate and sucrose could promote pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme responsible for pullulan synthesis and promoted effective potato starch hydrolysate conversion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan.(AU)

Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose.

Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullulan is more costly than other exopolysaccharides, which limits its effective application. The purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullulan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mixtures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose=80:20) was 0.212h-1, which was significantly higher than those of potato starch hydrolysate alone (0.146h-1) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch hydrolysate:glucose:fructose=80:10:10, 0.166h-1) with 100gL-1 total carbon source. The results suggest that mixtures of potato starch hydrolysate and sucrose could promote pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme responsible for pullulan synthesis and promoted effective potato starch hydrolysate conversion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan.

Fungal endophytes in germinated seeds of the common bean, Phaseolus vulgaris.

We conducted a survey of fungal endophytes in 582 germinated seeds belonging to 11 Colombian cultivars of the common bean (Phaseolus vulgaris). The survey yielded 394 endophytic isolates belonging to 42 taxa, as identified by sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) region. Aureobasidium pullulans was the dominant endophyte, isolated from 46.7 % of the samples. Also common were Fusarium oxysporum, Xylaria sp., and Cladosporium cladosporioides, but found in only 13.4 %, 11.7 %, and 7.6 % of seedlings, respectively. Endophytic colonization differed significantly among common bean cultivars and seedling parts, with the highest colonization occurring in the first true leaves of the seedlings.

Yeasts from native Brazilian Cerrado plants: Occurrence, diversity and use in the biocontrol of citrus green mould.

Yeasts are some of the most important postharvest biocontrol agents. Postharvest oranges frequently deteriorate due to green mould (Penicillium digitatum), which causes significant losses. The aims of this study were to determine the composition and diversity of yeasts on plants of the Brazilian Cerrado and to explore their potential for inhibiting citrus green mould. Leaves and fruit of Byrsonima crassifolia and Eugenia dysenterica were collected from Cerrado conservation areas, and thirty-five yeasts were isolated and identified by sequencing the D1-D2 domain of the rDNA large subunit (26S). The isolates represented the Aureobasidium, Meyerozyma, Candida, and Pichia genera. Three isolates identified as Aureobasidium pullulans exhibited potential for the control of P. digitatum in both in vitro and in vivo tests; these isolates reduced the incidence of disease and increased the storage time of fruit. Aureobasidium. pullulans has immense potential for the biological control of filamentous fungi.

Characterization of pectinase activity for enology from yeasts occurring in Argentine Bonarda grape.

Pectinolytic enzymes are greatly important in winemaking due to their ability to degrade pectic polymers from grape, contributing to enhance process efficiency and wine quality. This study aimed to analyze the occurrence of pectinolytic yeasts during spontaneous fermentation of Argentine Bonarda grape, to select yeasts that produce extracellular pectinases and to characterize their pectinolytic activity under wine-like conditions. Isolated yeasts were grouped using PCR-DGGE and identified by partial sequencing of 26S rRNA gene. Isolates comprised 7 genera, with Aureobasidium pullulans as the most predominant pectinolytic species, followed by Rhodotorula dairenensis and Cryptococcus saitoi. No pectinolytic activity was detected among ascomycetous yeasts isolated on grapes and during fermentation, suggesting a low occurrence of pectinolytic yeast species in wine fermentation ecosystem. This is the first study reporting R. dairenensis and Cr. saitoi species with pectinolytic activity. R. dairenensis GM-15 produced pectinases that proved to be highly active at grape pH, at 12 °C, and under ethanol and SO2 concentrations usually found in vinifications (pectinase activity around 1.1 U/mL). This strain also produced cellulase activity at 12 °C and pH 3.5, but did not produce ß-glucosidase activity under these conditions. The strain showed encouraging enological properties for its potential use in low-temperature winemaking.

Characterization of pectinase activity for enology from yeasts occurring in Argentine Bonarda grape

Pectinolytic enzymes are greatly important in winemaking due to their ability to degrade pectic polymers from grape, contributing to enhance process efficiency and wine quality. This study aimed to analyze the occurrence of pectinolytic yeasts during spontaneous fermentation of Argentine Bonarda grape, to select yeasts that produce extracellular pectinases and to characterize their pectinolytic activity under wine-like conditions. Isolated yeasts were grouped using PCR-DGGE and identified by partial sequencing of 26S rRNA gene. Isolates comprised 7 genera, with Aureobasidium pullulans as the most predominant pectinolytic species, followed by Rhodotorula dairenensis and Cryptococcus saitoi. No pectinolytic activity was detected among ascomycetous yeasts isolated on grapes and during fermentation, suggesting a low occurrence of pectinolytic yeast species in wine fermentation ecosystem. This is the first study reporting R. dairenensis and Cr. saitoi species with pectinolytic activity. R. dairenensis GM-15 produced pectinases that proved to be highly active at grape pH, at 12 °C, and under ethanol and SO₂ concentrations usually found in vinifications (pectinase activity around 1.1 U/mL). This strain also produced cellulase activity at 12 °C and pH 3.5, but did not produce β-glucosidase activity under these conditions. The strain showed encouraging enological properties for its potential use in low-temperature winemaking.

Characterization of pectinase activity for enology from yeasts occurring in Argentine Bonarda grape

Pectinolytic enzymes are greatly important in winemaking due to their ability to degrade pectic polymers from grape, contributing to enhance process efficiency and wine quality. This study aimed to analyze the occurrence of pectinolytic yeasts during spontaneous fermentation of Argentine Bonarda grape, to select yeasts that produce extracellular pectinases and to characterize their pectinolytic activity under wine-like conditions. Isolated yeasts were grouped using PCR-DGGE and identified by partial sequencing of 26S rRNA gene. Isolates comprised 7 genera, with Aureobasidium pullulans as the most predominant pectinolytic species, followed by Rhodotorula dairenensis and Cryptococcus saitoi. No pectinolytic activity was detected among ascomycetous yeasts isolated on grapes and during fermentation, suggesting a low occurrence of pectinolytic yeast species in wine fermentation ecosystem. This is the first study reporting R. dairenensis and Cr. saitoi species with pectinolytic activity. R. dairenensis GM-15 produced pectinases that proved to be highly active at grape pH, at 12 °C, and under ethanol and SO2 concentrations usually found in vinifications (pectinase activity around 1.1 U/mL). This strain also produced cellulase activity at 12 °C and pH 3.5, but did not produce β-glucosidase activity under these conditions. The strain showed encouraging enological properties for its potential use in low-temperature winemaking. (AU)

Highly cold-active pectinases under wine-like conditions from non-Saccharomyces yeasts for enzymatic production during winemaking.

UNLABELLED: The influence of oenological factors on cold-active pectinases from 15 preselected indigenous yeasts belonging to Aureobasidium pullulans, Filobasidium capsuligenum, Rhodotorula dairenensis, Cryptococcus saitoi and Saccharomyces cerevisiae was investigated. Pectinolytic enzymes were constitutive or partially constitutive; and high glucose concentration (200 g l(-1) ) did not affect or increased pectinase production at 12°C and pH 3·5 (up to 113·9 U mg(-1) ) only in A. pullulans strains. SO2 (120 mg l(-1) ) slightly affected the growth of A. pullulans strains but did not affect pectinase production levels. Ethanol (15%) barely affected pectinase activity of A. pullulans strains but diminished relative activity to 12-79% of basidiomycetous yeasts. Moreover, non-Saccharomyces strains showed promising properties of oenological interest. This study demonstrates that cold-active pectinases from some A. pullulans strains were able to remain active at glucose, ethanol and SO2 concentrations usually found in vinification, and suggests their potential use as processing aids for low-temperature winemaking. SIGNIFICANCE AND IMPACT OF THE STUDY: Nowadays, there is increasing interest in low-temperature winemaking. Nevertheless, commercial oenological pectinases, produced by fungi, are rarely active at low temperatures. Cold-active pectinases that are stable under vinification conditions are needed. This study indicated that cold-active and acid-tolerant pectinases from non-Saccharomcyes yeasts were able to remain active at glucose, ethanol and SO2 concentrations usually found in winemaking. Furthermore, not only are these yeasts a source of cold-active pectinases, but the yeasts themselves are also potential adjunct cultures for oenology to produce these enzymes during cold-winemaking.

Aureobasidium pullulans infection in a patient with chronic lymphocytic leukemia

Saprophytic fungi are being increasingly recognized as etiologic agents of mycoses in immunosuppressed patients. We report a case of subcutaneous infiltration by Aureobasidium pullulans, likely due to traumatic inoculation, in a neutropenic patient during chemotherapy for chronic lymphocytic leukemia. The patient was treated with amphotericin B deoxycholate but was subsequently switched to itraconazole, which improved the lesion. This case highlights the importance of considering unusual fungal infections in critically ill patients such as those who are immunosuppressed due to chemotherapy. Diagnostic techniques and effective antifungal therapy have improved the prognosis of these cases.

Production of pullulan by a thermotolerant Aureobasidium pullulans strain in non-stirred fed batch fermentation process

Total 95 isolates of Aureobasidium pullulans were isolated from different flowers and leaves samples, out of which 11 thermotolerant strains produced pullulan. One thermotolerant non-melanin pullulan producing strain, designated as RG-5, produced highest pullulan (37.1±1.0 g/l) at 42ºC, pH 5.5 in 48h of incubation with 3% sucrose and 0.5% ammonium sulphate in a non-stirred fed batch fermentor of 6 liters capacity. The two liters of initial volume of fermentation medium was further fed with the 2 liters in two successive batches at 5 h interval into the fermentor. The sterile air was supplied only for 10h at the rate of 0.5 vvm.

Production of pullulan by a thermotolerant aureobasidium pullulans strain in non-stirred fed batch fermentation process.

Total 95 isolates of Aureobasidium pullulans were isolated from different flowers and leaves samples, out of which 11 thermotolerant strains produced pullulan. One thermotolerant non-melanin pullulan producing strain, designated as RG-5, produced highest pullulan (37.1±1.0 g/l) at 42(o)C, pH 5.5 in 48h of incubation with 3% sucrose and 0.5% ammonium sulphate in a non-stirred fed batch fermentor of 6 liters capacity. The two liters of initial volume of fermentation medium was further fed with the 2 liters in two successive batches at 5 h interval into the fermentor. The sterile air was supplied only for 10h at the rate of 0.5 vvm.

Production of pullulan by a thermotolerant Aureobasidium pullulans strain in non-stirred fed batch fermentation process

Total 95 isolates of Aureobasidium pullulans were isolated from different flowers and leaves samples, out of which 11 thermotolerant strains produced pullulan. One thermotolerant non-melanin pullulan producing strain, designated as RG-5, produced highest pullulan (37.1±1.0 g/l) at 42ºC, pH 5.5 in 48h of incubation with 3% sucrose and 0.5% ammonium sulphate in a non-stirred fed batch fermentor of 6 liters capacity. The two liters of initial volume of fermentation medium was further fed with the 2 liters in two successive batches at 5 h interval into the fermentor. The sterile air was supplied only for 10h at the rate of 0.5 vvm.