Production and characterization of extracellular liamocins produced from fungal strains of Aureobasidium spp.

Liamocins, a group of high-density glycolipids, are only produced by certain strains of the yeast-like fungi in the genus Aureobasidium. Until now, few studies have focused on the surfactant properties of liamocins produced from the highly diverse tropical strains of Aureobasidium. Therefore, the aims of this research were to screen the liamocin production from tropical strains of Aureobasidium spp. and to characterize their surfactant properties. A total of 41 strains of Thai Aureobasidium spp. were screened for their ability to produce liamocins, and the products were detected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thin-layer chromatography. Of those strains, 30 strains of Aureobasidium spp. tested were found to produce liamocins with yields ranging from 0.53 to 10.60 g/l. The nature of all crude liamocins was heterogeneous, with different compositions and ratios depending on the yeast strain. These liamocins exhibited relatively high emulsifying activity against vegetable oils tested, with an emulsification index of around 40-50%; the emulsion stability of some liamocins was up to 30 days. The obtained critical micelle concentration values were varied, with those ​​of liamocins produced from A. pullulans, A. melanogenum and A. thailandense falling in ranges from 7.70 to 119.78, 10.73 to > 1,000, and 68.56 to > 1,000 mg/l, respectively. The emulsification activity of liamocins was higher than that of the analytical grade rhamnolipids. These compounds showed strong surface tension reduction in a sodium chloride concentration range of 2-12% (w/v), pH values between 3 and 7, and temperatures between 4 and 121 °C. This is the first report of liamocins produced by A. thailandense.

Xylooligosaccharides produced from sugarcane leaf arabinoxylan using xylanase from Aureobasidium pullulans NRRL 58523 and its prebiotic activity toward Lactobacillus spp.

In an attempt to enhance the value of sugarcane leaf, xylan was extracted and used for xylooligosaccharide (XO) production via enzymatic hydrolysis using xylanase from the black yeast Aureobasidium pullulans. The xylan was extracted from sugarcane leaf using alkali extraction according to the response surface methodology. The highest xylan yield (99.42 ± 4.05 % recovery) was obtained using 14.32 % (w/v) NaOH, 13.25:1 liquid: solid ratio, at 121 °C and 15 lb.in2 for 32 min. Sugar composition and FTIR spectrum analyses confirmed its structure as arabinoxylan. The extracted arabinoxylan had a relatively high molecular weight compared to previous studies. Crude endoxylanase from A. pullulans NRRL 58523 was selected for enzymatic hydrolysis of the xylan. The enzyme hydrolyzed well at 50 °C, pH 4.0 and was relatively stable under this condition (87.38 ± 1.26 % of the activity remained after 60 h). XOs, especially xylobiose and xylotriose, were obtained at the maximum yield of 237.51 ± 17.69 mg/g xylan via endoxylanase hydrolysis under the optimum conditions (50 °C, pH 4.0, 65.31 U/g xylan, 53 h). XOs exhibited species-specific prebiotic activity toward three strains of Lactobacillus spp. but not toward Bifidobacterium spp.

Antiproliferative activity and apoptosis­inducing effects of Trametes polyzona polysaccharides against human breast cancer cells.

Fungal polysaccharides have garnered interest due to their biological activities in terms of anticancer properties and antioxidant activity. The present study aimed to evaluate the anticancer properties and antioxidant activity of a newly isolated white-rot fungus, Trametes polyzona CU07 from Thailand. Crude T. polyzona polysaccharides (CTPPs) were extracted from mycelia using hot water. The chemical properties, including total carbohydrates, molecular weight and protein content, and Fourier-transform infrared spectroscopy analysis, were then investigated. The antioxidant activity was determined against the radicals 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The anticancer properties were evaluated in MCF-7 breast cancer (BC) cells, whereas the 293 cell line was used as a control. The inhibitory effects of CTPPs on viability were determined by MTT assay, followed by BrdU incorporation assay to assess cell proliferation. The induction of apoptosis was determined by flow cytometry. CTPPs were considered polysaccharide-protein conjugates, which had molecular weights in the range of 0.3-22,528 kDa. They contained ~50 and 37% carbohydrate and protein, respectively, with glucose as the main monosaccharide component. Notably, CTPPs had high antioxidant activity against ABTS, and had a significant inhibitory effect on the MCF-7 cell line with a half-maximal inhibitory concentration value of 0.58 mg/ml. However, they exhibited little effect on the 293 cell line. The BrdU incorporation assay demonstrated that CTPPs inhibited proliferation by ~20% compared with that in untreated cells. CTPPs also induced early- and late-stage apoptosis of MCF-7 cells. These results indicated that the CTPPs may exhibit potential antiproliferative and antioxidant activity, and apoptosis-inducing effects against human BC cells.

In vitro antioxidant and anticancer activities of Smilax corbularia extract combined with Phellinus linteus extract against breast cancer cell lines.

Treatment with extracts from whole herbs has been reported to synergistically enhance the anticancer activities of therapeutic agents in recent studies. The present study evaluated the antioxidant and anticancer activities of Smilax corbularia Kunth (S. corbularia) and Phellinus linteus (P. linteus) crude extracts individually and in combination. S. corbularia was extracted using ethanol, whereas P. linteus was extracted using hot water. Both crude extracts underwent physiochemical characterization. Subsequently, the possible antioxidant activities of both crude extracts, individually and in combination, were evaluated using 2,2-Diphenyl-1-picrylhydrazyl and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assays. Their effects on breast cancer cell cytotoxicity, proliferation and apoptosis were then assessed. The crude S. corbularia extract obtained was found to have a high level of total phenolic content, whilst the crude P. linteus extract had high levels of total polysaccharide content. The total phenolic content and total polysaccharide content results of the combinations depended on the respective ratios of the individual extracts. S. corbularia alone and combination 3 (which contained 75% S. corbularia: 25% P. linteus) demonstrated the greatest radical scavenging activity, followed by combination 1 (50% S. corbularia: 50% P. linteus), combination 2 (25% S. corbularia: 75% P. linteus) and P. linteus. The toxicity results of the extract samples on the cancer cells corresponded with their antioxidant activity. In particular, certain combinations demonstrated clearer inhibitory effects on cell proliferation against three types of breast cancer cells compared with those exerted by the two individual extracts. However, induction of apoptosis was limited, with the degree of apoptosis observed to be #x003C;5%. These findings suggested that treatment with combinations of these two extracts could confer enhanced antioxidant and antiproliferative effects on breast cancer cells. Therefore, the potential of these two extracts in combination as anticancer agents warrants further investigation.

Effect of Smilax spp. and Phellinus linteus combination on cytotoxicity and cell proliferation of breast cancer cells.

BACKGROUND: Although the prevalence of breast cancer (BC) has been reduced in recent years, proficient therapeutic regimens should be further investigated with the aim of further reducing the mortality rate. To obtain more effective treatment, the present study aimed to observe the effects of PL synergistically combined with Smilax corbularia and S. glabra extracts (PSS) on BC cell lines, MCF7, T47D, MDA-MB-231, and MDA-MB-468. METHODS: The half-maximal inhibition (IC50) concentrations of PSS and PL were determined in a dose- and time-dependent manner using MTT assay. The activity of PSS and PL on anti-BC proliferation was evaluated using BrdU assay, and colony formation assay. Moreover, cell cycle analysis and apoptosis induction as a result of PSS and PL exposure were investigated using propidium iodide (PI) staining and co-staining of annexin V DY634 and PI combined flow cytometric analysis, respectively. Finally, changes in the mRNA expression of genes involved in proliferative and apoptotic pathways (MKI67, HER2, EGFR, MDM2, TNFα, PI3KCA, KRAS, BAX, and CASP8) were explored using RT-qPCR following PSS and PL treatment. RESULTS: The PSS and PL extracts exhibited significant potential in BC cytotoxicity which were in were in dose- and time-dependent response. This inhibition of cell growth was due to the suppression of cell proliferation, the cell cycle arrest, and the induction of apoptosis. Additionally, an investigation of the underlying molecular mechanism revealed that PSS and PL are involved in downregulation of the MKI67, HER2, EGFR, MDM2, TNFα, and PI3KCA expression. CONCLUSIONS: This present study has suggested that PSS and PL possess anti-BC proliferative activity mediated via the downregulation of genes participating in the relevant pathways. PSS or PL may be combined with other agents to alleviate the adverse side effects resulted from conventional chemotherapeutic drugs.

In vitro and in vivo antiplasmodial activities of leaf extracts from Sonchus arvensis L.

BACKGROUND: Malaria continues to be a global problem due to the limited efficacy of current drugs and the natural products are a potential source for discovering new antimalarial agents. Therefore, the aims of this study were to investigate phytochemical properties, cytotoxic effect, antioxidant, and antiplasmodial activities of Sonchus arvensis L. leaf extracts both in vitro and in vivo. METHODS: The extracts from S. arvensis L. leaf were prepared by successive maceration with n-hexane, ethyl acetate, and ethanol, and then subjected to quantitative phytochemical analysis using standard methods. The antimalarial activities of crude extracts were tested in vitro against Plasmodium falciparum 3D7 strain while the Peter's 4-day suppressive test model with P. berghei-infected mice was used to evaluate the in vivo antiplasmodial, hepatoprotective, nephroprotective, and immunomodulatory activities. The cytotoxic tests were also carried out using human hepatic cell lines in [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. RESULT: The n-hexane, ethyl acetate, and ethanolic extracts of S. arvensis L. leaf exhibited good in vitro antiplasmodial activity with IC50 values 5.119 ± 3.27, 2.916 ± 2.34, and 8.026 ± 1.23 µg/mL, respectively. Each of the extracts also exhibited high antioxidant with low cytotoxic effects. Furthermore, the ethyl acetate extract showed in vivo antiplasmodial activity with ED50 = 46.31 ± 9.36 mg/kg body weight, as well as hepatoprotective, nephroprotective, and immunomodulatory activities in mice infected with P. berghei. CONCLUSION: This study highlights the antiplasmodial activities of S. arvensis L. leaf ethyl acetate extract against P. falciparum and P. berghei as well as the antioxidant, nephroprotective, hepatoprotective, and immunomodulatory activities with low toxicity. These results indicate the potential of Sonchus arvensis L. to be developed into a new antimalarial drug candidate. However, the compounds and transmission-blocking strategies for malaria control of S. arvensis L. extracts are essential for further study.

In silico anti-SARS-CoV-2, antiplasmodial, antioxidant, and antimicrobial activities of crude extracts and homopterocarpin from heartwood of Pterocarpus macrocarpus Kurz.

Natural products play an essential role in new drug discovery. In the present study, we determined the anti-SARS-CoV-2 (severe acute respiratory syndrome-related coronavirus-2), antioxidant, antiplasmodial, and antimicrobial activities of Pterocarpus macrocarpus Kurz. heartwood and structurally characterized the bioactive compounds. P. macrocarpus Kurz. heartwood was macerated with n-hexane, ethyl acetate, and ethanol, respectively, for 7 days, three times. The compounds were isolated by recrystallization with n-hexane and evaluated by thin-layer chromatography (TLC), gas chromatography-mass spectrophotometry (GC-MS), Fourier transform infrared spectroscopy (FITR), and nuclear magnetic resonance (NMR) spectroscopy. Ethyl acetate, ethanol, n-hexane extracts, and homopterocarpin exhibited antiplasmodial activity at 1.78, 2.21, 7.11, and 0.52 µg/ml, respectively, against P. falciparum 3D7 with low toxicity (selectivity index/SI ≥ 28.46). GC-MS identified compound showed in silico anti-SARS-CoV-2 binding affinity with stigmasterol and SARS-CoV-2 helicase of -8.2 kcal/mol. Ethyl acetate extract exhibited the best antioxidant activity against DPPH (0.76 ± 0.92 µg/ml) and ABTS (0.61 ± 0.46 µg/ml). They also demonstrated antimicrobial activity against B. subtilis, ethanol and ethyl acetate extracts against E. coli and C. albicans, and ethanol extract against S. aureus with diameter zone of inhibition of more than 1 cm. The results highlighted antiplasmodial activity of extracts and homopterocarpin from P. macrocarpus Kurz. heartwood and its potent binding in silico to anti-SARS-CoV-2 proteins with low toxicity. This study also confirmed that extracts exhibited antioxidant and antimicrobial activities. Further studies are needed to assess the safety and clinical trial of P. macrocarpus Kurz. for development as new drug candidate.

Production and characterization of thermostable acidophilic ß-mannanase from Aureobasidium pullulans NRRL 58524 and its potential in mannooligosaccharide production from spent coffee ground galactomannan.

The maximum yield of the crude mannanase from Aureobasidium pullulans NRRL 58524 was 8.42 ± 0.18 U mL-1 when cultured for 72 h in the optimized medium containing 3% (w v-1) defatted spent coffee grounds (SCG) and 0.67% (w v-1) ammonium sulphate. Two forms of mannanase were observed in the crude enzyme and the principal mannanase was enriched to apparent homogeneity via sequential filtration and anion exchange chromatography. The molecular mass of the enzyme was approximately 63 kDa as determined by SDS-PAGE. The enriched mannanase was active at high temperatures (45-75 °C) and a pH range from 3 to 6 with the maximum activity at 55 °C and pH 4.0. The enzyme was relatively thermostable with more than 75% of its initial activity remained after a 12 h incubation at 55 °C. The half-lives of the enriched mannanase were over 8 and 6 h at 60 and 65 °C, respectively. The enzyme was not adversely affected by chelator and most ions tested. This enzyme could hydrolyze both glucomannan and galactomannan and exhibited limited catalytic activity on beta-glucan. When the crude mannanase was used to hydrolyze galactomannan extracted from SCG, the maximum yield of reducing sugars mainly comprising of mannobiose (16.27 ± 0.84 mg 100 mg-1), and mannotriose (2.85 ± 0.20 mg 100 mg-1) was obtained at 58.22 ± 2.04 mg 100 mg-1 dry weight, under optimized condition (84.87 U g-1 mannanase, 41 h 34 min incubation at 55 °C and pH 4.0). These results suggested the prospect of the enzyme in mannan hydrolysis and mannooligosaccharide production at a larger scale. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03301-4.

Antioxidant and UV-Blocking Properties of a Carboxymethyl Cellulose-Lignin Composite Film Produced from Oil Palm Empty Fruit Bunch.

Oil palm empty fruit bunch (EFB) pulp with the highest cellulose content of 83.42% was obtained from an optimized process of acid pretreatment (0.5% v/v H2SO4), alkaline extraction (15% w/w NaOH), and hydrogen peroxide bleaching (10% w/v H2O2), respectively. The EFB cellulose was carboxymethylated, and the obtained carboxymethyl cellulose (CMC) was readily water-soluble (81.32%). The EFB CMC was blended with glycerol and cast into a composite film. Lignin that precipitated from the EFB black liquor was also incorporated into the film at different concentrations, and its effect on the UV-blocking properties of the film was determined. Interestingly, the EFB CMC film without lignin addition completely blocked UV-B transmittance. The incorporation of lignin at all concentrations significantly enhanced the UV-A blocking and other physical properties of the film, including the surface roughness, thickness, and thermal stability, although the tensile strength and water vapor permeability were not significantly affected. Complete UV-A and UV-B blocking were observed when lignin was added at 0.2% (w/v), and the film also exhibited the highest antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals with an half-maximal inhibitory concentration (IC50) value of 3.87 mg mL-1.

Production of cutinase from Fusarium falciforme and its application for hydrophilicity improvement of polyethylene terephthalate fabric.

Among 23 isolates of cutinase-producing fungi from Thailand, one strain of Fusarium falciforme PBURU-T5 exhibited the greatest cutinase activity (3.36 ± 0.12 U ml-1) against p-nitrophenyl butyrate. This strain was found to produce an inducible cutinase when cultivated in the liquid mineral medium containing cutin from papaya peel as the sole carbon source. By optimizing the production condition based on the central composite experimental design, the maximal cutinase activity up to 4.82 ± 0.18 U ml-1 was attained under the condition: 0.4% (w/v) papaya cutin as the carbon source, 0.3% (w/v) peptone as the nitrogen source, incubation temperature at 30 °C for 4 days, and initial pH 7.0. The crude enzyme was optimally active at 35 °C and pH 9.0 which was suitable for textile industrial application. The treatment with the crude PBURU-T5 cutinase (100 U g-1 dry weight of fabric) could enhance the wetting time, water adsorption and moisture regain of polyethylene terephthalate fabric up to 1.9-, 1.2- and 1.3-fold, respectively, comparing with the conventional 1M NaOH treatment. The increment of these fabric properties by enzymatic treatment could facilitate the dyeing process and enhance the fabric softness. Thus, F. falciforme PBURU-T5 is the promising source of cutinase for the modification of the PET fabric surface.

Enzymatic hydrolysis of tropical weed xylans using xylanase from Aureobasidium melanogenum PBUAP46 for xylooligosaccharide production.

The maximum yield of xylanase from Aureobasidium melanogenum PBUAP46 was 5.19 ± 0.08 U ml-1 when cultured in a production medium containing 3.89% (w/v) rice straw and 0.75% (w/v) NaNO3 as carbon and nitrogen sources, respectively, for 72 h. This enzyme catalyzed well and was relatively stable at pH 7.0 and room temperature (28 ± 2 °C). The produced xylanase was used to hydrolyze xylans from four tropical weeds, whereupon it was found that the highest amounts of reducing sugars in the xylan hydrolysates of cogon grass (Imperata cylindrical), Napier grass (Pennisetum purpureum), and vetiver grass (Vetiveria zizanioides) were at 20.44 ± 0.84, 17.50 ± 0.29, and 19.44 ± 0.40 mg 100 mg xylan-1, respectively, but it was not detectable in water hyacinth (Eichhornia crassipes) hydrolysate. The highest combined amount of xylobiose and xylotriose was obtained from vetiver grass; thus, it was selected for further optimization. After optimization, xylanase digestion of vetiver grass xylan at 27.94 U g xylan-1 for 92 h 19 min gave the highest amount of reducing sugars (23.65 ± 1.34 mg 100 mg xylan-1), which were principally xylobiose and xylotriose. The enriched XOs exhibited a prebiotic property, significantly stimulating the growth of Lactobacillus brevis and L. casei by a factor of up to 3.5- and 6.5-fold, respectively, compared to glucose.

Enzymatic Hydrolysis of Black Liquor Xylan by a Novel Xylose-Tolerant, Thermostable ß-Xylosidase from a Tropical Strain of Aureobasidium pullulans CBS 135684.

From three cell-associated ß-xylosidases produced by Aureobasidium pullulans CBS 135684, the principal enzyme was enriched to apparent homogeneity and found to be active at high temperatures (60-70 °C) over a pH range of 5-9 with a specific activity of 163.3 units (U) mg-1. The enzyme was thermostable, retaining over 80% of its initial activity after a 12-h incubation at 60 °C, with half-lives of 38, 22, and 10 h at 60, 65, and 70 °C, respectively. Moreover, it was tolerant to xylose inhibition with a K i value of 18 mM. The K m and V max values against p-nitrophenyl-ß-d-xylopyranoside were 5.57 ± 0.27 mM and 137.0 ± 4.8 µmol min-1 mg-1 protein, respectively. When combining this ß-xylosidase with xylanase from the same A. pullulans strain, the rate of black liquor xylan hydrolysis was significantly improved by up to 1.6-fold. The maximum xylose yield (0.812 ± 0.015 g g-1 dry weight) was obtained from a reaction mixture containing 10% (w/v) black liquor xylan, 6 U g-1 ß-xylosidase and 16 U g-1 xylanase after incubation for 4 h at 70 °C and pH 6.0.

The current status of Aureobasidium pullulans in biotechnology.

Different strains of the saprophytic yeast-like fungus Aureobasidium pullulans (Ascomycota: Dothideales) exhibit different biochemical characteristics, while their ubiquitous occurrence across diverse habitats and environmental conditions makes them an easily accessible source for biotechnological exploitation. They are useful in agricultural and industrial applications. Their antagonistic activities against postharvest pathogens make them suitable bioagents for the postharvest preservation of fruits and vegetables, while they possess antimicrobial activities against bacteria and fungi. Additionally, A. pullulans appears to be a potent source of single-cell protein. Many strains of A. pullulans harbor a wide range of industrially important enzymes, while the trademark exopolysaccharide pullulan that they produce has been extensively studied and is currently used in many applications. They also produce poly (ß-L-malic acid), heavy oil liamocins, siderophore, and aubasidan-like ß-glucan which are of interest for future applications. Ongoing studies suggest that A. pullulans holds many more interesting properties capable of further potential biotechnological applications.

Effect of polyols on thermostability of xylanase from a tropical isolate of Aureobasidium pullulans and its application in prebleaching of rice straw pulp.

In an attempt to find a thermostable xylanase enzyme for potential application in the pretreatment prior to H2O2 bleaching of paper pulp for industry, an extracellular xylanase from Aureobasidium pullulans CBS 135684 was purified 17.3-fold to apparent homogeneity with a recovery yield of 13.7%. Its molecular mass was approximately 72 kDa as determined by SDS-PAGE. The optimal pH and temperature for activity of the purified enzyme were pH 6.0 and 70°C, respectively. The enzyme was relatively stable at 50°C, retaining more than half of its original activity after 3-h incubation. The thermostability of the enzyme was improved by the addition of 0.75 mM sorbitol prolonging the enzyme's activity up to 10-fold at 70°C. When the potential of using the enzyme in pretreatment of rice straw pulp prior to bleaching was evaluated, the greatest efficiency was obtained in a mixture containing xylanase and sorbitol. Treatment of the rice straw pulp with xylanase prior to treatment with 10% (v/v) H2O2 and production of hand sheets increased the ISO sheet brightness by 13.5% and increased the tensile and tear strengths of the pulp by up to 1.16 and 1.71-fold, respectively, compared with pulps treated with H2O2 alone. The results suggested the potential application of the enzyme before the bleaching process of paper pulp when the maintenance of high temperature and enzyme stability are desirable.