The objectives of this study were to determine the efficacy of metabolites of a Streptomyces strain AS1 on (a) spore germination, (b) mycelial growth, (c) control of mycotoxins produced by Penicillium verrucosum (ochratoxin A, OTA), Fusarium verticillioides (fumonisins, FUMs) and Aspergillus fumigatus (gliotoxin) and (d) identify the predominant metabolites involved in control. Initial screening showed that the Streptomyces AS1 strain was able to inhibit the mycelial growth of the three species at a distance, due to the release of secondary metabolites. A macroscopic screening system showed that the overall Index of Dominance against all three toxigenic fungi was inhibition at a distance. Subsequent studies showed that the metabolite mixture from the Streptomyces AS1 strain was very effective at inhibiting conidial germination of P. verrucosum, but less so against conidia of A. fumigatus and F. verticillioides. The efficacy was confirmed in studies on a conducive semi-solid YES medium in BioScreen C assays. Using the BioScreen C and the criteria of Time to Detection (TTD) at an OD = 0.1 showed good efficacy against P. verrucosum when treated with the Streptomyces AS1 extract at 0.95 and 0.99 water activity (aw) when compared to the other two species tested, indicating good efficacy. The effective dose for 50% control of growth (ED50) at 0.95 and 0.99 aw were approx. 0.005 ng/ml and 0.15 μg/ml, respectively, with the minimum inhibitory concentration (MIC) at both aw levels requiring > 40 μg/ml. In addition, OTA production was completely inhibited by 2.5 μg/ml AS1 extract at both aw levels in the in vitro assays. Ten metabolites were identified with four of these being predominant in concentrations > 2 μg/g dry weight biomass. These were identified as valinomycin, cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val) and brevianamide F.
This study was conducted to evaluate the potential of pineapple peel (PP) and pineapple crown leaves (PCL) as the substrate for vanillic acid and vanillin production. About 202 ± 18 mg L-1 and 120 ± 11 mg L-1 of ferulic acid was produced from the PP and PCL respectively. By applied response surface methodology, the ferulic acid yield was increased to 1055 ± 160 mg L-1 by treating 19.3% of PP for 76 min, and 328 ± 23 mg L-1 by treating 9.9% of PCL for 36 min in aqueous sodium hydroxide solution at 120 °C. The results revealed that PP extract was better than PCL extract for vanillic acid and vanillin production. Furthermore, the experiment also proved that large volume feeding was more efficient than small volume feeding for high vanillic acid and vanillin yield. Through large volume feeding, about 7 ± 2 mg L-1 of vanillic acid and 5 ± 1 mg L-1 of vanillin was successfully produced from PP extract via Aspergillus niger fermentation.
In this study, a selected γ-aminobutyric acid (GABA)-rich Malaysian strain Aspergillus oryzae NSK was collected from soy sauce koji. The strain was used to explore the effect of using renewable native sugar syrup, sugarcane, nipa, and molasses as fermentable substrates for developing a novel functional GABA soy sauce. We evaluated the strain using the chosen native sugars for 7 days using shake flask fermentation at 30 °C. The results showed optimum GABA concentration was achieved using cane molasses as the fermentable substrate (354.08 mg/L), followed by sugarcane syrup (320.7 mg/L) and nipa syrup (232.07 mg/L). Cane molasses was subsequently utilized as a substrate to determine the most suitable concentration for A. oryzae NSK to enhance GABA production and was determined as 50% g/L of glucose standard cane molasses. Our findings indicate that cane molasses can be used as a GABA-rich ingredient to develop a new starter culture for A. oryzae NSK soy sauce production.
In submerged-liquid fermentation, seven key parameters were assessed using one-factor-at-a-time to obtain the highest GABA yield using an industrial soy sauce koji Aspergillus oryzae strain NSK (AOSNSK). AOSNSK generated maximum GABA at 30 °C (194 mg/L) and initial pH 5 (231 mg/L), thus was able to utilize sucrose (327 mg/L of GABA) for carbon source. Sucrose at 100 g/L, improved GABA production at 646 mg/L. Single nitrogen sources failed to improve GABA production, however a combination of yeast extract (YE) and glutamic acid (GA) improved GABA at 646.78 mg/L. Carbon-to-nitrogen ratio (C8:N3) produced the highest cell (24.01 g/L) and GABA at a minimal time of 216 h. The key parameters of 30 °C, initial pH 5, 100 g/L of sucrose, combination YE and GA, and C8:N3 generated the highest GABA (3278.31 mg/L) in a koji fermentation. AOSNSK promisingly showed for the development of a new GABA-rich soy sauce.
To review invasive aspergillosis (IA) in developing countries, we included those countries, which are mentioned in the document of the International Monetary Fund (IMF), called the Emerging and Developing Economies List, 2009. A PubMed/Medline literature search was performed for studies concerning IA reported during 1970 through March 2010 from these countries. IA is an important cause of morbidity and mortality of hospitalized patients of developing countries, though the exact frequency of the disease is not known due to inadequate reporting and facilities to diagnose. Only a handful of centers from India, China, Thailand, Pakistan, Bangladesh, Sri Lanka, Malaysia, Iran, Iraq, Saudi Arabia, Egypt, Sudan, South Africa, Turkey, Hungary, Brazil, Chile, Colombia, and Argentina had reported case series of IA. As sub-optimum hospital care practice, hospital renovation work in the vicinity of immunocompromised patients, overuse or misuse of steroids and broad-spectrum antibiotics, use of contaminated infusion sets/fluid, and increase in intravenous drug abusers have been reported from those countries, it is expected to find a high rate of IA among patients with high risk, though hard data is missing in most situations. Besides classical risk factors for IA, liver failure, chronic obstructive pulmonary disease, diabetes, and tuberculosis are the newly recognized underlying diseases associated with IA. In Asia, Africa and Middle East sino-orbital or cerebral aspergillosis, and Aspergillus endophthalmitis are emerging diseases and Aspergillus flavus is the predominant species isolated from these infections. The high frequency of A. flavus isolation from these patients may be due to higher prevalence of the fungus in the environment. Cerebral aspergillosis cases are largely due to an extension of the lesion from invasive Aspergillus sinusitis. The majority of the centers rely on conventional techniques including direct microscopy, histopathology, and culture to diagnose IA. Galactomannan, β-D glucan test, and DNA detection in IA are available only in a few centers. Mortality of the patients with IA is very high due to delays in diagnosis and therapy. Antifungal use is largely restricted to amphotericin B deoxycholate and itraconazole, though other anti-Aspergillus antifungal agents are available in those countries. Clinicians are aware of good outcome after use of voriconazole/liposomal amphotericin B/caspofungin, but they are forced to use amphotericin B deoxycholate or itraconazole in public-sector hospitals due to economic reasons.
Lipases are enzyme with versatile industrial applications can be produced by the solid-state fermentation (SSF) method and is an economical alternative for enzyme production assisted by fungus. In Malaysia, 5 million of copra waste were generated annually. Large amount of copra waste produced will cause an increasing amount of the waste dumped to the landfill. Copra waste is one of the potential substrates to produce lipase enzyme through SSF. Thus, the aim of this study is to optimize the lipase production by SSF associated by Aspergillus niger using the 23 full factorial design approach. In this study the factors affecting parameters that involved in the production of lipase enzyme such as temperature (25˚ and 35˚), substrates concentration (40% and 60%) and inoculum size of Aspergillus niger (1 and 9 petri dish) were determined. The maximum production of lipase was obtained after 120-hour incubation in SSF. The optimum condition for inoculum size of Aspergillus niger was 9 plates, 30°C of incubation temperature and 60 % moisture contents. The range of the concentration of lipase enzyme produced varied from 105 U/ml to 170 U/ml. When applied to the wastewater treatment, the reducing percentage of fat, oil and grease (FOG) in food processing wastewater is reduced from 219.4925mg/l to 169.467mg/l accounted to the amount of 34 % FOG removal. Lipase produced using copra waste as a substrate using SSF has the potential value to be developed in the future for various industry including wastewater treatment industry.
The present study aimed to investigate the removal efficiency of cephalexin (CFX) by a novel Cu-Zn bionanocomposite biosynthesized in the secondary metabolic products of Aspergillus arenarioides EAN603 with pumpkin peels medium (CZ-BNC-APP). The optimization study was performed based on CFX concentrations (1, 10.5 and 20 ppm); CZ-BNC-APP dosage (10, 55 and 100 mg/L); time (10, 55 and 100 min), temperature (20, 32.5 and 45 °C). The artificial neural network (ANN) model was used to understand the CFX behavior for the factors affecting removal process. The CZ-BNC-APP showed an irregular shape with porous structure and size between 20 and 80 nm. The FTIR detected CC, C-O and OH groups. ANN model revealed that CZ-BNC-APP dosage exhibited the vital role in the removal process, while the removal process having a thermodynamic nature. The CFX removal was optimized with 12.41 ppm CFX, 60.60 mg/L of CZ-BNC-APP, after 97.55 min and at 35 °C, the real maximum removal was 95.53% with 100.52 mg g-1 of the maximum adsorption capacity and 99.5% of the coefficient. The adsorption of CFX on CZ-BNC-APP was fitted with pseudo-second-order model and both Langmuir and Freundlich isotherms models. These findings revealed that CZ-BNC-APP exhibited high potential to remove CFX.
In this study, pineapple cannery waste materials were used as substrate for the microbial production of vanillic acid and vanillin by Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL 39533. Biotransformation of ferulic acid from pineapple waste by A. niger I-1472 to vanillic acid was optimized using Response Surface Methodology (RSM). A central composite rotatable design was used to allocate treatment combinations and factors tested for their influence on vanillic acid production were inoculum size, yeast extract concentration, diammonium tartrate concentration and initial medium pH. The amount of vanillic acid produced was used as the response for the fermentation study and was assumed to be under the influence of the four factors tested. The estimated conditions for optimal vanillic acid production were inoculum size, 3.08 ×105 CFU mL-1; yeast extract, 0.37 gL-1; diammonium tartrate, 3.88 gL-1 and initial pH, 4.3. Subsequent biotransformation of vanillic acid by P. cinnabarinus MUCL 39533 to vanillin was enhanced with the addition of resin. Under these optimal conditions, 141.00 mgL-1 of vanillin was produced from 5 g of pineapple cannery waste.
A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg(++), Fe(++), Zn(++), Cu(++), and Pb(++)) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries.
This work aims at optimizing the media constituents for citric acid production from oil palm empty fruit bunches (EFB) as renewable resource using artificial neural networks (ANN) approach. The bioconversion process was done through solid state bioconversion using Aspergillus niger. ANN model was built using MATLAB software. A dataset consists of 20 runs from our previous work was used to develop ANN. The predictive and generalization ability of ANN and the results of RSM were compared. The determination coefficients (R2-value) for ANN and RSM models were 0.997 and 0.985, respectively, indicating the superiority of ANN in capturing the non-linear behavior of the system. Validation process was done and the maximum citric acid production (147.74 g/kg-EFB) was achieved using the optimal solution from ANN which consists of 6.1% sucrose, 9.2% mineral solution and 15.0% inoculum.
Microfungi isolated from Malay traditional vegetables such as Centella asiatica, Cosmos caudatus, Oenanthe javanica, Persicaria odorata and Psophocarpus tetragonolobus are well diverse. A total of 40 isolates of the fungi were identified and classified into four genera such as Aspergillus, Fusarium, Penicillium and Trichoderma. Five species of Fusarium were morphologically identified as F. oxysporum, F. semitectum, F. proliferatum, F. solani and F. konzum. Three species of Aspergillus were identified as A. niger, A. fumigatus and A. flavus. The highest number of microfungi was isolated from Cosmos caudatus (12 isolates), followed by Persicaria odorata (9 isolates), Oenanthe javanica (8 isolates), Centella asiatica (6 isolates) and Psophocarpus tetragonolobus (5 isolates). Four isolates of Fusarium species were able to produce moniliformin (MON) and five isolates were able to produce fumonisin B1 (FB1). This is the first report on diversity of microfungi associated with some Malay traditional vegetables.
The present study deals with optimizing, producing, characterizing, application and techno- economic analysis of oxidative enzymes [Laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP)] from Aspergillus iizukae EAN605 in submerged fermentation process using pumpkin peels as a production substrate. The best operating parameters for producing Lac, MnP and LiP (6.15, 2.58 and 127.99 U mg-1 respectively) were recorded with 20 g 100 mL-1 of substrate, 4.6 mL 100 mL-1 of inoculum size at pH 5.5 after 10 days. The crude enzyme exhibited high stability at pH (3-9) and temperatures (20-60 °C). Km (Michaelis-Menten) of Lac, MnP and LiP crude enzyme was 2.25, 1.79 and 0.72 mM respectively. The decolourization of Remazol Brilliant Blue R by the crude enzyme was 84.84 %. The techno-economic analysis was assessed for a production unit with an annual operating time for enzymatic production and application is 7920 h/year and 100 m3 of the capacity. The process would produce 27,000 cm3 of crude enzyme with a price of USD 0.107 per cm3 compared to USD 1 per cm3 of the current commercial enzyme. The findings indicated that pumpkin peels have potential as a production substrate for oxidative enzymes from A. iizukae EAN605 and is economically feasible.
The conventional treatment process of palm oil mill effluent (POME) produces a highly colored effluent. Colored compounds in POME cause reduction in photosynthetic activities, produce carcinogenic by-products in drinking water, chelate with metal ions, and are toxic to aquatic biota. Thus, failure of conventional treatment methods to decolorize POME has become an important problem to be addressed as color has emerged as a critical water quality parameter for many countries such as Malaysia. Aspergillus fumigatus isolated from POME sludge was successfully grown in POME supplemented with glucose. Statistical optimization studies were conducted to evaluate the effects of the types and concentrations of carbon and nitrogen sources, pH, temperature, and size of the inoculum. Characterization of the fungus was performed using scanning electron microscopy, Fourier transform infrared (FTIR) spectroscopy, and Brunauer, Emmet, and Teller surface area analysis. Optimum conditions using response surface methods at pH 5.7, 35 °C, and 0.57 % w/v glucose with 2.5 % v/v inoculum size resulted in a successful removal of 71 % of the color (initial ADMI of 3,260); chemical oxygen demand, 71 %; ammoniacal nitrogen, 35 %; total polyphenolic compounds, 50 %; and lignin, 54 % after 5 days of treatment. The decolorization process was contributed mainly by biosorption involving pseudo-first-order kinetics. FTIR analysis revealed that the presence of hydroxyl, C-H alkane, amide carbonyl, nitro, and amine groups could combine intensively with the colored compounds in POME. This is the first reported work on the application of A. fumigatus for the decolorization of POME. The present investigation suggested that growing cultures of A. fumigatus has potential applications for the decolorization of POME through the biosorption and biodegradation processes.
Clitoria ternatea is known for its antimicrobial activity but the antifungal effects of leaf extract on growth and morphogenesis of Aspergillus niger have not been observed. The extract showed a favorable antifungal activity against A. niger with a minimum inhibition concentration 0.8 mg/mL and minimum fungicidal concentration 1.6 mg/mL, respectively. The leaf extract exhibited considerable antifungal activity against filamentous fungi in a dose-dependent manner with 0.4 mg/mL IC50 value on hyphal growth of A. niger. The main changes observed under scanning electron microscopy after C. ternatea extract treatment were loss of cytoplasm in fungal hyphae and the hyphal wall and its diameter became markedly thinner, distorted, and resulted in cell wall disruption. In addition, conidiophore alterations were also observed when A. niger was treated with C. ternatea leaf extract.
Fungal endophthalmitis is rare but may complicate glaucoma drainage device surgery. Management is challenging as the symptoms and signs may be subtle at initial presentation and the visual prognosis is usually poor due to its resistant nature to treatment. At present there is lesser experience with intravitreal injection of voriconazole as compared to Amphotericin B. We present a case of successfully treated Aspergillus endophthalmitis following Baerveldt glaucoma drainage device implantation with intravitreal and topical voriconazole.
A novel concept for the synthesis of a stable polymer hybrid matrix bead was developed in this study. The beads were further applied for enzyme immobilization to produce stable and active biocatalysts with low enzyme leakage, and high immobilization efficiency, enzyme activity, and recyclability. The immobilization conditions, including PEI concentration, activation time and pH of the PEI solution were investigated and optimized. All formulated beads were characterized for its functionalized groups, composition, surface morphology and thermal stability. Compared with the free β-glucosidase, the immobilized β-glucosidase on the hybrid matrix bead was able to tolerate broader range of pH values and higher reaction temperature up to 60 °C. The immobilized β-glucosidase was then used to hydrolyse pretreated macroalgae cellulosic residue (MCR) for the production of reducing sugar and a hydrolysis yield of 73.4% was obtained. After repeated twelve runs, immobilized β-glucosidase retained about 75% of its initial activity.
This study evaluated the potential of bioflocculant production from Aspergillus niger using palm oil mill effluent (POME) as carbon source. The bioflocculant named PM-5 produced by A. niger showed a good flocculating capability and flocculating rate of 76.8% to kaolin suspension could be achieved at 60 h of culture time. Glutamic acid was the most favorable nitrogen source for A. niger in bioflocculant production at pH 6 and temperature 35 °C. The chemical composition of purified PM-5 was mainly carbohydrate and protein with 66.8% and 31.4%, respectively. Results showed the novel bioflocculant (PM-5) had high potential to treat river water from colloids and 63% of turbidity removal with the present of Ca(2+) ion.
The ease with which auxotrophic strains and genes that complement them can be manipulated, as well as the stability of auxotrophic selection systems, are amongst the advantages of using auxotrophic markers to produce heterologous proteins. Most auxotrophic markers in Aspergillus oryzae originate from chemical or physical mutagenesis that may yield undesirable mutations along with the mutation of interest. An auxotrophic A. oryzae strain S1 was generated by deleting the orotidine-5'-monophosphate decarboxylase gene (pyrG) by targeted gene replacement. The uridine requirement of the resulting strain GR6 pyrGΔ0 was complemented by plasmids carrying a pyrG gene from either Aspergillus nidulans or A. oryzae. β -Galactosidase expression by strain GR6 pyrGΔ0 transformed with an A. niger plasmid encoding a heterologous β -galactosidase was at least 150 times more than that obtained with the untransformed strain. Targeted gene replacement is thus an efficient way of developing auxotrophic mutants in A. oryzae and the auxotrophic strain GR6 pyrGΔ0 facilitated the production of a heterologous protein in this fungus.
The study is to identify the extraction of intracellular inulinase (exo- and endoinulinase) and invertase as well as optimization medium composition for maximum productions of intra- and extracellular enzymes from Aspergillus niger ATCC 20611. From two different methods for extraction of intracellular enzymes, ultrasonic method was found more effective. Response surface methodology (RSM) with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. The effect of five main reaction parameters including sucrose, yeast extract, NaNO₃, Zn⁺², and Triton X-100 on the production of enzymes was analyzed. A modified quadratic model was fitted to the data with a coefficient of determination (R²) more than 0.90 for all responses. The intra-extracellular inulinase and invertase productions increased in the range from 16 to 8.4 times in the optimized medium (10% (w/v) sucrose, 2.5% (w/v) yeast extract, 2% (w/v) NaNO₃, 1.5 mM (v/v) Zn⁺², and 1% (v/v) Triton X-100) by RSM and from around 1.2 to 1.3 times greater than in the medium optimized by one-factor-at-a-time, respectively. The results of bioprocesses optimization can be useful in the scale-up fermentation and food industry.
The effect of heat treatment below the gelatinization temperature on the susceptibility of corn, mung bean, sago, and potato starches towards granular starch hydrolysis (35°C) was investigated. Starches were hydrolyzed in granular state and after heat treatment (50°C for 30 min) by using granular starch hydrolyzing enzyme for 24 h. Hydrolyzed heat-treated starches showed a significant increase in the percentage of dextrose equivalent compared to native starches, respectively, with corn 53% to 56%, mung bean 36% to 47%, sago 15% to 26%, and potato 12% to 15%. Scanning electron microscopy micrographs showed the presence of more porous granules and surface erosion in heat-treated starch compared to native starch. X-ray analysis showed no changes but with sharper peaks for all the starches, suggested that hydrolysis occurred on the amorphous region. The amylose content and swelling power of heat-treated starches was markedly altered after hydrolysis. Evidently, this enzyme was able to hydrolyze granular starches and heat treatment before hydrolysis significantly increased the degree of hydrolysis.