Clinical presentation and fungal species distribution in otomycosis in a tertiary care hospital

Objective: Otomycosis is a common fungal ear infection in tropical and subtropical regions worldwide. This study aimed to perform mycological analysis on fungal debris from the external auditory canals of the patients to study the most common clinical presentation and fungal species distribution in otomycosis. Materials and Methods: Patients who met the inclusion criteria for this cross-sectional study were included and evaluated after providing written informed consent. After obtaining a thorough medical history, ear swabs for culture, sensitivity, and potassium hydroxide were provided. Patients with positive culture results were studied between September 2019 and March 2021. Results: Among 103 cases observed for 18 months in the Department of ENT, Rajarajeswari Medical College, and Hospital, Bengaluru, India, we found that males (56.31%) were more affected than females (43.68%). Itching (67.96%) was the most primary and common symptom that was observed, followed by pain (20.38%), and the most common predisposing factor was the usage of earbuds (26.21%) followed by water in the ear (23.3%) and oil in the ear (16.50%). Unilateral infection was most common (96%), and the left ear was most affected (64.07%). Aspergillus niger was the most common fungal isolate (60.19%), and otomycosis was very common in postmonsoon (October–December) (58.25%). Conclusion: The most frequent fungal isolates in otomycosis are from the Aspergillus and Candida species. The left ear was typically affected by otomycosis, which frequently had a unilateral predominance. The most common clinical symptoms were itching and pain.

Expression of β-glucosidase An-bgl3 from Aspergillus niger for conversion of scopolin / 生物工程学报

Scopoletin is a coumarin compound with various biological activities including detumescence and analgesic, insecticidal, antibacterial and acaricidal effects. However, interference with scopolin and other components often leads to difficulties in purification of scopoletin with low extraction rates from plant resource. In this paper, heterologous expression of the gene encoding β-glucosidase An-bgl3 derived from Aspergillus niger were carried out. The expression product was purified and characterized with further structure-activity relationship between it and β-glucosidase analyzed. Subsequently, its ability for transforming scopolin from plant extract was studied. The results showed that the specific activity of the purified β-glucosidase An-bgl3 was 15.22 IU/mg, the apparent molecular weight was about 120 kDa. The optimum reaction temperature and pH were 55 ℃ and 4.0, respectively. Moreover, 10 mmol/L metal ions Fe2+ and Mn2+ increased the enzyme activity by 1.74-fold and 1.20-fold, respectively. A 10 mmol/L solution containing Tween-20, Tween-80 and Triton X-100 all inhibited the enzyme activity by 30%. The enzyme showed affinity towards scopolin and tolerated 10% methanol and 10% ethanol solution, respectively. The enzyme specifically hydrolyzed scopolin into scopoletin from the extract of Erycibe obtusifolia Benth with a 47.8% increase of scopoletin. This demonstrated that the β-glucosidase An-bgl3 from A. niger shows specificity on scopolin with good activities, thus providing an alternative method for increasing the extraction efficiency of scopoletin from plant material.

Methods of plant growth-promoting fungi application to enhance the growth and yield of wheat var. Ibaa99

Aims@#The main aim of the study was to evaluate some methods of application of Aspergillus niger AD1 and Trichoderma hamatum T-113 for enhancing the growth and yield of wheat var. Ibaa99 in pots and field conditions.@*Methodology and results@#Plant growth-promoting fungi (PGPF) loaded with peat moss were used at a rate of 100, 150 and 200 mL pot-1 or m-2 in filed soil; seed treatment (coating) with fungi suspension 19 × 107, soil treatment and combination of all the three methods was employed in the study. Wheat seeds were sown in pots and field plots during 2018-2019, and data regarding various growth and yield attributes were recorded. In both pot and field trials, the results revealed that the best treatments for the desired plant growth and yield attributes were peat moss 150 mL alone or in combination with soil and seed treatments. The soil physicochemical parameters were also improved after inoculation with selected fungal isolates in different application methods compared with un-inoculated control treatment in both pot and field conditions.@*Conclusion, significance and impact of study@#The PGPF play a vital role represented phytoremediation, phytostimulation and bio-fertilization. The isolates of PGPF, which were applied with peat moss at 150 mL to the pot and in the field alone or combined with seed treatment and soil application, were significantly the best effective method for improving wheat attributes.

An efficient marker-free genome editing method for Aspergillus niger / 生物工程学报

Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.

Process optimization for enhanced production of cellulases form locally isolated fungal strain by submerged fermentation

Cellulase has myriad applications in various sectors like pharmaceuticals, textile, detergents, animal feed and bioethanol production, etc. The current study focuses on the isolation, screening and optimization of fungal strain through one factor at a time technique for enhanced cellulase production. In current study sixteen different fungal cultures were isolated and the culture which quantitatively exhibits higher titers of cellulase activity was identified both morphologically and molecularly by 18S rDNA and designated as Aspergillus niger ABT11. Different parameters like fermentation medium, volume, temperature, pH and nutritional components were optimized. The highest CMCase and FPase activities was achieved in 100ml of M5 medium in the presence of 1% lactose and sodium nitrate at 30 oC, pH5 after 72 hours. The result revealed A. niger can be a potential candidate for scale up studies.

CRISPR/Cas-based genome editing in Aspergillus niger / 生物工程学报

Aspergillus niger is a vital industrial workhouse widely used for the production of organic acids and industrial enzymes. This fungus is a crucial cell factory due to its innate tolerance to a diverse range of abiotic conditions, high production titres, robust growth during industrial scale fermentation, and status as a generally recognized as safe (GRAS) organism. Rapid development of synthetic biology and systems biology not only offer powerful approaches to unveil the molecular mechanisms of A. niger productivity, but also provide more new strategies to construct and optimize the A. niger cell factory. As a new generation of genome editing technology, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) system brings a revolutionary breakthrough in targeted genome modification for A. niger. In this review, we focus on current advances to the CRISPR/Cas genome editing toolbox, its application on gene modification and gene expression regulation in this fungal. Moreover, the future directions of CRISPR/Cas genome editing in A. niger are highlighted.

Molecular characterization of phosphate solubilizing fungi Aspergillus niger and its correlation to sustainable agriculture

Aim: The present study aimed to investigate the phosphate solubilization potential of agriculturally important fungi, i.e., Aspergillus sp. isolated from the rhizosphere of healthy plants in Abha city, Saudi Arabia.Methodology: Sixteen Aspergillus sp. isolated and tested for phosphate solubilization potential were identified by 5.8S-ITS region sequencing and characterized by 11 ISSR-PCR markers. Finally, the highest phosphate solubilization potential isolates were used in field experiments on cucumber and tomato plants. Results: All Aspergillus niger isolates showed 96–100% similarity to A. niger strains available at GenBank database, Isolate ASAB-5 was most efficient at solubilizing phosphate on Pikovskaya’s medium, with a solubilization index of 2.67, and 235.22 mg l-1 of solubilized phosphate. ISSR-PCR markers revealed is total 142 bands in all isolates, with about 32.3% showing monomorphism and 67.6% polymorphism. Based on genetic similarity and intraspecies variability, the Aspergillus isolates were grouped into two different clusters with about 67.9% genetic similarity. The results of field experiments showed no significant difference between seeds treated with culture filtrate or conidial suspension of ASAB-5; however, both differed remarkably from untreated seeds. Interpretation: The current study confirms the existence of several useful phosphate solubilizing fungi in plants, which may serve as potential biological fertilizers. They are safer than chemical fertilizers and increase the bioavailability of soil phosphates for plants

Albizia lebbeck fruit pods as a substrate for the production of lignocellulolytic enzymes by Aspergillus niger

Aspergillus niger isolated from the soil was investigated for its capability to produce various lignocellulolyticenzymes, such as LiP, endoglucanase, FPase, xylanase by solid-state fermentation, using Albizia lebbeck fruitpods as a substrate. The chemical composition of the fruit pods was studied, and the production pattern of theenzymes was examined by growing the fungi for 25 days. The LiP activity was low, whereas a good productionof endoglucanase, FPase, and xylanase enzymes was noted. A dye decolorization capacity of the A. niger wasalso studied with Congo red. Therefore, A. lebbeck fruit pods which are considered as waste and burnt off canbe utilized for the production of holocellulolytic enzymes using A. niger.

Improved protease activity of Pixian broad bean paste with cocultivation of Aspergillus oryzae QM-6 and Aspergillus niger QH-3

BACKGROUND: The preparation of broad bean koji is a key process in the production of Pixian broad bean paste (PBP). Protease is essential for the degradation of proteins during PBP fermentation. To obtain broad bean koji with high protease activity using the cocultivated strains of Aspergillus oryzae QM-6 (A. oryzae QM-6) and Aspergillus niger QH-3 (A. niger QH-3), the optimization of acid and neutral protease activities was carried out using Box­Behnken design with response surface methodology (RSM). RESULTS: The optimum conditions were found to be as follows: inoculation proportion (X1), 3:1 (A. oryzae QM-6: A. niger QH-3, w/w); culture temperature (X2), 33°C; inoculum size (X3), 0.5% (w/w); incubation time (X4), 5 d. The acid and neutral protease activities were 605.2 ± 12.4 U/g and 1582.9 ± 23.7 U/g, respectively, which were in good agreement with the predicted values. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles revealed that the broad bean koji extracellular proteins in the case of cocultivation were richer compared to those in the case of A. oryzae QM-6 or A. niger QH-3 strain only. In addition, the free amino acids (FAAs) in the fermentation product were 55% higher in the cocultivation process than in that involving only A. oryzae QM-6, further confirming the diversity of proteases in the fermentation products. CONCLUSIONS: The optimal conditions of koji-making in PBP were obtained using RSM. The cocultivation of A. oryzae and A. niger increases the overall enzyme activities in the culture medium and the FAAs content, which would thus have potential application in the PBP industry.

Synthesis And Biological Evaluation Of Thiosemicarbazone Analogues Of Ornidazole As Antifungal Agents

To synthesize a series of ornidazole thiosemicarbazone analogues on the basis of literature reviews of 2-Methyl-5-nitroimidazoles and thiosemicarbazones and to evaluate all the analogues in vitro for their activity against Aspergillus niger and fumigatus. Thiosemicarbazone analogues were synthesized from oxidising ornidazole with potassium dichromate and refluxing the oxidised product with substituted thiosemicarbazide using ethanol as solvent in the acidic medium overnight. All the synthesized analogues of ornidazole showed good antifungal action against fumigatus and niger except compound C-4. Unsubstituted amine analogue C-2 has shown highest percentage inhibition (96.6%, 500 μg/ml) against fumigatus while aromatic amine with or without electronegative atom analogues C-3 and C-5 has shown highest activity against Aspergillus niger which is two times than standard drug ornidazole (100%, 1000 μg/ml).

A simple and accurate method for specific quantification of biomass in mixed cultures of filamentous fungi by quantitative PCR / Un método simple y preciso para la cuantificación específica de biomasa en cultivos mixtos de hongos filamentosos por PCR cuantitativa

Abstract Production of lignocellulolytic enzymes by filamentous fungi have a great potential at industrial level due to their widespread applications. Mixed fungal cultures and particularly mixed fungal biofilms constitute a promising fermentation system for an enhanced enzyme production. However, it has not been addressed how much of this enhancement depends on the mixed biomass proportion. In this sense, the aim of this study was to develop a method to specifically and accurately quantify mixed fungal biomass. For this purpose, mixed biofilm cultures composed of Aspergillus niger and Trichoderma reesei, two filamentous fungi used industrially for cellulase production, were collected from 48 to 120 h of growth; mycelia were pulverized, and DNA was extracted for qPCR assays with specific primers for each fungus. Primers were designed from non-conserved regions of sequences of actin and β-tubulin genes of both A. niger and T. reesei. Specificity of these primers was tested in silico and experimentally. A statistically significant correlation was obtained between qPCR-calculated biomass and dry weight biomass data. By this method, it was possible to detect changes on mycelia proportions in biofilms over time, suggesting a competitive interaction between these two fungi. In conclusion, this method allows a specific and accurate quantification of mixed fungal biomass and could be also applied to different mixed culture systems for studying microbial interactions.

Resumen La producción de enzimas lignocelulolíticas por hongos filamentosos tiene un gran potencial a nivel industrial debido a sus diversas aplicaciones. Los cultivos fúngicos mixtos y particularmente las biopelículas fúngicas mixtas constituyen un sistema de fermentación prometedor para una mayor producción enzimática. Sin embargo, no se ha abordado cuánto de esta mejora depende de la proporción de biomasa mixta. En este sentido, el objetivo de este estudio fue desarrollar un método para cuantificar de forma específica y precisa la biomasa fúngica mixta. Para este propósito, se recolectaron cultivos mixtos de biopelículas de 48 a 120 h de crecimiento compuestos por Aspergillus niger y Trichoderma reesei, dos hongos filamentosos utilizados industrialmente para la producción de celulasas; el micelio se pulverizó y el ADN se extrajo para ensayos de qPCR con cebadores específicos para cada hongo. Los cebadores se diseñaron a partir de regiones no conservadas de las secuencias de los genes de actina y β-tubulina de A. niger y T. reesei. La especificidad de estos cebadores se probó in silico y experimentalmente. Se obtuvo una correlación estadísticamente significativa entre la biomasa calculada mediante qPCR y los datos de biomasa en peso seco. Mediante este método, fue posible detectar cambios en las proporciones de los micelios en las biopelículas a lo largo del tiempo, lo que sugiere una interacción competitiva entre estos dos hongos. En conclusión, este método permite una cuantificación específica y precisa de la biomasa fúngica mixta y también podría aplicarse a diferentes sistemas de cultivo mixto para estudiar interacciones microbianas.

A comparative study on enzyme production ability of microorganism producing γ-aminobutyric acid in processing of Sojae Semen Praeparatum at different pH and temperatures / 中草药

Objective: To lay a foundation for elucidating the mechanism of microbial regulation of γ-amino butyric acid (GABA) formation in the processing of Sojae Semen Praeparatum. Methods: The ability of glutamic acid decarboxylase and protease (neutral protease, alkaline protease and acid protease) production of Bacillus subtilis, Enterococcus avium, Enterococcus faecium, Bacillus amyloliquefaciens, Aspergillus niger, Aspergillus flavus, Aspergillus tamarii, Penicillium citrinum, Cladosporium tenuissimum, Phanerochaete sordida, Rhizopus oryzae, Sporidiobolus salmonicolor in different pH and temperature conditions were determined by Berthelot colorimetry and Folin phenol method. Results: The GAD and protease activities of the twelve strains were stronger at pH 5-7 and temperature 28-37 ℃. The highest GAD enzyme activity from A. flavus was 41.97 U/h, the optimum pH was 7 and the temperature was 28 ℃, followed by S. salmonicolor, A. niger, R. oryzae, and B. subtilis, respectively. The enzyme activities were 29.04, 25.78, 22.42 and 19.43 U/h. The highest neutral protease activity of B. subtilis was 24.80 U/mL, the optimum pH was 7 and the temperature was 37 ℃, followed by B. amyloliquefaciens, R. oryzae and E. avium. The enzyme activities were 16.86, 12.51 and 9.18 U/mL. The highest alkaline protease activity of B. amyloliquefaciens was 13.29 U/mL, the optimum pH was 7 and the temperature was 34 ℃, followed by B. subtilis and R. oryzae. The enzyme activities were 8.86 and 6.20 U/mL, respectively. The ability of 12 kinds of microorganisms to produce acid protease was generally poor. Conclusion: The optimal pH and temperature of the 12 kinds of microorganisms selected for this experiment are basically consistent with the natural processing of Sojae Semen Praeparatum. Among them, fungi have stronger GAD production capacity and bacteria have stronger neutral protease production capacity. The high GABA concentration of Sojae Semen Praeparatum is caused by the joint action of multiple strains.

Production Of Lignocelluloses From Aspergillus Niger Using Areca Nut Spade As Biomass

Areca palm (ChrysalidoCarpus lutescenes) a widely used plant having feathery arching brands with 100 leaflets. All these plants produce much of waste in additions to greeny and nuts. This waste of spade is used for the production of various molecules that are used in industry and pharma sector. Fermentation techniques are used to generate economically important enzymes for industrial and pharmaceutical purposes. Cellulase enzyme degrades the cellulose in between β-1, 4 glucosidic link found in lignocellulosic complex which under physical treatment is slower to degrade. The present study of Aspergillus niger for cellulose production was carried in solid state (SS) and submerged (SM) fermentations for production of cellulase enzyme. Cellulase production in SSF after 72 h of fermentation was 8.02 and in SMF activity was 2.98 per ml of cultured broth at H 6 and temperature at 30°C. Both SMF and SSF were supplemented with lactose and lactobionic acid, which acted as cellulase P production inducers. The aim of the present work was to study the effect of Areca palm spade as substrate for Aspergillus niger and its cellulase production under SMF and SSF.

Enhanced production of ß- glucosidase by locally isolated fungal strain employing submerged fermentation / Aumento da produção de ß-glucosidase por estirpe fúngica isolada localmente, por fermentação submersa

ß-glucosidase has wide spectrum of biotechnological applications in different industries including food, textile, laundry detergents, pulp and paper, pharmaceutical and biofuel industry. The present investigation related to isolation, screening, and process optimization of fungal strain for enhanced production of ß-glucosidase (BGL). For this purpose, different fungal stains were isolated from different sources including soil, fruits, bark of tree as well as from the compost. The screening of fungal strain for BGL production was carried out via submerged fermentation. All the tested strains were identified on the basis of micro and macroscopic features. The fungal strain having greater ability for BGL synthesis among tested ones wasidentified as Aspergillus niger and given the code SBT-15. The process parameter including fermentation media, temperature, pH, rate of fermentation, carbon and nitrogen sources, volume of media were optimized. Five different fermentation media were evaluatedM3medium gave maximum production. The optimal conditions for BGL production was 72 hours of incubation at 40°C, pH 6 and 50 ml fermentation medium. Glucose (1%) and ammonium sulphate(3%) were optimized as best carbon and nitrogen sources, respectively.

A ß-glicosidase possui amplo espectro de aplicações biotecnológicas em diferentes indústrias, incluindo alimentos, têxteis, detergentes para lavanderia, papel e celulose, indústria farmacêutica e de biocombustíveis, etc. A presente investigação relaciona-se ao isolamento e triagem e otimização de processos de cepas fúngicas para produção aumentada de ß- glucosidase (BGL). Para este efeito, diferentes manchas fúngicas foram isoladas a partir de diferentes fontes, incluindo solo, frutos, casca de árvore, bem como a partir do composto. A triagem da linhagem fúngica para produção de BGL foi realizada via fermentaçãosubmersa. Todas as cepas testadas foram identificadas com base em características micro e macroscópicas. A linhagem fúngica com maior capacidade de síntese de BGL entre os testados foi identificada como Aspergillus niger e recebeu o código SBT-15. O parâmetro do processo, incluindo meios de fermentação, temperatura, pH, taxa de fermentação, fontes de carbono e nitrogênio, volume de mídia foram otimizados. Cinco meios de fermentação diferentes foram avaliados. O meio M3 deu a produção máxima. As condições ótimas para a produção de BGL foram 72 horas de incubação a 40 ° C, pH 6 e 50ml de meio de fermentação. Glicose (1%) e sulfato de amônio (3%) foram otimizados como melhores fontes de carbono e nitrogênio, respectivamente.

Inhibitory Effect of the Stem Bark Extracts of Albizia chevalieri Harms on the Activity of Alpha-glucosidase Obtained from Aspergillus niger

The aim of the current study is to evaluate the inhibition of α-glucosidase activity by stem bark extract of Albizia chevalieri. The activity of alpha glucosidase was assayed in vitro using 50 mM acetate buffer pH 6.0 (prepared from acetic acid and sodium acetate) and various concentration of maltose (0.5 mM to 10 mM). Five test tubes, labeled TA – TE, each containing 1.5 ml of acetate buffer, 0.5 ml of alpha glucosidase and 0.5 ml of a known concentration of plant extract and control tubes (CA – CE) were assessed for Alpha glucosidase activity. The results showed that hexane, ethyl acetate and methanol extracts inhibited α-glucosidase activity. The results further indicated that the extracts act by competitive inhibition with inhibition constant of 232 mg/ml, 157 mg/ml and 67 mg/ml for hexane, ethyl acetate and methanol extracts, respectively. The value for the inhibition constants shows that there is a strong binding of the enzyme to the inhibitor as the polarity of solvent increases. The inhibitory activity of Albizia chevalieri may be due one or more of the phytochemicals present in the extracts.

Produção de ácido cítrico por Aspergillus niger AN 400 a partir de resíduo agroindustrial / Citric acid production by Aspergillus niger AN 400 from agroindustry waste

RESUMO A agroindústria gera grandes volumes de resíduos com carga poluidora elevada, o que exige o desenvolvimento de tecnologias para minimização de impactos causados pela disposição inadequada desses resíduos no ambiente. A produção de ácido cítrico utilizando resíduos agroalimentares como substrato para fermentação é uma solução para a redução da carga orgânica desses poluentes, além de agregar valor econômico pela geração de produto rentável. Aspergillus niger AN 400 foi utilizado para produzir ácido cítrico a partir de soro de queijo. A pesquisa foi dividida em três fases, conforme adição de açúcar extra (50, 100 e 150 g.L-1): Fase I, com glicose; Fase II, com sacarose; e Fase III, apenas com o soro de queijo, sem adição extra de açúcar. Os reatores permaneceram sob agitação de 150 rpm e a 30ºC, por 10 dias. A maior concentração de ácido cítrico (2.379 mg.L-1) foi observada quando da adição de 100 g.L-1 de glicose. Porém, em termos de produtividade, os maiores valores foram registrados nos reatores com 50 (458 mg.L-1.dia-1) e 100 g.L-1 (745 mg.L-1.dia-1) de sacarose, seguido pelo reator que continha apenas soro de queijo, sem adição de açúcar extra (313 mg.L-1.dia-1), demonstrando o potencial desse resíduo para a obtenção desse ácido de grande interesse comercial.

ABSTRACT The agro-industry generates large volumes of waste with high organic load, which requires the development of technologies to minimize the impacts caused by the improper disposal of this waste on the environment. The citric acid produced by agro food wastes as substrate for fermentation is a solution to reduce the organic load of these pollutants and add economic value by generating a profitable product. Aspergillus niger AN 400 was used to produce citric acid from cheese whey. The study was divided in three phases according to the addition of extra sugar (50, 100, 150 g.L-1): Phase I, with glucose; Phase II, with sucrose, and Phase III, with cheese whey only, without adding extra sugar. The reactors remained under agitation 150 rpm and at 30ºC for 10 days. The highest concentration of citric acid (2,379 mg.L-1) was observed upon the addition of 100 g.L-1 of glucose. However, the greatest yields were recorded in the reactors with 50 (458 mg.L-1.day-1) and 100 g.L-1 (745 mg.L-1. day-1) of sucrose, followed by the reactor that contained only cheese whey, without adding extra sugar (313 mg.L-1.day-1), demonstrating the potential of this waste to obtain citric acid with a great commercial interest.

Invitro antibacterial activity of Cassia fistula Linn methanolic leaf extracts

Background: Cassia fistula Linn is a plant which is widely grown in India and is used for medicinal purposes. The study was carried out with an objective to demonstrate the antimicrobial activity of leaves of Cassia fistula Linn. The aim of the study is to assess antibacterial and antifungal activity of methanolic leaf extract of Cassia fistula Linn against selected clinical isolates.Methods: The antimicrobial activity of methanolic extract of Cassia fistula was evaluated using agar well diffusion method and to zone of inhibition of extract was determined. Clinical isolates of Staphyloccocus aureus, MRSA, Pseudomonas aeruginosa, E. coli and Proteus were screened.Results: The methanolic extracts exhibited antibacterial activity against Staphylococcus aureus. The extract was not active against E. coli, Proteus, MRSA, Pseudomonas aeruginosa. The extract also failed to demonstrate antifungal activity against Candida albicans and Aspergillus niger.Conclusions: The global emergence of multidrug resistant bacterial strains is increasing, limiting the effectiveness of current drugs and treatment failure of infections. A novel approach to the prevention of antibiotic resistance of pathogenic species is the use of new compounds that are not based on existing synthetic antimicrobial agents.

High-cell density cultivation of recombinant Pichia pastoris harboring fungal endoinulinase and prebiotics applications of inulo-oligosaccharides

Aim: The objective of the present study was to clone and express A. niger endoinulinase gene in P. pastoris for high-level expression. Further to explore high cell density cultivation, biochemical characterization of recombinant endoinulinase and application of inulo-oligosaccharides (IOS) as prebiotics was also studied. Methodology: Molecular cloning of A. niger endoinulinase gene in P. pastoris, screening of positive clones by genomic DNA PCR, shake flask studies, high cell density fermentation performed with both conventional and temperature shift approach, biochemical characterization of endoinulinase and in-vitro fermentation of IOS was carried out to confirm prebiotic efficacy. Results: The endoinulinase gene of 1482 bp from Aspergillus niger was genetically engineered in the GS115 host and was secreted extracellularly using α signal sequence. As a result of fermentation with the conventional approach, recombinant endoinulinase activity was enhanced to 65.7 U ml-1. Recombinant endoinulinase showed absolute substrate specificity for inulin, hydrolyzing inulin to IOS with the DP range 3-4. Interpretation: Hydrolysis of inulin by recombinant endoinulinase was characterized. In-vitro fermentation of IOS by lactic acid and bifidogenic bacteria was studied as a part of industrial application and functional properties of IOS was similar to commercial prebiotics.

Purification and characterization of tannase from the local isolate of Aspergillus niger

Fungi of genus Aspergillus are active producers of extracellular tannase, and characteristics of enzyme production by local Aspergillus species have been well studied. The aim of this experimental study is isolation and partial purification of tannase enzyme from the Aspergillus niger fungus species. The purification was achieved by applying respectively ammonium sulfate (50-70%), dialysis with a specific activity 1227.08 (unit/mg protein), then passed the enzymatic extract through superdex 200 column in the gel filtration chromatography by using ÄKTA Pure 25 apparatus, the activity and the specific activity reached to 525.12 (unit/ml) and 2917.33 (unit/mg protein) respectively, with purification fold of 11.629 and yield 18.40%. One peak was obtained from an enzyme purity, which diagnosed by the absence of denaturation substances for protein SDS. Characteristics of pure enzyme showed that the molecular weight of the pure enzyme was determined, and it was found that the weight of enzyme was 95.49 KDa by using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, the enzyme was active in pH range 3 to 6 and temperature of 20 to 50°C. The optimum pH and the temperature for tannase activity were recorded to be pH 5 and 35°C, respectively. Moreover, the tannase activity was inhibited by Cu, Fe and Hg ions while increased by Mg and K ions when added at 1 and 5 mM

Preliminary Research on Dynamic Change of Microbial Population in Fermentation Process of Pinelliae Rhizoma Fermentata / 中国实验方剂学杂志

Objective:To detect the colony number of bacteria, yeasts and molds in fermentation process of Pinelliae Rhizoma Fermentata (PRF), microbial flora species, and quantitatively analyze the dynamic changes of four dominant microorganisms at different fermentation time points of PRF, so as to provide experimental basis for exploring the processing mechanism of PRF. Method:According to Pharmaceutical Standard Preparation of Traditional Chinese Medicine Prescription of Ministry of Health of the People's Republic of China (the 10th volume), PRF was processed. The samples at five different fermentation time points (0, 30, 60, 90, 120 h) of PRF were taken, the culturing, isolation and purification of bacteria, yeasts and molds were carried out with selective media, and the colonies were counted. Fluorescence quantitative polymerase chain reaction (PCR) technique was employed to conduct absolute quantification of Bacillus subtilis, Paecilomyces variotii, Byssochlamys spectabilis and Aspergillus niger. The recombinant plasmids of these 4 microorganisms were used as the standard substances, and the standard curves were prepared after dilution of multiple ratios, quantitative analysis was performed on these 4 microorganisms in five samples at different processing time points (0, 30, 60, 90, 120 h) of PRF. Result:During the fermentation process of PRF, the number of bacteria was low with smooth change, while molds and yeasts grew dramatically at the late stage of fermentation and reached 1×106 CFU·mL-1 at the end of fermentation. At 5 different fermentation time points, the copy numbers of Bacillus subtilis were 3.53×105, 7.56×104, 1.58×105, 1.90×106, 1.85×106 copies·g-1, the copy numbers of Paecilomyces variotii were 0, 0, 0, 3.45×107, 4.15×108 copies·g-1, the copy numbers of Byssochlamys spectabilis were 0, 0, 0, 1.04×108, 2.28×108 copies·g-1, the copy numbers of Aspergillus niger were 0, 0, 9.48×105, 1.47×106, 7.56×106 copies·g-1, respectively. Conclusion:The change trend of microflora in the fermentation process of PRF can be reflected by the dynamic change of four dominant microorganisms, and molds may play an important role in the processing of PRF. Fluorescence quantitative PCR technique has the advantages of rapidity, sensitivity, good repeatability and high specificity, it is suitable for exploring processing mechanism of PRF.