Response surface methodology-based optimisation of chitin production and its antioxidant activity from Aspergillusniger.

- In this study, we focused on isolating fungi capable of producing extracellular chitin, a critical component of fungal cell walls. Aspergillus niger was chosen as the candidate, and we aimed to optimise chitin production. Initially, one variable at a time (OVAT) method was used to enhance chitin yield under the best fermentation conditions. Subsequently, the Plackett-Burman design was employed to identify the key medium components influencing chitin production. These factors were then fine-tuned using the Central Composite Design, resulting in the optimal concentrations of dipotassium hydrogen phosphate (0.7 mg/l), calcium chloride (0.5 mg/l), thymine hydrochloride (0.5 mg/l), and pH (4), as confirmed by ANOVA. The application of response surface methodology (RSM) led to a remarkable improvement in chitin yield, increasing it from 1.14 g/l to an impressive 4.42 g/l, a substantial 3.34-fold enhancement compared to unoptimized conditions. Additionally, we explored the antioxidant activity of the produced chitin, revealing its promising properties with a scavenging activity ranging from 32% to 55% at concentrations of 1-2 mg/ml, surpassing the control. In conclusion, our study successfully optimized chitin production from Aspergillus niger and demonstrated the remarkable antioxidant potential of the produced chitin, highlighting its significance in various applications.

Cutinase production from Fusarium verticillioides using response surface methodology and its application as potential insecticide degrader.

Cutinase, a multifunctional enzyme, has shown great potential in environmental applications such as degradation of plastics and some commonly used insecticides. To overcome these environmental threatening problems, attempts should be made to enhance enzyme production. In the present study, a cutinolytic fungus was isolated from the soil. Based on 18 s rDNA sequencing, it was found that isolate AR08 belongs to the genus Fusarium and clades with Fusarium verticillioides. Optimization of medium composition for enhancement in cutinase production was done using. classical and statistical methods. Firstly, key factors were selected by one variable at a time (OVAT) method, then by Plackett- Burman design. Concentration of these important factors was optimized by Central Composite design. A total of 30 experiments were conducted and the optimized concentration of sodium nitrate, dipotassium hydrogen phosphate, flaxseed oil and zinc sulphate were found to be 0.455%, 0.305%, 2% and 0.0355% respectively. The result of ANOVA (analysis of variance) test revealed that p value was significant for the model. Interaction between flaxseed oil and sodium nitrate was found to have a positive effect on cutinase production. A 14.57 fold increase in enzyme activity was found under optimized conditions with the maximum cutinase activity of 626.6 IUml-1.

Amelioration of sperm immobilisation factor-induced infertility by bacterial antigenic determinants cross-reacting with spermatozoa.

A strain of Staphylococcus aureus, capable of invitro immobilisation of human and mouse spermatozoa, was already present in our laboratory. Therefore, in the present study, the factor responsible (sperm immobilisation factor, SIF) was isolated and purified. It was found to compromise not only motility, but also viability, morphology and Mg2+-ATPase activity of mouse spermatozoa. Also, SIF (250µgmL-1), when administered intravaginally in female BALB/c mice before mating, showed 100% contraceptive effect. Moreover, fluorescein isothiocyanate-labelled SIF was also found to bind mouse spermatozoa and various motile as well as non-motile bacteria, indicating the presence of common SIF-binding receptors on spermatozoa and bacteria. Further, to demonstrate molecular mimicry, the amelioration of SIF-induced impairment of sperm function by a SIF-binding bacterial receptor was compelling. For this, the SIF-binding receptor from Escherichia coli (E-SBR) was purified and evaluated for its ameliorative effect on SIF-induced sperm impairment invitro and invivo. Interestingly, upon the addition of mouse spermatozoa to SIF pre-incubated with E-SBR, an ameliorative effect against SIF-induced impairment of sperm function could be observed through analysis of normal sperm parameters (motility, viability, morphology, Mg2+-dependent ATPase levels). E-SBR also blocked binding of labelled SIF to spermatozoa and bacteria and alleviated SIF-induced infertility in female BALB/c mice. This provided evidence for molecular similarities between bacteria and spermatozoa, owing to which anti-bacterial antibodies cross-reacting with spermatozoa might be produced and infertility might follow.

An indigenous hyperproductive species of Aureobasidium pullulans RYLF-10: influence of fermentation conditions on exopolysaccharide (EPS) production.

In recent years, a significant interest has been generated in discovering and developing exopolysaccharides (EPS) produced by microorganisms, especially fungi due to their multifaceted industrial and pharmacological applications. A number of filamentous and cellular fungi have been explored for this; however, according to the existing literature, the work on exopolysaccharide production by indigenous culture on this aspect is still very less and requires a serious attention. The present work is an attempt in this regard and aims to optimize the submerged culture conditions to produce the exopolysaccharides from an indigenous yeast Aureobasidium pullulans RYLF-10 with respect to several operating parameters in shake flask fermentation. The yeast A. pullulans RYLF-10 was identified by 18s RNA sequencing and detailed study on its nutritional requirements, and environmental conditions for submerged culture have been optimized. The optimal temperature and pH for both the vegetative growth and EPS production were found to be 28 ± 1 °C and 5.0, respectively, while the agitation speed and inoculum size were reported to be 150 rpm and 1 % (v/v), respectively. Sucrose (50 g/l) and yeast extract (1 g/l) were found to be the most suitable carbon and nitrogen sources which worked best in the ratio of 60:1 and resulted in the maximum EPS yield. Similarly, the other variables like growth regulator (riboflavin) and minerals (NaCl + K2HPO4 + MgSO4) altogether resulted in a noteworthy EPS yield of 45.24 g/l which is the maximum yield from this indigenous isolate of A. pullulans RYLF-10.