Browsing by Author "Prasanna, B.D."

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Biotechnology and Biochemical Engineering
(2016) Prasanna, B.D.; Gummadi, Sathyanarayana N; Vadlani, Praveen V.
Development of novel refining techniques and enzymatic synthesis of antioxidant esters for improving the oxidative stability of sardine oil
(National Institute of Technology Karnataka, Surathkal, 2018) Vaisali, C.; Prasanna, B.D.; Regupathi., I.
The growing market for n-3 polyunsaturated fatty acids (PUFA) has lead to the increasing amount of research in fish oil as they are excellent sources of n-3 PUFA. However, the highly unstable nature of these compounds has to be addressed. Hence, the present work deals with improving the stability of Indian sardine oil obtained from the western coastal region. This study aimed at developing novel refining strategies for removing impurities that caused poor stability and quality. Novel technologies involving simultaneous degumming and deacidification and membrane assisted free fatty acid removal were employed in the present work to eliminate the impurities in crude sardine oil that might increase the rate of oxidation. A novel degumming method involving triethanolamine resulted in simultaneous degumming and deacidification with phospholipid reduction from 303 ppm to 37 ppm, while the free fatty acids were reduced from 3.75% to 1.21%. Further removal of impurities was done by membrane assisted deacidification followed by bleaching using activated charcoal, resulting in refined sardine oil. The n-3 PUFA content in sardine oil was retained without much loss The best and convenient method for improving the oxidative stability of fish oils is by adding antioxidants which delays the oxidation process. In the current study, several natural antioxidants were studied for their effect in reducing the oxidation process in Indian sardine oil. Quercetin, rutin, gentisic acid and caffeic acid showed maximum effect in imparting oxidative stability. Further, the effect of antioxidants in stabilizing sardine oil in the presence of metal ions and trace water were tested. Though the effectiveness of antioxidants was not affected when sardine oil possessed trace metals, their efficiency reduced drastically with increase in trace water levels. Considering the relatively high antioxidant activity of rutin in Indian sardine oil and the complex heterogeneous nature of the oil, rutin was enzymatically modified toimprove its solubility in oil. It was identified that rutin was more effective during oxidation of refined sardine oil, whereas rutin ester showed higher activity in sardine oil containing trace water indicating the importance of antioxidant polarity and oil heterogeneity.
Studies on a Fibrinolytic Enzyme Produced From Marine Serratia marcescens subsp. sakuensis
(National Institute of Technology Karnataka, Surathkal, 2018) Krishnamurthy, Anusha; Prasanna, B.D.
Fibrinolytic enzymes are agents/drugs that are responsible for the breakdown of fibrin in the blood clots. They find application in treatment of myocardial infarctions, ischemic strokes, cardiac and respiratory failure. This research work was aimed at isolating a fibrinolytic enzyme producing microorganism and to assess its suitability as a potential drug candidate for therapeutic applications. Six out of the eight bacteria isolated from the sea water sample tested positive for the fibrinolytic enzyme production during the initial screening experiments. Based on the results from screening experiments, one out of the six bacteria was chosen for further work and was identified as Serratia marcescens subsp. sakuensis (KU296189.1). The medium components were optimised by one-factor-at-a-time approach and Plackett-Burman design for enhanced production of fibrinolytic enzyme. A 3.4 fold increase in fibrinolytic enzyme activity was obtained with optimised production medium. The crude enzyme solution was then purified by a three step process involving ammonium sulphate precipitation, dialysis and size exclusion chromatography. Properties of the purified enzyme such as the molecular weight, optimum pH and temperature, stability at different pH and temperature, in vitro half-life, effect of metal ions and chemical reagents, in vitro clot lysing potential and thrombolytic mechanism, proteolytic activity against several substrates and its partial amino acid sequence were determined. Chemical modification of the purified enzyme was carried out using amino acid specific modifiers and modification with 2.5 mM EDAC resulted in a 9- fold increase in the fibrinolytic activity. The circular dichroism spectrum analysis of the modified and native enzyme revealed a similar structure except a few minor changes in a- helix and ß-sheet conformation of the enzymes. The findings suggest that the fibrinolytic enzyme produced in the present work could be considered as a potential candidate, safe for human use.
Studies on the production of n-3 polyunsaturated fatty acid glyceride concentrate from Indian sardine oil
(National Institute of Technology Karnataka, Surathkal, 2018) Sampath, Charanyaa; Prasanna, B.D.; Regupathi., I.
Indian oil sardine (Sardinella longiceps) is a chief pelagic fishery resource of India and one of the richest and cheapest sources of n-3 polyunsaturated fatty acids (n-3 PUFA) such as Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA). The beneficial effects of n-3 PUFA in the prevention and treatment of coronary, neuromuscular, immunological disorders and allergic conditions are well documented. The crude oil extracted from Sardines contains mainly glycerides in addition to several undesirable components viz., free fatty acids, primary oxidation products, metal ions, pigments, moisture, phospholipids, phospholipases and insoluble impurities. Due to this, oil is highly susceptible to spoilage during storage and transhipment. Through a comprehensive study, a tailor made strategy was developed consisting of degumming, deacidification by solvent extraction, bleaching with granular activated carbon to eliminate all the aforesaid undesirable components, while retaining n-3 PUFA glycerides. This strategy was able to produce sardine oil of superior quality with minimal oil loss, without any loss of n-3 PUFA content. The effect of various extrinsic factors (light, temperature and moisture content) and intrinsic factors (metal ions, phosphotidylcholine, phospholipase-A and oleic acid) on storage stability of refined oil was undertaken to identifying the most detrimental factor during five-week storage. Moisture, ferric ions, oleic acid and sunlight were found to cause highest oxidative and hydrolytic instability and highest reduction in n- 3 PUFA content. Interestingly, even in the presence of ferric ions and oleic acid, phosphotidylcholine and phospholipase-A exhibited n-3 PUFA protection in spite of high oxidative and hydrolytic instability. In order to enhance n-3 PUFA content in the refined sardine oil, lipase mediated hydrolytic removal of unwanted fatty acids was attempted using Candida rugosa lipase (CRL) and Pseudomonas cepacia lipase. In order to facilitate reuse of CRL and to achieve higher efficiency and thermal stability, CRL was bioimprinted and immobilized. The preparation of immobilized enzyme and the hydrolysis of oil were optimized. The n-3 PUFA content in the deacidified oil was enriched up to 2.83-fold using bioimprinted immobilized lipase. The resultant oil had negligible di- and triglycerides content with the increase in the monoglyceride content. This proves higher efficiency in the hydrolysis of ester bonds of fatty acids,other than n-3 PUFA. The LC-MS data analysis of oil hydrolyzed by CRL-CLEA revealed the presence of increased quantities of monoglycerides of EPA and Palmitic acid (PA). Reusability studies showed the bioimprinted – immobilized lipase could be reused up to 5 runs without a substantial reduction in its performance.