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ThesisItem Open Access Influence of P. aeruginosa and glutathione on physio-chemical changes in Vigna mungo cultivars under PEG-6000 induced stress(Department of Biochemistry & Biochemical Engineering Jacob Institute of Biotechnology and Bioengineering Sam Higginbottom University of Agriculture, Technology and Sciences Allahabad, 2019) KESHARWANI, KOMAL; Sushma, Dr. (Mrs.)In the present study, the physiochemical changes by P. aeruginosa and glutathione under PEG (polyethylene glycol) induced stress were compared in two Vigna mungo varieties. Vigna mungo seeds were subjected to PEG solutions of two different concentration, 7% and 12%, glutathione solution of two different concentration, 50 ml L-1 and 100 ml L-1 and in combination with P. aeruginosa. Various physiological parameters, proline and chlorophyll content and various enzymatic antioxidants invertase, amylase, catalase, superoxide dimutase, glutathione reductase ascorbate peroxidase and non-enzymatic antioxidants GSH (reduced) and ascorbate were measured. Decrease in root length, fresh weight and dry weight was observed under different treatments but P. aeruginosa enhanced the root length, fresh and dry weight. Relative water content, chlorophyll, invertase, amylase, catalase decreased under PEG-6000, but showed enhancement under P. aeruginosa and where as decreased under glutathione. PEG stress reduced ascorbate content while glutathione and P. aeruginosa showed similar effect as control while GSH suddenly increase in both varities. Overall IPU2-43 showed better result than Shekhar-2 in all the parameters studied.ThesisItem Open Access Antioxidant and Polyphenolic Changes in Tomato during storage under different conditions(Department of Biochemistry and Biochemical Engineering, Jacob Institute of Biotechnology and Bio-Engineering, Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad, U.P. (India), 2020) MISHRA, SAUMYA; Prakash, Pfro. VeeruTomato, Lycopersicum esculentum, corresponds to the Solanaceae family of vegetables. L. esculentum belongs to a class of climacteric fruit which has a very short shelf life of usually 1-2 weeks. The present study was carried out to determine the optimum storage temperature and treatments by taking a record of various kinds of biochemical changes during the post-harvest storage for ensuring better quality for consumption and its shelf-life. Treatment of Calcium Chloride (1%, 2% and 5%), Hydrogen Peroxide (1%, 2% and 4%), Controlled atmosphere storage (1% CO2 & 0.5% O2; 2%CO2 & 1% O2; and 3% CO2 & 1 % O2) and 1-MCP (0.5 μL/L, 1.0 μL/L and 1.5 μL/L) were given to the tomato fruit of the Hisar Arun and Kashi Vishesh cultivar, harvested at three stages of ripening (Mature Green, Breaker, and Mature Red) and stored under three different temperatures (5°C, 10°C and 15°C). Changes in non-enzymatic and enzymatic activity as well as nutritive substances were evaluated after every 7 days up to a total of 21 days of the storage period. When treated with CaCl2 or H2O2, the non-enzymatic antioxidants (except lycopene) in both the cultivars of tomato and for all the treating conditions provided, showed an increasing trend until 14 days of storage after which, it started declining. On the other hand, the same exhibited a gradual, yet increasing trend all the way up to 21 days of the storage when treated with 1-MCP or provided Controlled Atmospheric Storage (CAS) condition. Particularly, corresponding to the mature green stage of Kashi Vishesh, the maximum (39%; 233.184) change in antioxidant activity was seen for 5% CaCl2 treated fruits after 14 days while minimum (16%; 181.824) change was perceived by the fruits treated with 0.5 μL/L 1-MCP after 21 days stored at 15°C. Nutritionally, ascorbic acid is very important for the human diet; and tomatoes are a rich source of ascorbic acid. The trend of change in ascorbic acid on the basis of maturity stages was different as compared to the other studied parameter. Highest changes were seen in mature green, followed by mature red. However, the breaker stages displayed least changes in due course of the storage at all conditions. The quality of fruits is decided by the phenolics, thus maintenance of phenolics during storage is crucial. Changes in total phenol content during storage was found to be temperature dependent, notably at 5°C storage temperature, the increase in the total phenol content was minimum, followed by that corresponding to 10°C, and 15°C. Initially, phenolic content was the highest in mature green fruits, but the rate of change was higher in mature red stages, followed by the breaker, and then in the mature green stage. The contents of carotenoids, as well as other chemoprotective substances, are highly conditioned by the genotype and environmental conditions. Percentage change in carotenoids increased up to 14 days in CaCl2 and H2O2 treated fruits; while the same increased consistently up to 21 days in CAS and 1-MCP treatment. Lowest carotenoid content was recorded in 5% CaCl2 (51.831 mg/100gm) in the mature red Hisar Arun cultivar corresponding to the storage temperature of 15°C. Lycopene is a natural plant nutrient with antioxidative properties and great health benefits. In general, a continuous rise in lycopene content was found up to 21 days of storage. A higher amount of change was recorded corresponding to the mature green fruit when treated with CaCl2 or H2O2. Whereas for the CAS and 1-MCP treated fruits, mature red fruits showed the maximum changes. Changes in Lycopene content of tomato fruits is found to vary in the range of 1.5-58% in the mature green of Hisar Arun. The enzymatic antioxidants assessed in the present study viz. Superoxide Dismutase (SOD), Glutathione reductase (GR), Ascorbate Peroxidase (APX) and Catalase (CAT) shown an increasing trend up to 21 days of storage. Specific activity of SOD (78.633 unit/mg/protein FW); APX (212.819 unit/mg/protein FW); GR (125.506 unit/mg/protein FW) and CAT (203.486 unit/mg/protein FW) were found to be maximum in the mature green fruits of Kashi Vishesh cultivar when treated with 5% CaCl2 stored at lower temperature (5°C) after 21 days of storage among all the studied conditions. In contrast to the aforementioned biochemical activities in tomatoes, which, in general, increased during the storage, Protein showed a totally different trend i.e. decreased from day one. Fruits of Kashi Vishesh harvested at the mature red stage when treated with 5% CaCl2 displayed the highest decrement while 1.5 μL/L 1-MCP showed the lowest change after 21 days of storage when stored under 15°C temperature. Sugars constitute an important component of tomato fruit as they determine sweetness and influence the overall tomato flavor. Reducing sugar in all the treated fruits samples were increased up to 21 days and the rate of change was highly dependent on the provided treatment. Reducing sugar content of local cultivar varied in the range of 2.245-3.201 mg/100gm FW; 3.146- 4.619 mg/100gm FW and 4.733-6.088 mg/100gm FW in the mature green, breaker and mature red fruits, respectively. Summarily, we found tomato fruits, harvested at the breaker stage, retained a significantly higher amount of non-enzymatic antioxidants after 14 days of storage compared to fruit harvested at the mature green and red stage. In addition, our investigations suggest the 0.5 μL/L 1-MCP treatment to be the most effective in terms of offering a definitive control in the rate of evolution of non-enzymatic antioxidants of the tomatoes such as lycopene and carotenoids. Further, although Hisar Arun (Local) variety is found to retain more nutritional content than Kashi Vishesh (hybrid) variety, the latter showed a higher shelf life.ThesisItem Open Access In vivo assessment of immunomodulatory and hepatoprotective potentials of Tinospora cordifolia in mice(Department of Biochemistry and Biochemical Engineering Jacob Institute of Biotechnology and Bioengineering Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad-211007, India, 2019) Srivastava, Radhika; Sushma, Dr. (Mrs.)The present study was carried out to of immunomodulatory and hepatoprotective potentials of aqueous extract Tinospora cordifolia. In the immunomodulatory and hepatoprotective potentials studies and immunomodulatory studies were conducted in mice which were divided into seven groups. The different groups in this study included group I control, group II reference control (silymarin 25 mg/kg. The mice were administered carbon tetrachloride orally at doses of 200 and 400 mg/kg mice body. Finally 200 mg/kg b.w. was taken as the therapeutic oral dose for aqueous extracts. The animals were examined daily for any clinical symptoms, blood samples were collected at different intervals like 7th, 14th, and 21st-day post induction of toxicity and levels of neutrophilic adhesion, DTH, serum Immunoglobin, cell-mediated immune response and HA titre measured.The results confirm the tough effect of carbon tetrachloride on the liver cause while enzymes consisting of SGOT, SGPT had been found eminent in the serum. In conclusion with these results, it can be said that the leaf extracts of T. cordifolia exhibited a hepatoprotective effect against carbon tetrachlorideinduced hepatotoxicity.ThesisItem Open Access Partial purification and industrial applications of alkaline protease from selected bacterial strains(DEPARTMENT OF BIOCHEMISTRY AND BIOCHEMICAL ENGINEERING JACOB INSTITUTE OF BIOTECHNOLOGY AND BIOENGINEERING SAM-HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY AND SCIENCES PRAYAGRAJ (ALLAHABAD) -211007, 2019) ASH, KRISHNA; Sushma, Dr. (Mrs.)Protease enzymes have immense commercial value and play a pivotal role in application of various industrial sectors. Microbial proteases are one of the important groups of industrially and commercially produced enzymes which have several applications. In this study 148 bacterial strains were isolated from 50 different soil samples of slaughter house, fish market and sewage wastes of Lucknow, Uttar Pradesh, India. Out of which ninety four strains competent of secreting extracellular alkaline protease. In preliminary screening the isolates SSB1 and SSB2 showed highest ability to hydrolyze casein and skimmed milk which was done on skim milk agar media. Based on biochemical tests the isolate showed positive for casein, starch, catalase and negative for gram staining, indole, methyl red, voges proskauer, gelatin, urea, oxidase, hemolysis and triple sugar iron test and found to be non motile. Strain SSB1 and SSB2 with the maximum yield alkaline protease was identified as Pseudomonas aeruginosa MH298778 and Acinetobacter variabilis MG650110 based on nucleotide homology and phylogenetic analysis (16S rDNA sequencing). Protease production was enhanced by optimizing the culture conditions. Many physical and chemical parameters were studied to optimize the maximum yield of alkaline protease. The maximum enzyme activity were observed with optimum incubation time, temperature, pH, carbon, nitrogen sources, NaCl and metallic ions were determined as 36 hrs, 37°C, pH 11.0, 1% glucose, 1% yeast extract, 1M NaCl, and 1mM Zn2+, respectively for protease production further the P.aeruginosa protease was purified to homogeneity using ammonium sulphate precipitation, dialysis and ion exchange chromatography with a fold purification of 2.28 and a recovery of 35.91%. The enzyme has a relative molecular weight of 30 kDa, pH and temperature optima for this protease were 9.0 and 40ᵒC. The activity was stable between a pH ranged of 8.0-10.0. Metal ions such as Ca2+ and Mg2+ stimulated the protease activity up to 115 and 109% respectively. The activity was completely inhibited by EDTA indicating the presence of metalloprotease. The protease was found to be stable in some commonly used commercial detergents tested (Wheel, Rin, Ariel and Tide). It retained 70% residual activity after 3hrs of incubation with Wheel, 71% with Rin, 73% with Ariel and 52% with Tide. The maximum stability was observed with Ariel. The 9 supplementation of the enzyme preparation in detergents could significantly improve the cleansing performance towards the blood and grass stains and suggest its possible use as a laundry additive. The protease was degrading 60% of poultry feathers in 48hrs showing its keratin degrading potential and it can be used as an additive in poultry industry as waste treatment. The A. Variabilis protease was purified to homogeneity using ammonium sulphate precipitation, dialysis and ion exchange chromatography with a fold purification of 2.01 and a recovery of 41.27%. The enzyme has a relative molecular weight of 28.5 kDa, pH and temperature optima for this protease were 11.0 and 40ᵒC. The activity was stable between a pH ranged of 9.0-12.0. Metal ions such as Ca2+ and Mg2+ stimulated the protease activity up to 110 and 104% respectively. The activity was completely inhibited by EDTA indicating the presence of metalloprotease. The protease was found to be stable in some commonly used commercial detergents tested (Wheel, Rin, Ariel and Tide). It retained 66% residual activity after 3hrs of incubation with Wheel, 72% with Rin, 76% with Ariel and 66% with Tide. The maximum stability was observed with Ariel. The supplementation of the enzyme preparation in detergents could significantly improve the cleansing performance towards the blood and grass stains and suggest its possible use as a laundry additive. The protease was degrading 60% of poultry feathers in 48hrs showing its keratin degrading potential and it can be used as an additive in poultry industry as waste treatment. The results of the application studies viz, compatibility with detergents, washing performance and feather degradation revealed that the bacterial strains P.aeruginosa and A. variabilis are potent source of alkaline protease. In consequence, these two strains have immense scope in various industries.ThesisItem Open Access Prediction of Drug Induced Nephrotoxicity Using Traditional Markers and Urinary Biomarkers in Sprague Dawley Rats(Department of Biochemistry and Biochemical Engineering Jacob School of Biotechnology and Bioengineering Sam Higginbottom University of Agriculture, Technology And Sciences, Allahabad-211007 (U.P.) India, 2019) Udupa, Venkatesha; Prakash, Prof. (Dr.) VeeruConsistent, sensitive biomarkers of acute kidney injury in animal models and humans have historically represented a poorly met need for investigators and clinicians. This research work was undertaken to evaluate novel renal markers use in identifying acute kidney injury (AKI) compared to traditional serum biomarkers which show changes when 30-40% of kidney is damaged in experimentally induced nephrotoxicity in male Sprague Dawley rats. Cisplatin and Gentamicin, a known nephrotoxicant is used at 2.5 and 5 mg/kg dose (single dose, intraperitoneal) and 30 and 100 mg/kg/day (subcutaneous) level for 7 consecutive days, respectively. On day 4 and day 8 post treatment, serum and urine samples from these rats were analysed for traditional (serum: blood urea nitrogen and creatinine, urine: protein, albumin and micro albumin (mALB) and novel biomarkers [Clusterin, kidney injury molecule-1 (Kim-1), Lipcalin-2/ Neutrophil gelatinase associated lipocalin (NGAL) and Cystatin C], light and electron (transmission and scanning) microscopic evaluation of kidneys were carried out. Further immunohistochemical localization of Kim-1 was carried out on day 4 and 8 kidney samples as well. Urinary biomarkers were increased several folds as compared to traditional markers at both doses of cisplatin and gentamicin treated rats and were duration and dose dependent. Histological and electron microscopic changes seen on Day 4 and Day 8 were of minimal to mild and moderate to severe in nature at both doses, respectively. Current experimental data suggests urine Clusterin and IP 10 are highly sensitive biomarkers of detecting nephrotoxicity with Cisplatin and Gentamicin in Sprague Dawley rats. Similarly, Kim-1 is also considered highly sensitive renal biomarker for Cisplatin and Gentamicin (only at 100 mg/kg) as the levels increased on day 4 that persisted until day 8 too. Urinary Kim-1 level, immunoreactivity and histological changes further confirms to several published reports and FDA and EMA recommendation that Kim-1 is highly sensitive biomarker for detecting drug induced acute renal injury. The results indicated that the novel urinary biomarkers are sensitive than traditional biomarkers in identifying kidney damage. Kim-l level and immunohistochemical localization demonstrated the correlation of the renal injury. Renal biomarkers is useful in identifying early kidney damage and thus assessing adversity in toxicology studies. In addition, this noninvasive method is useful in diagnosing acute kidney damage in patients when the renal damage is less than 30% which is not detected using traditional biomarkers.ThesisItem Open Access Nutritional and Antioxidative changes during maturation process of Dillenia pentagyna fruit(DEPARTMENT OF BIOCHEMISTRY AND BIOCHEMICAL ENGENEERING JACOB INSTITUTE OF BIOTECHNOLOGY AND BIOENGEENERING SAM-HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY AND SCIENCES ALLAHABAD-211007, 2017) Yadav, Ranjeet Kumar; SRIVASTAVA, Dr. Shailendra KumarEnzymatic, non-enzymatic antioxidants and nutritional compounds in Dillenia pentagyna fruits of four time harvests during maturation process were studied. The present study aims to pattern the antioxidant potential and also to identify the amino acid compounds in the fruit extracts were evaluated using Gas chromatography. The results revealed that the plant fruits contained appreciable amounts of proximate content was at the maximum level on the 55th days, i.e. moisture, ash, crude fiber and malic acid content and then significantly decreased on the 10th day and trace elements i.e. copper (Cu), nickel (Ni), manganese (Mn) and iorn (Fe) were decreases of 37.9%, 39.3%, 28.7% and 24.6% in the respect of 10th day fruit. The total phenol content in the fruit extracts (10D, 25D, 40D and 55D) expressed as gallic acid equivalent (GAE) was in the range of 0.352 to 0.703 mg GAE/g DW. Activity of Ascorbate peroxidase, guiacol peroxidase and glutathione reductase content in the fresh fruit extract (10D, 25D, 40D and 55D) expressed as μM/g fw were in the range of 406.90 to 708.66 μM/g fw, 0.372 to 0.207 μM/g fw and 0.117 to 0.184 μM/g fw while SOD, expressed as U/g fw was in the range 165.53 to 253.92 U/g fw. The results indicated that the Dillenia pentagyna fruits are nutrient-dense foods that can be good sources of ascorbic acid, -Tocopherol as well as carbohydrates, dietary fiber and trace elements can be utilized as a natural source of antioxidant. Keywords: Antioxidants, Dillenia pentagyna, Gas chromatography and Medicinal plantThesisItem Open Access Biochemical study on marine diatoms for biodiesel production(DEPARTMENT OF BIOCHEMISTRY AND BIOCHEMICAL ENGINEERING, JACOB INSTITUTE OF BIOTECHNOLOGY AND BIOENGINEERING, SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY AND SCIENCES, ALLAHABAD- 211007, 2019) Upadhyay, Mr. Shailesh; Lawrence, Dr. KapilSample (marine diatoms) were collected and isolated from Vellar estuary, Southeast coast of India (Bay of Bengal) in July-2014. Pure cultures of two species were subjected to morphological identification and DNA barcoding (rbcL gene sequencing). From the data two new strains of Odontella aurita (Accession number KR733098) and Chaetoceros muelleri (Accession number KR733100) were identified. Highest cell density for both the diatoms cultured in Guilard‟s f/2 nutrient medium under Si stress (53, 106 and 159μM Si) was observed on day 11. The highest increase the total lipid content in O. aurita and C. muelleri was 23.7±0.79 % and 25.6 ±0.48 % of dry biomass respectively under 53 μM Si stress. However, highest increase in cell density of 85.63 % and 45.93% as compared to the control was reported under 159 μM Si stress in O. aurita and C. muelleri respectively. Lipids extracted from both diatoms under 53 μM Si stress were transesterified (FAME) and analysed via GC-MS. In O. aurita FAME constituted: 43.61% SFA methyl esters, 33.22% MUFA methyl esters and 23.17% PUFA methyl esters whilst C. muelleri lipids contained was 69.62% SFA methyl esters, 16.53% MUFA methyl esters and 13.85% PUFA methyl esters. Three key enzymes involved lipid biosynthesis (acetyl Co-A carboxylase-EC 6.4.1.2, pyruvate dehydrogenase complex-EC 1.2.4.1 and malate dehydrogenase-EC 1.1.1.37) were also assayed in the crude extracts of the two species. As compared to control, activities of all three enzymes increased under 53μM Si stress. A similar trend was observed in the specific activities as well. The highest activity amongst the three enzymes assayed was reported for malate dehydrogenase at 53 μM Si stress in both species (737.46±14.5 for O. aurita and 777.46±22.1 for C. muelleri). This could be interpreted as a response to cope with onset of Si stress. The order of enzyme activity across treatments was: malate dehydrogenase > pyruvate dehydrogenase complex > acetyl Co-A carboxylase. A pH and temperature dependence was also observed. The results suggest that regulation of lipid biosynthesis under Si stress in O. aurita and C. muelleri is effected by the activity of these enzymes. However, a more detailed study is required to understand the underlying complex mechanisms. As compared to Odontella aurita (KR733098), Chaetoceros muelleri (KR733100) under 53μM Si stress is a probable indigenous species for biodiesel production.ThesisItem Open Access In vitro studies on the variations of biochemical metabolites in Glycyrrhiza glabra by using various elicitors(Department of Biochemistry & Biochemical Engineering, Jacob Institute of Biotechnology & Bioengineering, Sam Higginbottom University of Agriculture, Technology & Sciences Allahabad-211007, 2018) Jaiswal, Nancy; Verma, Dr. (Mrs.) YashodharaThe Glycyrrhiza glabra, of the Fabaceae family, is a medicinal and edible herbs that contain a wide range of phytochemicals which are used pharmaceutically and commercially. Glycyrrhiza glabra is under the threat of overexploitation and depletion therefore, there is an urgent need for conservation. It is advantageous to develop in vitro techniques not only for propagation, multiplication and preservation but also for elicitation of secondary metabolite production. The present study provides information on the micropropagation of G. glabra related to the use of different explants, the combination of plant growth regulators with different sterilization strategies, the culture conditions, and additional factors influencing in vitro propagation (such as light, temperature, humidity and pH). Conservation of germplasm from G. glabra, through encapsulation have also been studied which avail the germplasm for commercial cultivation over the long run. Qualitative and quantitative screening for phytoconstituents and the evaluation of their antimicrobial activity have also been presented. Enhanced production of glycyrrhizin, main bioactive component of G. glabra by different elicitors is deliberated. Successful multiplication and elicitation will lead to the production of not only greater quantities of planting material with improved quality but also commercially desired metabolites. This study will be helpful in future studies on somaclonal variation, genetic transformation and drug discovery.ThesisItem Open Access Optimization and Characterization of Purified Laccase from Bacterial and Fungal Sources(DEPARTMENT OF BIOCHEMISTRY AND BIOCHEMICAL ENGINEERING JACOB INSTITUTE OF BIOTECHNOLOGY AND BIOENGINEERING SAM-HIGGINBOTTOM UNIVERSITY OF AGRICULTURE AND TECHNOLOGICAL SCIENCES ALLAHABAD-211007, 2018) Mishra, Sarvesh Kumar; Srivastava, Shailendra KumarLaccase is an extensively studied enzyme because of its apparent use in several areas such as food, textile, paper and pulp industries. Laccase is the model enzymes for multi-copper oxidases can be used in bioremediation, beverage processing, ascorbic acid determination, baking, as a biosensor and to improve food sensory factors. Laccases have become significant industrial enzymes that can be used for a number of diverse applications. Laccase is broadly dispersed in the wide range of fungi as well as in bacteria. Laccase is more important because it oxidizes both toxic and nontoxic substrates. Laccase producing microorganism, Bacillus subtilis (MTCC 2057), Streptomyces lavendulae (MTCC 6821), Ganoderma lucidum (MTCC 1039) and Pleurotus ostreatus (MTCC 1802), were subjected to laccase production. The laccase production was optimized in the presence 2% carbon, 3% nitrogen, temperature 30°C, incubation time 60 hr and pH 5. The laccases were partially purified using ammonium salt precipitation method at an of 80% saturation. The 3% nitrogen sources, peptone and beef extract and 2% carbon sources, glucose and sucrose in the medium considerably increased the laccase production. Purified laccase showed highest laccase activity by ammonium sulfate precipitation at a saturation of 80%. The molecular weights of partially purified laccase were ~ 80kDa. The dialysis of bacterial fraction showed 1.43U/ml and 1.59U/ml enzyme activity, total protein 0.034mg/ml and 0.036mg/ml. The dialyzed fungal fraction showed 1.86U/ml and 2.03U/ml enzyme activities, total protein 0.031mg/ml and 0.032mg/ml. Subsequently, laccase was purified by DEAE-cellulose anion exchange ion-exchange chromatography. After each step of purification of the bacterial and fungal enzyme, the specific activity was increased. In this study, it was also identified that the purified laccase showed comparatively higher activity at pH 7 and high concentrations of metal CuSO4 and FeSO4 and organic solvent methanol. Enzymes were able to decolorize several dyes with over 80% and 90% reduction in color in case of methyl orange and trypan blue. This investigation thus conveys the higher yields of laccase produced from B. subtilis (MTCC 2057), S. lavendulae (MTCC 6821), G. lucidum (MTCC 1039) and P. ostreatus (MTCC 1802) strains with good potential for several industrial applications.