Process Optimization for Mass Production of Marine Yeast Candida sp. S 27 and its Nutritional Characterization

The main source of protein for human and animal consumption is from the agricultural sector, where the production is vulnerable to diseases, fluctuations in climatic conditions and deteriorating hydrological conditions due to water pollution. Therefore Single Cell Protein (SCP) production has evolved as an excellent alternative. Among all sources of microbial protein, yeast has attained global acceptability and has been preferred for SCP production. The screening and evaluation of nutritional and other culture variables of microorganisms are very important in the development of a bioprocess for SCP production. The application of statistical experimental design in bioprocess development can result in improved product yields, reduced process variability, closer confirmation of the output response to target requirements and reduced development time and overall cost.The present work was undertaken to develop a bioprocess technology for the mass production of a marine yeast, Candida sp.S27. Yeasts isolated from the offshore waters of the South west coast of India and maintained in the Microbiology Laboratory were subjected to various tests for the selection of a potent strain for biomass production. The selected marine yeast was identified based on ITS sequencing. Biochemical/nutritional characterization of Candida sp.S27 was carried out. Using Response Surface Methodology (RSM) the process parameters (pH, temperature and salinity) were optimized. For mass production of yeast biomass, a chemically defined medium (Barnett and Ingram, 1955) and a crude medium (Molasses-Yeast extract) were optimized using RSM. Scale up of biomass production was done in a Bench top Fermenter using these two optimized media. Comparative efficacy of the defined and crude media were estimated besides nutritional evaluation of the biomass developed using these two optimized media.

Studies on Production of Bacterial Xylanases

Xylanases with hydrolytic activity on xylan, one of the hemicellulosic materials present in plant cell walls, have been identified long back and the applicability of this enzyme is constantly growing. All these applications especially the pulp and paper industries require novel enzymes. There has been lot of documentation on microbial xylanases, however, none meeting all the required characteristics. The characters being sought are: higher production, higher pH and temperature optima, good stabilities under these conditions and finally the low associated cellulase and protease production. The present study analyses various facets of xylanase biotechnology giving emphasis on bacterial xylanases. Fungal xylanases are having problems like low pH values for both enzyme activity and growth. Moreover, the associated production of cellulases at significant levels make fungal xylanases less suitable for application in paper and pulp industries.Bacillus SSP-34 selected from 200 isolates was clearly having xylan catabolizing nature distinct from earlier reports. The stabilities at higher temperatures and pH values along with the optimum conditions for pH and temperature is rendering Bacillus SSP-34 xylanase more suitable than many of the previous reports for application in pulp and paper industries.Bacillus SSP-34 is an alkalophilic thertmotolerant bacteria which under optimal cultural conditions as mentioned earlier, can produce 2.5 times more xylanase than the basal medium.The 0.5% xylan concentration in the medium was found to the best carbon source resulting in 366 IU/ml of xylanase activity. This induction was subjected to catabolite repression by glucose. Xylose was a good inducer for xylanase production. The combination of yeast extract and peptone selected from several nitrogen sources resulted in the highest enzyme production (379+-0.2 IU/ml) at the optimum final concentration of 0.5%. All the cultural and nutritional parameters were compiled and comparative study showed that the modified medium resulted in xylanase activity of 506 IU/ml, 5 folds higher than the basal medium.The novel combination of purification techniques like ultrafiltraton, ammonium sulphate fractionation, DEAE Sepharose anion exchange chromatography, CM Sephadex cation exchange chromatography and Gel permeation chromatography resulted in the purified xylanase having a specific activity of 1723 U/mg protein with 33.3% yield. The enzyme was having a molecular weight of 20-22 kDa. The Km of the purified xylanase was 6.5 mg of oat spelts xylan per ml and Vmax 1233 µ mol/min/mg protein.Bacillus SSP-34 xylanase resulted in the ISO brightness increase from 41.1% to 48.5%. The hydrolytic nature of the xylanase was in the endo-form.Thus the organism Bacillus SSP-34 was having interesting biotechnological and physiological aspects. The SSP-34 xylanase having desired characters seems to be suited for application in paper and pulp industries.

Distribution and bioactive potential of soil actinomycetes from different ecological habitats

Emergence of drug resistance among pathogenic bacteria to currently available antibiotics has intensified the search for novel bioactive compounds from unexplored habitats. In the present study actinomycetes were isolated from two relatively unexplored and widely differing habitats such as mountain and wetlands and their ability to produce antibacterial substances were analyzed. Pure cultures of actinomycetes were identified by morphological and biochemical tests. Various genera of actinomycetes encountered included Nocardia, Pseudonocardia, Streptomyces, Nocardiopsis, Streptosporangium, Micromonospora, Rhodococcus, Actinosynnema, Nocardiodes, Kitasatosporia, Gordona, Intrasporangium and Streptoalloteichus. The frequency of occurrence of each genus was found to vary with sample. About 47% of wetland isolates and 33% of mountain isolates were identified as various species of Nocardia. The isolated strains differed among themselves in their ability to decompose proteins and amino acids and also in enzyme production potential. Antibiotic activities of these actinomycetes were evaluated against 12 test pathogenic bacteria by well diffusion method using agar wells in glycerol-yeast extract agar. About 95% of actinomycete isolates from wetland ecosystem and 75% of highland isolates suppressed in different degrees the growth of test pathogens. Relatively high antibacterial activity among these isolates underlined their potential as a source of novel antibiotics.

Impact of process parameters on chitinase production by an alkalophilic marine Beau6eria bassiana in solid state fermentation

A chitinolytic fungus, Beau6eria bassiana was isolated from marine sediment and significant process parameters influencing chitinase production in solid state fermentation using wheat bran were optimised. The organism was strongly alkalophilic and produced maximum chitinase at pH 9·20. The NaCl and colloidal chitin requirements varied with the type of moistening medium used. Vegetative (mycelial) inoculum was more suitable than conidial inoculum for obtaining maximal enzyme yield. The addition of phosphate and yeast extract resulted in enhancement of chitinase yield. After optimisation, the maximum enzyme yield was 246·6 units g 1 initial dry substrate (U gIDS 1). This is the first report of the production of chitinase from a marine fungus.

Utilization of prawn waste for chitinase production by the marine fungus Beauveria bassiana by solid state fermentation

Prawn waste, a chitinous solid waste of the shell®sh processing industry, was used as a substrate for chitinase production by the marine fungus Beauveria bassiana BTMF S10, in a solid state fermentation (SSF) culture. The process parameters in¯uencing SSF were optimized. A maximum chitinase yield of 248.0 units/g initial dry substrate (U/gIDS) was obtained in a medium containing a 5:1 ratio (w/v) of prawn waste/sea water, 1% (w/w) NaCl, 2.5% (w/w) KH2PO4, 425±600 lm substrate particle size at 27 °C, initial pH 9.5, and after 5 days of incubation. The presence of yeast extract reduced chitinase yield. The results indicate scope for the utilization of shell®sh processing (prawn) waste for the industrial production of chitinase by using solid state fermentation.

Extracellular production of L-glutaminase by alkalophilic Beauveria bassiana BTMF S10 isolated from marine sediment

Beauveria sp. BTMF S10 isolated from marine sediment produced extracellular L-glutaminase. Maximal L- glutaminase yield (46.9 U/ml) was obtained in a medium supplemented with 1% (w/v) yeast extract and sorbitol, 9% (w/v) sodium chloride and 0.2% (w/v) methionine, initial pH 9.0 and at 27 °C after 108 h. This enzyme was inducible and growth-associated.

Immunostimulatory effect of a marine yeast Candida sake S165 in Fenneropenaeus indicus

The efficacy of a marine yeast Candida sake as source of immunostimulant to Indian white shrimp Fenneropenaeus indicus was estimated. Biomass of C. sake was prepared using malt extract agar and incorporated at graded levels into a standard diet to prepare yeast diets of varying biomass concentrations (1%, 10% and 20%). F. indicus were fed on these diets for a period of 28 days and challenged orally with white spot syndrome virus (WSSV) and immune parameters such as total haemocyte count, phenoloxidase and nitroblue tetrazolium reduction (NBT) were determined. Ten per cent C. sake in the diet was found to support an optimum immune response in the animals in general and their enhancement could be observed on the second and third day following challenge with the virus. The study has demonstrated that marine yeast C. sake at 10% in diet (w/w) may be used as an effective source of immunostimulants in F. indicus

Effect of leaf extract of Aegle marmelose in diabetic rats

Alloxan induced animal model was used to evaluate the potential antidiabetic effect of A . marmelose leaf extract. The diabetic animals were given insulin injection and another group A . marmelose leaf extract orally. It maintained the weight of the animals near to the control rats but a significant decrease in weight was noted it diabetic animals without any treatment . The blood elucose level in 'treated animals were near to that of control ones. Also a significantly increased glucose tolerance was observed in animals orally given the leaf extract prior to the experiment . A significant decrease in liver glycogen ( 1.24 -.07 s!/ 100 g of wet tissue) was observed in diabetic rats which was brought to almost the normal level (I.84- . 14 g; 100 g) with Icaf extract treatment. Blood urea and serum cholesterol increased ( 62.66 - 3.50 and 192.67± 13.64 mg' dl) significantly in alloxxan diabetic rats . The leaf extract treatment decreased the blood urea and serum . cholesterol (37.83 - 3.97 and 99?0±8.43 mg/dl ) to that of control ones . A similar effect was seen with insulin treatment . The results indicate that the active principle in .4. marmelose leaf extract has similar hypoglycaemic activity to insulin treatment.

Effect of Aegle Marmelose Leaf Extract on Allbxan Induced Diabetic Rats

Alloxan induced diabetic animal model was sed to evaluate the potential antidiabetic effect of ie aqueous leaf extract of Aegle marmelose. Insuinjection and oral treatment of Aegle marmelose of extract to the alloxan induced rats maintained ;e animal weight near to the control rats whereas ,,ere was a significant decrease in weight in alloxan •duced rats. Insulin injection and oral treatment the leaf extract reversed the decrease of protein )ntent in all the organs studied except the pancas. Glucose level was maintained near to the co- :rol levels in the insulin injected and leaf extract Bated alloxan diabetic rats. A significant increased ucose tolerance was observed in animals orally ven the leaf extract prior to the experiment. The suits indicate that there is an increased utilization glucose in the Aegle marmelose leaf extract treed rats.

Kinetic Studies of Purified malate Dehydrogenase in Liver of Streptozotocindiabetic Rats and the Effect of Leaf Extract of Aegle Marmelose (L.) Correa ex Roxb

The functional basis of diabetes-mellitus to a certain extent, can be elucidated by studying diabetes-induced changes in metabolic enzymes. Malate dehydrogenase (MDH), is an enzyme directly involved in glucose metabolism. The kinetic parameters of MDH and its purified cytosolic isozyme, S-MDH, have been studied in the liver of streptozotocin- diabetic rats; also the potential of the leaf extract of A. marmelose as an was investigated. The Km of the liver enzyme increased significantly, in both crude and purified preparations in the diabetic state when compared to Lhe respective controls. Insulin as well as leaf- •extract treatment of the diabetic rats brought about a reversal of K. values to near normal. Vmax of purified S-MDH was significantly higher in the diabetic state when compared to the control. Insulin and leaf extract treatment did not reverse this change. Since MDH is an important enzyme in glucose metabolism, the variation in its quantitative and qualitative nature may contribute to the pathological status of diabetes. The fact that leaf extract of A. marmelose was found to be as effective as insulin in restoration of blood glucose and body weight to normal levels, the use of A. marmelose as potential hypoglycemic agent is suggested.

Down-regulation of cerebellar 5-HT2C receptors in pilocarpine-induced epilepsy in rats: Therapeutic role of Bacopa monnieri extract

Epilepsy is a syndrome of episodic brain dysfunction characterized by recurrent unpredictable, spontaneous seizures. Cerebellar dysfunction is a recognized complication of temporal lobe epilepsy and it is associated with seizure generation, motor deficits and memory impairment. Serotonin is known to exert a modulatory action on cerebellar function through 5HT2C receptors. 5-HT2C receptors are novel targets for developing anticonvulsant drugs. In the present study, we investigated the changes in the 5-HT2C receptors binding and gene expression in the cerebellum of control, epileptic and Bacopa monnieri treated epileptic rats. There was a significant down regulation of the 5-HT content (pb0.001), 5-HT2C gene expression (pb0.001) and 5-HT2C receptor binding (pb0.001) with an increased affinity (pb0.001). Carbamazepine and B. monnieri treatments to epileptic rats reversed the down regulated 5-HT content (pb0.01), 5-HT2C receptor binding (pb0.001) and gene expression (pb0.01) to near control level. Also, the Rotarod test confirms the motor dysfunction and recovery by B. monnieri treatment. These data suggest the neuroprotective role of B. monnieri through the upregulation of 5-HT2C receptor in epileptic rats. This has clinical significance in the management of epilepsy

Serotonin- 5-HT2A and Glutamate Receptors Gene Expression in Streptozotocin induced diabetic rats:Effects of pyridoxine and Aegle marmelose leaf extract

Department of Biotechnology, Cochin University of Science and Technology

Marine yeast isolates from Arabian Sea and Bay of Bengal :Biochemical, Molecular and Phylogenetic Analysis

the present study was undertaken with the following objectives: 1. Isolation and identification of yeasts from Arabian Sea and Bay of Bengal. 2. Molecular characterization of yeast isolates and phylogenetic analysis 3. Physiological and biochemical characterization of the isolates. 4. Proximate composition of yeast biomass and bioactive compounds. The Thesis is comprised of six chapters. A general introduction to the topic is given in Chapter1. Isolation and identification of marine yeasts are presented in Chapter 2. Chapter 3 deals with molecular identification and physiological characterization of Non- pigmented yeasts. Molecular identification and physiological characterization of pigmented yeast is presented in Chapter 4. Proximate composition of yeast biomass of various genera and their bioactive compounds are illustrated in Chapter 5. A summary of the results of the present study is given in Chapter 6. References and Appendices are followed

Characterisation and bioprospecting of cold adapted yeast from water samples of kongsfjord ,Norwegian Artic

A total of 34 yeast isolates were characterized from 4 water samples collected from Kongsfjord at Ny Alseund region of Norwegion Artic during the Indian Artic summer expedition of 2009.They were studied for the effect of tempereture and salt concentration on growth as well as for their ability to produce various hydrolytic enzymes at two different temperatures. Result showed that 5 out of 8 genera were common to all the stations. Cryptococcus was the predominant genera folowed by Trichosporan and Rhodotorula 82% of the yeast isolates were oxidative in nature and except filobasidium all the isolates used nitrate as a nitrogen source for growth. Yeast isolates from all the ststions showed growth at 4 and 20 degree centigarade. These temperatures were chosen as most of the bacterial and yeast isolates showed psychrotrop[hic nature. 94% of the yeast isolates showed growth at 2.0M and lipolytic activity were marginally less than 4.None of the isolates produced amylase enzymes when incubated at 4 and 20. The present study highlights the wide tolerence of the psychrotrophic yeast isolates to temperature and salinity as well as their potential in biotechnology