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ThesisItem Open Access DEVELOPMENT OF HYBRIDS OF PLEUROTUS FLORIDA AND VOLVARIELLA VOLVACEA THROUGH PROTOPLAST FUSION(2013) Zenebe Teka MengeshaProtoplast fusion using Polyethylene glycol (PEG) and electrofusion was carried out between strains of Pleurotus florida and Volvariella volvacea to develop improved hybrids. Preliminary screening was carried out based on responses to growth at different temperature regimes, colony radial growth, morphology and growth patterns of colony, hyphal size, presence or absence of clamp connections, and other unique characters like production of reddish exudates. Following the rigorous screening, eight intergeneric hybrids (Pv14, Pv19, Pv21, Pv27, Pv45, Pv66, Pv102 and Pv127) were selected and further evaluated for fruit body formation. Pv45, Pv66 and Pv102 were unable to give primordia after 25 days of growth and were considered sterile. Among those which developed fruit bodies, Pv21 and Pv127 had brownish circular caps with regular margins and central stipes. Other strains showed cap morphologies closely related to PF5. Isozyme (alcohol dehydrogenase, malate dehydrogenase, esterase and super oxide dismutase) pattern analyses indicated presence of bands in all the strains which verified hybridization between the two parents. Besides, two bands observed neither in the parents nor in the other strains were noticed in pv27 and pv127. Amplifications of genomic DNA from all the strains using 14 arbitrary decamer RAPD primers resulted in 98 bands, with size range of 160bps to 2500bps, of which 57.14 per cent were polymorphic. The bands were obtained only from 8 primers. A combined clustering analysis of these bands using NTSYSpc software indicated that Pv127 exhibited marked divergence from the other strains in the phylogenetic clusters and had better performance. Therefore, the salient investigation of the current work was screening of a hybrid strain (Pv127) with high BE (52.71%) and low temperature tolerance compared to V. volvaceaThesisItem Restricted EXTRACELLULAR ENZYME PRODUCTION BY Pleurotus SPECIES AS A POTENTIAL SELECTABLE MARKER FOR MUSHROOM YIELD(PAU Ludhiana, 2011) Simerjit Kaur; P. P., JohlPleurotus is a sub- tropical mushroom which can be grown under natural climatic conditions in North Africa, Southern Europe and Central Asia. Pleurotus has gained broad acceptability due to its proliferation on wide range of suitable substrates and a simple cultivation technique. During the present study four species of Pleurotus namely P. eryngii, P. flabellatus, P. florida and P. sajor-caju were evaluated for their mycelial extension rate, lignocellulolytic enzyme activity, total protein content and yield potential. Maximum mycelial extension rate was observed in P. sajor-caju on paddy straw (PS 11.4 cm) and wheat straw+paddy straw (WS+PS 11.4 cm). Screening of Pleurotus species for lignocellulolytic enzyme activity indicated that P. sajor caju showed maximum cellulase activity (Fpase, endoglucanase and cellobiohydrolase, 1.55, 4.64 and 6.00 (U/mg protein) followed by P. florida whereas reverse was true for endoxylanase (9.66 U/mg protein) and laccase (1.45 U/mg protein). Under solid state fermentation, P. sajor-caju showed maximum cellulase activity (Fpase-3.21 on WS+PS, endoglucanase-6.96 on WS+PS, cellobiohydrolase-20.2 on PS) and endoxylanase activity-30.9 on WS (U/mg protein) followed by P.florida. Maximum laccase activity (1.64 on PS) was observed in P.florida. Maximum protein content (1.72 mg) was observed in culture filtrate of P. eryngii. SDS-PAGE of extracellular proteins showed two bands in P. flabellatus and P. eryngii at molecular weight of 15.0 and 14.7 KDa which were similar to that found in P. florida and P. sajor-caju. It appeared that these bands showed their species specificity Among the four species, it was observed that P. florida had a maximum potential to show biological efficiency 102.5% followed by P. sajor-caju 87.2% on WS+PS. Pinhead appeared between 35-45 days and average weight of a fruit body ranged between 9.8-19.3 gm. The maximum number of fruit bodies were harvested from P. florida followed by P. sajor-caju with lowest in P. eryngii on WS, PS and WS+PS. P. sajor-caju was the best strain in terms of enzyme activity whereas P. florida was better in terms of enzyme activity and yield potential but this correlation pattern was not followed in other species-P. eryngii and P. flabellatus which could be the probable effect of biotic and abiotic factors involved in fruiting morphogenesis of mushrooms.ThesisItem Open Access OPTIMIZATION OF FERMENTATION CONDITIONS FOR PRODUCTION OF WINE FROM GUAVA (Psidium guajava L.)(PAU Ludhiana, 2011) Pooja; G.S., KocherGuava (Psidium guajava L.) varieties (Punjab pink, Arka amulya and Lucknow-49) were evaluated for wine production. A prefermentation treatment of guava pulp with Pectinase (3.5 units/mg) was optimized using Response Surface Methodology (RSM) whereby a 47.2, 55.6 and 59.5% decrease in OD550 nm was observed in the statistical combination of 45°C, 6h and 0.50 mg/100ml, 45°C, 6 h and 0.84 mg/100 ml and 45°C, 6 h and 0.84 mg/100 ml for Punjab pink, Arka Amulya and Lucknow-49, respectively. S. cerevisiae strain 35 was found to be significantly better (fermentation efficiency 80.0%) than S. cerevisiae Y-2034 (fermentation efficiency 73.0%) in terms of ethanol production and sugar catabolic rate. The effect of different fermentation parameters viz. sugar, temperature, inoculum size and DAHP supplementation revealed 25°B, 25°C, 9% and 300 mg/100ml as optimum respectively in all the three varieties, with an ethanol production of 13.8, 13.6 and 13.6% in 6, 8 and 8 days for Punjab pink, Arka Amulya and Lucknow-49, respectively. Post fermentative storage of wine (at 15°C) for 90 days freed the wine off viable yeast cells but also led to reduction in ascorbic acid, total phenolic content along with significant decrease in % ethanol levels. . The prepared wine was subjected to the sensory analysis (at 15 and 90 days of storage). Wines prepared from Punjab pink and Arka amulya varieties was of standard quality whereas that of Lucknow-49 was below standard at young wine stage. After the storage of 90 days, wine from Punjab pink scored a superior quality wine score (68.8 + 3.27) whereas wines from Arka amulya and Lucknow-49 remained the same i.e of standard and below standard quality (54.2 + 3.11 and 47.2 + 2.38 respectively). The scale up studies (var. Punjab pink) at 5L scale validated the optimized fermentation parameters with the ethanol production of 13.6 + 0.15 % and having an ethanol yield of 0.492g/g. Hence, Guava can act as a suitable substrate for production of wine with all the important properties of wine having high content of phenols and ascorbic acid.ThesisItem Open Access ENHANCEMENT OF NUTRITIONAL AND FUNCTIONAL PROPERTIES OF RICE FLOUR BY MICROBIAL ENZYMES(PAU Ludhiana, 2011) Shruti Puri; Maninder, AroraFilamentous fungi have been widely used to produce hydrolytic enzymes for industrial applications. Amylase and glucoamylase were produced using Aspergillus oryzae under solid state fermentation. Different agricultural residues such as rice bran, wheat bran, rice bran: wheat bran (1:1) and rice bran: paddy husk (1:1) were used to produce amylase and glucoamylase. Rice bran yielded maximum amylase (2.72 IU) and glucoamylase (4.11 IU) activity among all other residues. Various cultural conditions were optimized and temperature of 30 °C, moisture content (80 %), pH 5.0, spore suspension (1 ml) of 1x107 spores/ml and incubation period of 5 days were found optimum for maximum production of enzymes. The enzymes obtained were partially purified using isopropanol and rice flour was treated with these hydrolysing enzymes. Enzymatic pretreatment conditions were optimized using Response Surface Methodology (RSM). Effect of different parameters such as slurry concentration, enzyme concentration, time and temperature was studied. Enriched rice flour was obtained by treating 7 % slurry with 1 ml of enzyme at 55 °C for 40 min. This enriched rice flour was analysed for its nutritional, functional, toxic residues and microbiological parameters. Protein content of the enzymatically treated rice flour increased from 8.03 % to 20.79 % (2.5 fold enhancement) and ash from 0.69 % to 1.40 %. Fat content of both, untreated and treated rice flours, differed insignificantly but crude fiber was hydrolysed from 2.50 to 0.42. Moisture content of the treated rice flour decreased from 12.40 % to 11.85 %. The digestibility coefficient of in vitro digestibility also increased from 644.63 to 1351.67 in treated rice flour. Microbiological count of treated rice flour decreased whereas contaminating organisms (coliforms and salmonella) and toxic residues (aflatoxins) were not detected.ThesisItem Open Access “STUDIES ON GROWTH PARAMETERS OF ENTOMOPATHOGENIC FUNGI AND ITS EVALUATION AGAINST Tetranychus urticae KOCH(PAU Ludhiana, 2012) Neha Sharma; Neelam, JoshiEight fungal isolates were studied for the growth parameters on different substrates, effect of pesticides on fungal growth and pathogenicity against Tetranychus urticae. In the study of effect of pesticides on fungal growth at different concentrations radial growth of fungal isolates on the growth media supplemented with pesticides was recorded and was found that the radial growth of fungal isolates varied among genera and species. Among all fungal isolates, Metarhizium anisopliae MTCC 4104 @ 750 ppm was most compatible to propargite and showed 33.6 per cent growth reduction over control. In media supplemented with ethion, endosulfan and chlorpyriphos, Hirsutella thompsonii PDBC-1 caused 32.9 per cent @ 1000 ppm, 6.3 per cent @ 2500 ppm and 20.1 per cent @ 2500 ppm per cent growth reduction over control, respectively and was most compatible with these pesticides. Further, in all pesticides tested, radial growth varied in inverse proportion to the concentration. Lower the concentration, more is the radial growth. Seven different substrates were evaluated for growth parameters of all fungal isolates and variability of the mean conidial count, colony forming unit and biomass was observed both among substrates and various fungal isolates. Among the isolates, mean conidial count and colony forming unit was maximum in H. thompsonii PDBC-1 recording 26.79 x 106conidia/g and 26.6 x 106 CFU/g, respectively whereas Beauveria bassiana P isolate recorded maximum mean biomass production of 0.49 g. Among the substrates, sorghum recorded maximum mean conidial count and colony forming unit of 21.29 x 106conidia/g and 20.9 CFU/g, respectively whereas maximum biomass production was recorded in rice wash water (0.37 g). In bioassay studies against adult of T. urticae, the fungal isolate H. thompsonii PDBC-1 and Beauveria bassiana MTCC 6291 recorded 96.50 per cent mortality at 1011conidia/ml after seven days of treatment. Further in all treatments, higher concentration was significantly better than the middle and lower concentration. Similarly, H. thompsonii PDBC-1 recorded minimum LC50 value of 6.84 x 109, 1.20 x 107and 8.38 x 102 conidia/ml after 72, 120 and 168 hrs, respectively which was significantly better than all other isolates and was the most pathogenic isolate.ThesisItem Open Access Characterization of potential Bradyrhizobium/ Ensifer strains for improving biological nitrogen fixation and yield in soybean [Glycine max (L.) Merrill](PAU Ludhiana, 2013) Davinderdeep Kaur; Poonam Sharmahe present investigation was carried out to study the characterization of potential Bradyrhizobium/Ensifer strains for improving biological nitrogen fixation and yield in soybean [Glycine max (L.) Merrill]. Nine authentic strains of rhizobia were collected from different AICRP centres of soybean in India. Out of nine strains, 5 were identified as slow grower Bradyrhizobium strains (IND1, LSBR3, PANT1, SB271 & DS1) and 4 strains (IND2, LSER7, LSER8 & PANT2) as fast grower on the basis of their morphological and biochemical characteristics. Further, These strains were investigated for their functionality traits viz. indole acetic acid (IAA), phosphate (P) solubilization hydrogenase activity and intrinsic antibiotic spectra. Significantly high IAA was recorded with LSER8 (25.75µg ml-1) in the presence of L-tryptophan (0.01%). Significantly high P solubilization of TCP (100 mg) in Pikovaskaya’s medium was observed with IND2 (5.80 mg 100 ml-1) at 12th day. In qualitative screening of hydrogenase activity 2 Bradyrhizobium strains (LSBR3 and DS1) and 3 Ensifer strains (LSER7, LSER8 and PANT2) showed red coloration on YEMA media amended with 0.01% TTC dye. Roots from soybean seeds bacterized with Bradyrhizobium strain LSBR3 produced significantly high amount of flavonoid like compounds. Potential of different rhizobial strains were further evaluated for symbiotic efficiency, growth improvement and grain yield in soybean (SL744) under field conditions. Significantly high growth parameters, symbiotic traits and grain yield was observed with two Bradyrhizobium strains (LSBR3 and PANT1) and Ensifer strain (LSER8). These rhizobial strains can be explored as future biofertilizer to promote growth and yield in soybean.ThesisItem Open Access STANDARDIZATION OF FERMENTATION CONDITIONS FOR THE PREPARATION OF DEBITTERED LOW ALCOHOLIC NATURALLY CARBONATED BEVERAGE FROM KINNOW(PAU Ludhiana, 2012) Sakshi Sharma; Param Pal, Sahotaexhibited mutagenesis with naringin yielded colony morphology variants characterized by raised, rough-surfaced colony of irregular outline in marked contrast to the flat, shiny, circular colonies of the parental strain. Naringin induced mutant has been isolated and examined by phase contrast microscopy. The pleomorphic form of Clavispora lusitaniae, developed in response to nutritional inducer naringin over a wide temperature 10-60°C and pH 2-8, assimilated fructose, maltose, dextrose, trehalose and sucrose. An inducible enzyme, naringinase showed maximum activity (0.56 IU/ml) with 0.1% naringin. Technology for the preparation of low-alcoholic naturally carbonated debittered beverage from kinnow with yeast under optimized fermentation conditions has been developed. Microbiological,physicochemical, mineral and sensory evaluation of kinnow beverage with 40 per cent juice revealed pH 3.4, TSS 12.6°B, acidity 0.54%, ascorbic acid 6.72 mg/100ml, reducing sugar 1.38%, total sugars 10.10%, limonin 3.3 ppm, naringin 161.9 ppm, total carotene 0.50 mg%, potassium 672.27 mg/kg, calcium 70.64 mg/kg, magnesium 61.89 mg/kg, sodium 19.24 mg/kg, iron 0.73 mg/kg, alcohol 0.86 (%v/v), CO2 1.36 bar and total plate count 3.3x108cfu/ml, ranked highest for taste (7.9), aroma (8.5), colour (7.8), astringency (8.25) and overall acceptability (7.8) during storage period of 3 months under refrigerated conditions (40C).The percentage decreased in limonin and naringin on storage was 54 and 64.8 percent respectively, much below the threshold level.The major elements (K, Mg, Na, Ca, Fe) were retained in kinnow beverage during storage for period of 3 months,while ascorbic acid content decreased significantly. Carbon dioxide developed in the beverage acts as supercritical solvent, having antimicrobial property, imparts tangy taste, effervescence, and has masking effect on bitterness.ThesisItem Open Access CHARACTERIZATION OF NARINGINASE ENZYME FOR PRODUCTION OF DEBITTERED LOW ALCOHOLIC NATURALLY CARBONATED KINNOW BEVERAGE(PAU Ludhiana, 2013) Navjot Kaur; Param Pal, SahotaNaringin, the bitter flavonone glycoside and primary bitter component in citrus fruit juices can be hydrolysed by naringinase enzyme into tasteless component, naringenin. A rapid detection test for naringinase producing microorganisms using FeCl3 has been developed. Naringin an inducer in kinnow juice, mutated yeast Clavispora lusitaniae and induced the production of crude enzyme naringinase with the activity of 0.0135 IU/ml. The parameters optimized for naringinase production were pH (4), temperature (50°C), naringin (0.8%) and rhamnose concentration (0.6%) with high substrate activity Km value of 1.00 mM and Vmax value of 28.56 mM. Divalent cations Cu2+ and Ca2+ competitively inhibited enzyme activity where as Mn2+ and Zn2+ stimulated enzyme activity of 0.024 IU/ml and 0.0079 IU/ml, respectively. The debittering of kinnow juice by Clavispora lusitaniae mutant prompted for utilization of kinnow juice for the production of low alcoholic naturally carbonated fermented kinnow beverage. Technology for the production of low alcoholic naturally carbonated fermented kinnow beverage with yeast Clavispora lusitaniae under optimized fermentation conditions were developed. Microbiological, physiochemical and sensory evaluation of kinnow beverage with 40 percent juice revealed pH 4.2, TSS 10.5°B, acidity 0.53%, ascorbic acid 9.2 mg/100ml, reducing sugar 8.83 percent, total sugars 9.4 percent, limonin 1.7 ppm, naringin 120.8 ppm, β-carotene 0.26μg/L, alcohol 0.28% (w/v), CO2 1.09 bar and plate count 9.3x108 cfu/ml, ranked highest for taste 7.9, aroma 8.5, colour 7.8, astringency 8.25 and overall acceptability 7.8 during storage period of 70 days under refrigerated conditions (4°C). The percentage decrease in limonin and naringin on storage was 54 and 64.8 percent, below the threshold level of limonin (6 ppm) and naringin (600 ppm) respectively. The pasteurization conditions standardized for kinnow beverage were 85°C for 3 sec.ThesisItem Open Access CHARACTERIZATION OF PLANT GROWTH PROMOTING RHIZOBACTERIA AND EVALUATION OF THEIR SYNERGISTIC POTENTIAL WITH Rhizobium IN LENTIL (Lens culinaris Medikus(PAU Ludhiana, 2011) Preeti Saini; Veena, KhannaForty-eight rhizobacterial isolates obtained from lentil rhizospheric soils from 34 locations were characterized and found belonging to genera Bacillus (23), Pseudomonas (23) and Serratia (2). The isolates were evaluated for their plant growth and antagonistic traits. Fifteen of the isolates produced siderophore as evidenced on Chrome-azurol-S agar, maximum catechol-type siderophore being produced by B-40 (97μg/ml), while maximum hydroxamate-type siderophore by B-20 (129.5μg/ml). Phosphorus solubilization was recorded with 39.5% isolates and the relative efficiency of P-solubilized in liquid medium ranged from 3.5-44mg/100ml. All these isolates produced the phytostimulator Indole-acetic-acid which ranged from 3-106.1μg/ml. Nine of the isolates were able to utilize ACC as the sole N-source, their ACC-metabolizing rate was measured as optical density (OD496) ranged from 0.54-1.39. The antagonistic ability against Fusarium oxysporum ranged from 3.3–16.6%, maximum being recorded with P-1 (16.6%), followed by B-36 (13.3%) and B-40 (3.3%). Similar trend was recorded in liquid medium. On the basis of their PGP traits B-40 and P-1 were selected for field studies. These isolates showed compatibility with Rhizobium leguminosarum under axenic conditions and also promoted root/shoot growth in lentil seedlings. The effect of selected PGPR and Rhizobium sp. along with check strain KBB-133 was studied on lentil crops in the field. The results showed that maximum increase in symbiotic parameters was observed in case of dual inoculations of PGPRs with Rhizobium. The grain yield with dual inoculations with B-40 (1703 kg/ha) and P-1 (1679 kg/ha) was statistically on par to dual inoculation with KBB-133 (1698 kg/ha).
