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ThesisItem 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 Restricted IMPROVING BIOMASS AND YIELD POTENTIAL IN Ganoderma lucidum STRAINS FOR THE PRODUCTION OF TERPENOIDS(PAU Ludhiana, 2011) Sheenu Gupta; P. K., KhannaThree strains of Ganoderma lucidum (GL-1, GL-2 and GL-3) were used in present studies for biomass and fruit body production and extraction of triterpenes. Mycelial biomass was raised in Mushroom Complete Medium according to central composite rotatable design using design expert statistical software. The conditions were optimized and were found to be 29.50C to 34.30C temperature, 6.1 to 7.0 pH, 30.5 to 32 days of incubation period and 50.9 to 52 rpm agitation for getting maximum mycelial biomass. Fruit bodies were raised on wheat straw supplemented with wheat bran and rice bran. Maximum biological efficiency of 16 per cent was obtained in GL-1 at 5 per cent level of supplementation of wheat bran. Triterpenes were extracted from biomass as well as powdered fruit bodies and separated in hexane, dichloromethane and ethylacetate in order of increasing polarity. Fruit bodies contained maximum amount of triterpenes and yielded about 7.8 to 7.9 per cent from GL-1 and GL-2 respectively. The colours of the obtained masses were ranged from yellow to dark brown. Thin layer chromatography (TLC) and mass spectroscopy (MS) was carried out for further resolution of extracted triterpenes in different solvents using benzene: ethylacetate (5: 1 v/v) and chloroform: benzene (7:1 v/v) by TLC. There is resolution of a no. of compounds in TLC. Their Rf values ranged from 0.11 to 0.96 in two different solvent systems. Lower Rf values represented bond forms of triterpenoid like compounds and higher Rf values showed the presence of released forms of triterpenes. MS of the samples reveals the presence of Ganoderic acid A, AM- 1, B, J, H, Lucidenic acid N & A, Ganolucidic acid A & D and two new triterpenes 3β- hydroxyl-4,4,14- trimethyl- 7,11,15- trioxochol- 8-en-24-oic acid and 7,15-dihydroxy- 4,4,14- trimethyl-3,11-dioxochol-8-en-24-oic acid.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 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).ThesisItem Restricted STANDARDIZING GROWTH CONDITIONS FOR CONIDIATION OF ENTOMOPATHOGENIC STRAINS OF BEAUVERIA BASSIANA (BALS.) VUILL. AND EVALUATION AS BIOPESTICIDE(PAU, 2012) Sushant DharThirteen fungal species were isolated from field soils of Punjab on Beauveria specific selective medium (BS-medium). Molecular screening of these strains using PCR amplification with B. bassiana specific primer set P1-P3 resulted in amplification of expected 524bp DNA band from only three strains (now named as BbR1, BbR2 and BbR3) and reference BbM1 strain (B. bassiana MTCC 2028), establishing only these to be true B. bassiana strains. Comparative RAPD-PCR amplification with 10 different RAPD primers identified many polymorphic markers which established that all the three strains were different strains. Genetic relatedness dendrogram as developed using RAPD-PCR data by UPGMA measured the quantitative description of genetic diversity. B. bassiana strains achieved their maximum growth during optimum incubation period of seven days. The optimum pH and temperature for maximal growth of B. bassiana strains was found to be 6-7 and 25-30ºC, respectively. All the tested carbon and nitrogen sources supported growth and development of the B. bassiana strains. Starch and peptone as carbon and nitrogen sources supported maximum radial growth and conidiospore count. Strain BbR2 was the fastest growing strain on almost all nutrient sources studied and possessed commendable growth rate and sporulation potential. Wheat bran (WB) and rice bran (RB) in the proportion of 3:1 supported maximum conidiospores yields (1.90×107 conidia/ml) for strain BbR2 in solid state fermentation conditions. Significant mortality of S. litura was observed at all the tested concentrations of conidial suspensions (103 to 107 conidia/ml) of B. bassiana and conidial concentrations of 106 and 107 conidia/ml being statistically at par with each other in causing maximum mortality. In comparison, under similar conditions, the standard strain BbM1 caused only least mortality (67.12%) and was significantly poorest in bio-efficacy amongst all the strains. In this study, B. bassiana strain BbR2 was concluded to be most potential locally isolated strains both for growth and conidiospore production as well as for causing maximum mean mortality (83.33%) of S. litura followed by BbR1 (72.22%), BbR3 (74.33%) which were better than the standard BbM1 strain.ThesisItem Open Access Microbiological preparation of non-alcoholic naturally carbonated blended beverage from guava and lemon(Punjab Agricultural University, Ludhiana, 2010) Davneet KaurA reliable, controllable and reproducible technology for preparation of non-alcoholic naturally carbonated fermented beverage from guava var. Allahabad Safeda, Lucknow-49, Punjab Pink and its blends with lemon var. Baramasi, Citrus latifolia after fermentation with yeast Clavispora lusitaniae under optimized fermentation conditions has been developed. The specific growth rate (h-1) and generation time (h) of yeast in guava and guava-lemon beverage (1:1) were 0.39, 1.77 and 0.35, 1.93 respectively. On the basis of physicochemical, microbiological and sensory evaluation the guava varieties Allahabad Safeda with 15 per cent juice, pH 3.9, TSS 11.7°B, acidity 0.53%, ascorbic acid 13.5 mg/100ml, alcohol (%v/v) 0.89, CO2 1.53 bar and total plate count 33x109 cfu/ml and Punjab Pink 15 per cent juice, pH 4.0, TSS 12.7°B, acidity 0.49%, ascorbic acid 9.7 mg/100ml, alcohol (%v/v) 0.81, CO2 1.56 bar and total plate count 98x108 cfu/ml, ranked highest for appearance (7.4), aroma (7.4), body (7.38), astringency (7.3) and overall acceptability (7.25) during storage period of 90 days under refrigerated conditions (40C). The best blend Punjab Pink-Citrus latifolia (1:1) 12.5 per cent juice, pH 2.7, TSS 12.14°B, acidity 0.51%, ascorbic acid 6.2 mg/100ml, alcohol (%v/v) 0.92, CO2 1.50 bar and total plate count 27x109 cfu/ml with shelf life of 90 days under refrigerated conditions (40C) was rated as liked very much. The optimized temperature and potassium metabisulphite concentration to inhibit the starter yeast culture was standardized as 55oC and 700ppm, respectively, organoleptically heat treated beverage was superior than potassium metabisulphite treated beverage.