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EXTRACELLULAR ENZYME PRODUCTION BY Pleurotus SPECIES AS A POTENTIAL SELECTABLE MARKER FOR MUSHROOM YIELD
(PAU Ludhiana, 2011) Simerjit Kaur; P. P., Johl
Pleurotus 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.
Open Access
OPTIMIZATION OF FERMENTATION CONDITIONS FOR PRODUCTION OF WINE FROM GUAVA (Psidium guajava L.)
(PAU Ludhiana, 2011) Pooja; G.S., Kocher
Guava (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.
Open Access
ENHANCEMENT OF NUTRITIONAL AND FUNCTIONAL PROPERTIES OF RICE FLOUR BY MICROBIAL ENZYMES
(PAU Ludhiana, 2011) Shruti Puri; Maninder, Arora
Filamentous 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.
Open Access
STRAIN IMPROVEMENT OF Calocybe indica THROUGH MUTAGENESIS AND ITS AGRONOMIC OPTIMIZATION
(PAU Ludhiana, 2011) JATINDER KAUR; H. S. Sodhi
Tremendous revolution in the mushroom technology in recent years with respect to types and strains of cultivated mushrooms has made Calocybe indica, a specialty mushroom, the third most popular and commercially grown mushroom in India. A lot of work has been done as far its cultivation technology is concerned but no systematic work has yet been undertaken for its genetic improvement. During present investigation nine strains of C. indica were cultivated on wheat straw using recommended technology. Strain Ci- 3 gave maximum biological efficiency of 81.28%. Basidiospores from all the nine strains were collected and subjected to germination trial on different media but no germination was observed. Improvement of high yielding strain, Ci-3 using mutagenic treatment on protoplasts was conducted. Four mutagenic treatments (one physical and three chemical) yielded 30 putative mutants. Growth studies of putative mutants indicated maximum growth rate of CMB-4 on wheat straw while CMN-11 gave maximum biomass on complete yeast extract medium. Mutant CMN-9 gave maximum endoglucanase (0.345 μg/min/ml) and xylanase (0.540 μg/min/ml) activity. Seven mutants were identified on the basis of growth and enzymes studies. A set of 14 primers were used for molecular characterization of mutants along with the parent using RAPD-PCR. Out of these only 6 primers resulted gave distinct amplification products. Six primers yielded 68 scorable bands ranging from 40bp to 280bp. Similarity coefficient of Ci-3 ranged from 0.706 for CMN-3 to 0.794 for CMU-2. Three mutants (CMU-2, CMU- 5 and CME-2) showed 4 bands each having similar relative mobility values (0.2, 0.35, 0.5, 0.65) during protein profiling using SDS-PAGE. Three bands were obtained for CMN-11, CMB-4 and Ci-3 and 2 bands for CMN-9. Only one band was obtained in case of CMN-3 with relative mobility of 0.2. Cultivation of the mutants was carried out on wheat straw following hot water treatment. Four mutants (CMN-3, CMN-9, CMN-11 and CMB-4) were found to give significantly higher yield than the parent while only three mutants (CMN-3, CMN-9 and CMN-11) gave better results on mixture of wheat straw and paddy straw and on steam sterilized wheat straw. During nutritional studies mutant CMN-9 was found to be nutritionally better with high protein, tocopherol, β- carotene and lycopene content. Postharvest treatment of mutants indicated cabinet drying as a better option in terms of colour and texture retention.
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TECHNOLOGY FOR QUALITY IMPROVEMENT AND POTENTIAL UTILIZATION OF CEREAL BRANS
(PAU Ludhiana, 2011) Satinder Kaur; Savita, Sharma
The present investigation was carried out to explore the processing treatments for cereal brans and to access its functionality. Phytic, trypsin inhibitor, oxalates, tannins and polyphenols, are the major undesirable constituents which restrict direct utilization of brans in diet. Various processing treatments for stabilization were applied for quality improvement. Brans (wheat, rice, barley and oat) were investigated for proximate composition, functional characteristics such as water absorption, fat absorption and bulk density. All brans have pleasing flavor. Significant variations were observed with regard to color values of brans. Defatting treatment given to cereal brans using hexane as solvent represented a marginal decrease in antinutritional components in all cereal brans. The effect of processing treatments viz. dry heating, wet heating, microwave heating, extrusion cooking and chemical treatment on reduction of antinutritional factors were studied. All the processing treatment resulted a decrease in all antinutritional factors. Dry heating at 110°C for 25 min resulted maximum reduction in all antinutritional compounds (phytic acid, polyphenols, trypsin inhibitors, oxalates and saponins). Wet heating at 110°C for 25 min reduced trypsin inhibitor activity (83.07%). Microwave heating (2450MHz for 2.5 min) resulted in 53.85%, 57.21%, 78.25%, 65.0% and 100% reduction in phytic acid, polyphenols, trypsin inhibitor, oxalates and saponins, respectively. In extrusion cooking, the highest reduction in antinutrients was observed at 140°C with 20% moisture content. Acetic acid (1%) @ 22% was the most effective in reducing these antinutritional factors followed by calcium hydroxide and hydrochloric acid. Bulk density increased significantly as compared to raw brans after all processing treatments and increase in redness and decreases in yellowness of brans were observed after treatments. Processed brans were stored in high density polyethylene (HDPE) pouches at ambient and refrigeration temperature. Quality assessment (moisture content, free fatty acids, water activity and physical quality) of brans were taken up to six months, at one month intervals. Free fatty acid content, moisture content and water activity remained stable during entire storage period. Processing treatments and storage temperature have the positive effect on extending the shelf life of all cereal brans. Bread, macaroni and extruded snacks were prepared after supplementation of processed cereal brans singly (wheat bran at the level of 0-15%) and in combination (5 and 10% of wheat, rice and oat). Resultant products were the most acceptable in terms of color, texture and sensory quality. Thus, the developed fortified cereal products could be regarded as a palatable, convenience and nutritious food product.
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IMPROVING BIOMASS AND YIELD POTENTIAL IN Ganoderma lucidum STRAINS FOR THE PRODUCTION OF TERPENOIDS
(PAU Ludhiana, 2011) Sheenu Gupta; P. K., Khanna
Three 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.
Open Access
“BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ALKALINE PROTEASE OF BACILLUS CIRCULANS MTCC 7906”
(PAU Ludhiana, 2011) Inderjeet Kaur; Gurvinder Singh, Kocher
The present study was conducted to characterize alkaline protease of Bacillus circulans MTCC 7906. Cotton deoiled meal (2.1%) supported optimum enzyme production in 144 hours of incubation. Among the agricultural byproducts used to improve alkaline protease production, the combination of Soybean meal and Glucose supported maximum enzyme production in 96 hours of incubation which was comparable with the control Reese medium (Casein + Glucose) in terms of alkaline protease activity as well as incubation time required for enzyme production. The alkaline protease was purified (16.2 fold) to homogeneity from the culture supernatant by the combination of ammonium sulphate precipitation (30-60%) and DEAE-cellulose anion exchange chromatography. The biochemical characterization of partially purified alkaline protease displayed maximum activity at a pH of 9.0, temperature of 60°C, an enzyme concentration of 0.1 ml/3.0 ml and substrate concentration of 12 mg/ml. The Km and Vmax of partially purified alkaline protease were found to be 4.5 mg/ml and 5555 nmole tyrosine min-1 ml-1, respectively. The enzyme activity was activated by divalent ions whereas a metal chelator EDTA and ammonium hydroxide reduced the activity. The molecular weight of the enzyme was estimated to be 46 kDa on SDS-PAGE. For, molecular analysis, full length alkaline protease gene (BcAP, 1329 bp) was PCR amplified from B. circulans DNA and cloned into pTrcHisA vector. Sequencing and alignment of nucleotide and predicted amino acid sequences of alkaline protease region identified a number of substitutions (mutation) which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The multiple alignment of nucleotide and amino acid sequences of alkaline protease gene of B. circulans established major matches with three closely related subspecies of B. subtilis (B. subtilis subspecies subtilis strain 168, B. subtilis BSn5 and B. subtilis subspecies spizizenii strain W23). Molecular evolution of B. circulans strain was determined by phylogenetic analysis of alkaline protease gene and predicted amino acid sequences. This analysis also suggested that alkaline protease gene is novel gene, which has been accessioned in GenBank with accession number JN645176.1. Recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the protein with estimated molecular size of 46 kDa as observed earlier.
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, Khanna
Forty-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).
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MICROBIAL ENZYMES FOR IMPROVING THE MILLING CHARACTERISTICS OF MOONG BEAN (Vigna radiata (L.) Wilczek)
(Punjab Agricultural University, Ludhiana, 2011) MONICA
Moong bean (Vigna radiata (L.) Wilczek) variety PAU-911 was enzymatically pretreated with fungal enzymes to improve its milling and cooking characteristics using Response Surface Methodology technique. The fungal enzymes cellulase and ligninase were produced by Phanerochaete chrysosporium and Trichoderma reesei by Solid State Fermentation. Three enzyme concentrations were used viz. 45, 55, 65 mg per 400 g moong bean. It was treated with 100 ml of the enzyme solution prior to milling for 15 mins, 20 mins and 25 mins at different temperatures i.e. 45°C, 50°C and 55°C. The optimized conditions according to response surface methodology were 54.03°C incubation temperature, enzyme concentration 55 mg/400 g and incubation period as 19.46 minutes. The enzymatically pretreated moong bean samples under optimized cultural conditions were compared with un treated samples in terms of percentage dhal yield, percentage broken, time of milling and optimal cooking time. Enzymatic pretreatment under the optimized conditions could increase dhal yield by 8.6%, decreased broken percentage by 6.9% and time of milling by 11.6 seconds and decreased optimal cooking time by 3 minutes. No significant effect was found on water uptake ratio, swelling index and cooking coefficient of enzymatically pretreated and non-treated samples. Hence, the enzymatic treatment prior to milling enhanced the milling performance and cooking quality of moong bean.

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