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2016 - 201922020 - 20231
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Subject: Microbiology × Author: Manmeet Kaur × Start date: 1947 × Type: Thesis ×
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    Characterization and industrial application of oxidative/hydrolytic enzymes of Pleurotus florida (Mont.) Sing. and Calocybe indica (Pur. & Chan.)
    (Punjab Agricultural University, 2023) Manmeet Kaur; Sharma, Shivani
    Pleurotus florida and Calocybe indica are the edible mushrooms that have been widely accepted due to their ability to grow on a variety of substrates and possess the potential to produce oxidative and hydrolytic enzymes. The present work involved the study and characterization of oxidative/hydrolytic enzymes of P. florida and C. indica for their potential applications in alcohol fermentation, dye decolorization, biobleaching and mushroom production. The oxidative and hydrolytic enzymes extracted from P. florida and C. indica were estimated at different mycelial growth stages for enzyme production. The intracellular and extracellular enzyme activity of P. florida and C. indica increased with time and maximum activity of lignocellulolytic enzymes was found to be on 14th day of incubation. In both mushrooms, ligninolytic enzyme activity increased during substrate colonisation but quickly decreased during fruiting body development. On the other hand, P. florida and C. indica showed relatively modest hydrolase activity during substrate colonisation. The activity of hydrolytic enzymes increased dramatically during primordial formation and peaked at the fruiting body stage. The purification of laccase, lignin peroxidase, manganese peroxidase and endoxylanase from the fruiting body of P. florida resulted in maximum purification fold of 21.49, 17.73, 16.81 and 12.78 with yield of 24.98%, 20.60%, 19.53% and 14.86%, respectively. However, the purification of laccase, lignin peroxidase, manganese peroxidase and endoxylanase from the fruiting body of C. indica resulted in maximum yield of 21.36%, 18.92%, 17.37% and 16.62% with purification fold of 25.6, 25.12, 20.88 and 19.97, respectively. The SDS-PAGE of the purified enzyme laccase and lignin peroxidase isolated from P. florida showed a single prominent band at 66 kDa and 55 kDa respectively. The SDS-PAGE of the purified enzyme laccase and lignin peroxidase isolated from C. indica showed a single prominent band at 64 kDa and 47 kDa, respectively. FTIR spectra of the purified enzymes indicated a secondary structure that reflected the amide I and amide II bands, respectively. The pretreatment of paddy straw and wheat straw with P. florida, C. indica and their ligninolytic enzyme resulted in decrease in lignin and hemicellulose content, respectively during the incubation period of 30 days. However, the decrease in cellulose content occurred during the pretreatment of substrates with fungus while relative increase in cellulose content during the treatment with ligninolytic enzymes was observed over the incubation period of 30 days. Under optimized conditions, saccharification of biologically pretreated paddy and wheat straw resulted in release of 0.415 and 0.389 g/gds reducing sugars, respectively. The fermentation of biologically pretreated and commercial cellulase saccharified paddy and wheat straw hydrolysate resulted in 0.129 and 0.119 g/g ethanol, respectively. The crude enzyme extract of P. florida and C. indica were able to degrade RBBR (25.74 %, 22.06%) and Amido Black (19.76%, 17.58%) dyes maximally after 96 hours of incubation at 30°C and pH 7.0. The paddy and wheat straw was treated with a ligninolytic crude enzyme, which stimulated faster mushroom growth and fructification. The present study thus revealed that expression of biosynthetic potential of P. florida and C. indica is highly dependent on the method of fungi cultivation. These ligninolytic enzymes showed a unique profile in terms of versatility, greenness, pollutant removal and efficiency in lignin degradation for the exploitation and valorization of agro-wastes.
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    ThesisItemOpen Access
    Enzymatic debittering and aroma enhancement of kinnow juice
    (Punjab Agricultural University, Ludhiana, 2017) Manmeet Kaur; Sahota, Param Pal
    The processing of acidic fruits to beverages has encountered commercial restrictions due to development of delayed and inherent bitterness by limonoids and flavonoids. The present study has been undertaken with purified debittering enzymes; limonin dehydrogenase, naringinase and β-glucosidase to meet the consumer palatability of citrus juices. The limonin dehydrogenase enzyme has been purified to 1.14- fold with 33.98% recovery, 29.25 IU activity and 0.525 IU/mg specific activity resulted in 86.43% degradation of limonin in juice by catalyzing oxidation of limonin –A-ring lactone to the non-bitter 17dehydroxylimonoate. The debittering naringinase enzyme 24.8IU activity,0.442 IU/mg specific activity, purified to1.68 fold could reduce 57.93% naringin which is hydrolysed by the enzyme into non-bitter bioactive compound naringenin.. The flavour enhancing βglucosidase with activity 14.68 IU, specific activity 0.344 IU/mg purified to 1.18 fold resulted in the increase in glucose content upto 4.25µg/ml in kinnow juice. The debittered fermented low alcoholic naturally carbonated (LANC) beverage has been developed by optimizing the bioprocess conditions as TSS 13ºB, yeast inoculum @ 0.75%(v/v), incubation temperature 28±2ºC and time 36hrs.The physicochemical changes were recorded as TSS 13ºB, acidity 0.313%, naringin 181.13 ppm, limonin 4.73 ppm, glucose 1.3µg/ml, total sugars 42.97 µg/ml, ascorbic acid 34.22 mg/ml in fermented kinnow beverage after 25 days of storage at 4ºC. In kinnow juice, maximum reduction in bitter component limonin 87.34%, naringin 58.41% whereas increase in glucose upto 4.38 µg/ml, acidity 30.13% and total sugar content 42.97 µg/ml were observed. The nutraceuticals recorded in fermented kinnow beverage were flavonoids (mg/L)- gallic acid 0.075, caffeic acid 0.002, rutin 0.001, ferulic acid 0.001; Organic acids (mg/L) – oxalic acid 0.243,tartaric acid 3.698, mallic acid 0.018, citric acid 0.432; Fat soluble vitamins (mg/L)-Vitamin A (Retinol) 0.060, cholecalciferol (D3) 0.038, α-tocopherol (Vitamin E) 0.001, Vitamin K 0.018 ; water soluble vitamins (mg/L)thiamine 0.782, pyridoxine hydrochloride 0.040, pantothenic acid 0.002, riboflavin 0.001, biotin 0.011; Protein 6.42g/100g,Carbohydrate 40.95g/100g.
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    ThesisItemOpen Access
    Optimization, production and partial purification of βglucosidase from yeast using citrus peel
    (Punjab Agricultural University, Ludhiana, 2016) Manmeet Kaur; Sahota, Param Pal
    The enzyme β-glucosidase hydrolyzes cellobiose and short chain cello-oligosaccharides to glucose. The study was conducted to evaluate the production of enzyme β-glucosidase using economically viable substrate citrus fruit waste with optimized batch scale fermentation using Clavispora lusitaniae strains KF633446 and KP131848.1 as yeast inoculum. The process parameters optimized during study are: substrate concentration (117.9g/L lemon peel powder or 46.875 g/L lime) or 16.8 g/L banana peel powder, temperature (35°C) and pH (5) , inoculum concentration of 0.75% v/v, time period of 36 h and 30h using yeast strains KP131848.1 and KF633446 respectively. Use of citrus fruit waste as substrate for enzyme production has proved to be economical; the maximum enzyme activity 0.49 IU/ml with lemon peel, followed by lime (0.28 IU/ml) and banana peel (0.337 IU/ml). The enzyme activity (IU) of β-glucosidase after partial purification was found to be 18.52 IU. The partially purified β-glucosidase was highly stable at temperature 45°C and pH 5. The value of Km and Vmax was 1.25mM and 17.98 IU/ml respectively using pNPG (para-nitrophenyl-β-Dglucopyranoside) as substrate. The addition of enzyme @ 0.4 ml per 100 ml of juice resulted in increased reducing sugar content in sweet lime juice (58.42%), grapefruit juice (57.36%), lemon juice (55.26%) and kinnow juice (53.19%), with storage time period of 15 days. Sensory analysis on 9 point hedonic scale revealed maximum score of 8.4±0.2 for aroma and 7.85±0.25 for flavour of lemon juice. This study revealed the commercial utilization of peel as substrate for β-glucosidase production and wide applicability of β-glucosidase enzyme in aroma and flavor enhancement along with debittering properties of enzyme Naringinase and Limonin dehydrogenase.