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2020 - 20225
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Subject: Microbiology × End date: 2022 × Start date: 2020 × Thesis Type: Ph.D ×
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    Development of lactic acid starter culture based fermented fruit and vegetable juices
    (Punjab Agricultural University, Ludhiana, 2021) Modi, Ritika; Sahota, Parampal
    The lactic acid fermented fruit and vegetable beverages have been developed through controlled fermentation using ten allochthonous high lactic acid producing (0.612-1.35%), phenotypically and genotypically characterized, homo-lactic- Lactic Acid Bacterial (LAB) strains as starter culture consortium. Value-added secondary metabolite enriched Turmeric, Amla, Black carrots (Kanji), and Black pearl grapes-based functional beverages were developed with improved nutritive value (15-35%) antioxidants, (21-63%) polyphenols, (20-42%) flavonoids and (24%) carotenoids; microbial quality (1010 LAB CFU/ml) and acceptability (8±0.2) with shelf life of 90 days. The optimized bioprocess parameters using Box-Behnken Design in Response Surface Methodology with 5% (v/v) (107 CFU/ml) active starter culture for Turmeric beverage -[turmeric 2%w/v), lemon juice (5%v/v), ginger juice (1.5%v/v); dilution ratio (1:3 with sterilised water); salt (1%w/v)]; Amla beverage -[amla juice: guava juice: ginger juice (1:1:1.5 %v/v); dilution ratio (1:3); salt (0.6%w/v)]; Kanji beverage -[black carrots juice (100ml); salt and rye (1.5%w/v); dilution ratio (1:3)]; Grapes beverage -[grapes juice (100ml), lemon juice (8% v/v); dilution ratio (1:1.5); salt (1.2%w/v)], pasteurized at 82ºC for 10-15 sec and fermentation at 37ºC for 28 hrs. A generic HACCP plan determining critical control points on the line was recommended as a food safety tool during the preparation of beverages. Unstructured kinetic model so developed depicts maximum LAB growth at 8th hour and highest ∆pH and Vmax on the 28th hour and 8th hour, respectively. The increase in lactic acid production (0.35, 0.55, 0.96, 0.63 %TA), reduction in pH (4.51, 4.96, 3.76, 2.64) with enhanced functional aspects based on total polyphenols (52.30, 45.58, 41.85, 52.29 gallic acid equivalents mg/100ml) and flavonoids (44.20, 31.13, 43.91, 46.96 quercitin equivalents mg/100ml) with significantly stronger scavenging activities for the 2,2diphenyl-1- picrylhydrazyl (DPPH) radical (74.25, 86.36, 86.91, 69.70%) and ferric reducing power (87.9, 94.4, 108.66, 100.3 μM FeSO4 equivalents) was observed for Turmeric, Amla, Kanji and Grapes fermented beverages, respectively. These bio-interventions showed antimicrobial activity against food borne pathogens Staphylococcus aureus MTCC3906, Listeria monocytogenes MTCC657, Klebsiella pneumonia MTCC109, Escherichia coli MTCC443, Aeromonas hydrophila MTCC173, as well arrested the initial phase of MOLT-4 and CaCo2 cancer cells lines, down regulating the expression of proto-oncogenes and up regulating the tumor suppressor gene exhibiting the antitumorigenic effect. Further, hepatoprotective and hypoglycemic effect studies showed administration of functional lactic acid fermented turmeric and amla beverages (10 ml/kg body weight) for 6 weeks, significantly reverse or reduce the physiological, metabolic damage, and histological alterations equivalent to the hepatoprotective drug Liv52 in alcohol-induced liver damaged and hypoglycemic drug glibenclamide in streptozotocin-induced diabetic Wistar rats, respectively. Further, lyophilised freeze-dried turmeric and amla fermented powders were developed which can be consumed as ready-to-use fermented beverages by reconstitution @2% and @3.2% (with sterilized water) with retention of all properties of freshly prepared beverage prepared.
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    Bioprocessing of corn stover for bioethanol production
    (Punjab Agricultural University, Ludhiana, 2022) Mandeep Kaur; Kocher, Gurvinder Singh
    The present study was conducted with the objective of standardization and evaluation of an efficient and ecofriendly, method for the pretreatment of corn stover and its subsequent conversion to bioethanol. The proximate analysis of corn stover (500 µ) revealed a composition of cellulose (57.4%), hemicellulose (17.5%), lignin (14.4) and ash (2.2%) in the raw corn stover. Among the different chemical pretreatment methods, acid (1.0% H2SO4) –autoclave (15 psi for 90 min) followed by alkali (2.0% NaOH) pretreatment resulted in 86.8 % and 79.4% decrease in lignin and hemicellulose, respectively and 66.0% increase in relative proportion of cellulose. Among the green solvent methods of pretreatment, organosolv pretreatment methods, acetic acid (40:60, 40%) resulted in 84.6 % and 53.6% decrease in lignin and hemicellulose, respectively and 53.7% increase in relative proportion of cellulose whereas deep eutectic solvent pretreatment, choline chloride-lactic acid (1:8 molar, ratio) resulted in 82.5 % and 33.0% decrease in lignin and hemicellulose, respectively and 69.3% increase in relative proportion of cellulose. For biological pretreatment of corn stover, a two fungal consortium of strains viz. Pleurotus ostreatus PAU03 and Phanerochaete chrysosporium MTCC787 was screened for ligninolytic enzyme production by plate assay on lignin modifying enzyme (LME)- basal medium (LBM), supplemented with 0.01 % (w/v) Azure B and 0.02 % (w/v) Remazole brilliant blue dye and inoculated with agar discs ( 10 mm) of active mycelia. Decolourization of the respective dyes was observed with ligninolytic index of 1.5 and 1.22 for Azure B and Remazole brilliant blue dyes, respectively. The consortium culture of two fungal strains viz. P. ostreatus and P. chrysosporium was used for ligninolytic enzyme production, by using corn stover as substrate. Maximum enzyme activity (U/ml) was recorded on 10th day as 48.33, 61.85 and 40.8 for LiP, MnP, laccase, respectively. The enzyme production was scaled upto 4000ml and the crude extract was concentrated (6.7 times) using acetone and 600 ml concentrated enzyme was produced having enzyme activities (U/ml) 68.89, 41.13 and 110.08 for Lacc, LiP and MnP enzymes, respectively. The latter was partially purified by Fast Performing Liquid Chromatograpgy (FPLC) technique. The enzyme activity (U/ml) of 131.96, 130.38 and 94.21 was recorded for Lacc, LiP and MnP enzymes, respectively in the partially purified enzyme, which was further concentrated (1.5 times) and the enzyme activities (U/ml) of 140.9, 168.53 and 98.05 for Lacc, LiP and MnP enzymes, respectively. This partially purified ligninozyme was used for nanoligninozyme synthesis. In the nanologninozyme (enzyme: sodium:silicate nanohydrate, 1:1) showed the enzyme activities (U/ml) of 128.93, 187.09 and 116.94 for for Lacc, LiP and MnP enzymes respectively. The pretreatment of corn stover with nanoligninolzyme under shake flask conditions (50 ml reaction volume) using optimized physico-chemical parameters viz. corn stover concentration, 2.5 g; enzyme volume, 8.0 ml; Mn2+ ions (0.5 mM) and incubation temperature, 45°C in 72 h of enzymatic action resulted in 87.2% and 67.4% decrease in lignin and hemicellulose, respectively and 80.3% increase in relative proportion of cellulose. The saccharification of pretreated corn stover at different concentrations of corn stover (1.0-10mg) with Arrowzyme (commercial cellulase) at enzyme loading of 30 FPU, resulted in maximum release of reducing sugars (0.396 g/gds) at 2.5g concentration of corn stover. Under these optimized conditions, saccharification of organosolv (acetic acid, 40:60) and biological (nanoligninozyme) pretreated corn stover resulted in release of 0.395 and 0.439 g/g, reducing sugars, respectively. The fermentation of organosolv as well as biological pretreated, and Arrowzyme saccharified corn stover hydrolysate resulted in 0.112 and 0.132 g/gds ethanol, respectively. The fermentation efficiency of 66.54 % [33.9 % yield (yps)] and 70.91 % [36.1 % yield (yps)] was recorded for organosolv and biological pretreatment, respectively.
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    ThesisItemOpen Access
    Mapping of quantitative trait loci (QTLs) associated with biological nitrogen fixation in an interspecific cross of chickpea
    (Punjab Agricultural University, Ludhiana, 2021) Nagpal, Sharon; Sharma, Poonam
    The current study was aimed at investigating the genetic variation responsible for nitrogen fixation efficiency, growth and yield attributing traits in Cicer. The phenotypic variation was studied at two different agroclimatic zones viz., Central plain zone (Ludhiana) and Sub mountain undulating zone (Gurdaspur) for 2 consecutive rabi seasons (2018-20). Both the experimental sites varied significantly for the climatic conditions and soil physicochemical and nutrient profiles over the years. Wild parent C. reticulatum ILWC292 showed significantly high performance in terms of biological nitrogen fixation (BNF) traits over the cultivated C. arietinum GPF-2. On pooled mean basis of 2 years across the zones, the nodulation (19.7nodules/plant), nodule dry weight (172.8 mg/plant) and leghaemoglobin content (4.06 mg/g fresh wt. of nodules) were higher in wild over cultivated Cicer species. The triple interaction of genotypes × locations × years was significant (P0.05) and were used for quantitative trait loci (QTL) analysis. Using QTL cartographer, markers- CAGM02697, CAGM09835, CAGM09777, CAGM09227, CAGM09021, CAGM08679 were found linked with QTLs for BNF. These markers can be validated further for identification of genes for BNF traits and marker assisted selection in chickpea. To the best of our knowledge this is the first report on identification of markers associated with key BNF traits in chickpea.
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    ThesisItemOpen Access
    Development of probiotic vegetable pickles
    (Punjab Agricultural University, Ludhiana, 2021) Kusam Lata; Sahota, Param Pal
    Fermented vegetables were evaluated as vector for administration of probiotic lactic acid bacterial cultures following the proficiency of the production of eight lactopickles (Ginger (Zingiber officinale), Garlic (Allium sativum), Cabbage (Brassica oleracea var. capitata), Cauliflower (Brassica oleracea var. botrytis), Cucumber (Cucumis sativus), Beans (Phaseolus vulgaris), Pea (Pisum sativum) and Green chilli (Capsicum annuum)) based fermented pickle products via lactic acid bacteria. The fermentation of eight vegetables (ginger, garlic, cabbage, cauliflower, cucumber beans, pea and green chilli) were carried by two pure lactic strains Lactobacillus acidophilus (MTCC10307) and Pediococcus acidilactici (MK028218) as functional starter cultures. These cultures were phenotypically and molecularly characterized for its probiotic potential (acid tolerance at pH 3: 6.56 and 6.52 log CFU/mL, bile tolerance at 0.5%: 0.16 and 0.5%, enzyme tolerance for trypsin: 6.0 and 5.3 log CFU/mL, cholesterol assimilation: 4.64 and 3.88 log CFU/mL) and bioprocess optimized from vegetables. The bioprocess for fermentation of above mentioned vegetables was optimized; inoculation rate 5.0% w/v (106 CFU/mL) with active starter culture, incubation temperature 37ºC for 24-72 hours, dilution ratio 1:3, brine 5.0%; pH 4.8 and 4.6, total soluble solids 4.8 and 5.2ºB, titrable acidity 0.25 and 0.35%, LAB count 8.82 and 8.91 log CFU/mL, and overall acceptability 8.4 and 8.6. Out of eight vegetables fermented ginger scored highest on hedonic scale and fermentation up scaled with promising strain; LAB count 5.54 and 5.03 log CFU/mL, pH 3.8 and 3.7, total soluble solids 4.6 and 4.5ºB, titrable acidity 1.60 and 1.52%, overall acceptability 8.3 and 8.5 viz. nutraceuticals; total polyphenolic content 50.4 and 49.14 mg GAE/100g, total flavonoids 45.11 and 43.11 mg/100g and total antioxidants 72.11 and 58.78%. The profile of amino acids in finished products in lactic acid fermented vegetable as pickle in L. acidophilus; L-threonine-1.05, L- phenylalanine-8.64, L- alanine2.42 and L- glycine-2.42mg/g while the P. acidilactici fermented vegetables were enriched with Lglutamic-13.28 and L-aspartic acid-26.31mg/g. Organic acids in lactic strains L. acidophilus and P. acidilactici were: Citric acid (0.08% and 0.06%) and acetic acids (1.10% and 1.13%). The scanning electron micrography of ginger pickle clearly depicted parenchyma cells had oval shape, expanded, middle lamella weakned and higher firmness during the process of salting and fermentation. During the PCR the amplified bands of 173 base pair of plasmid DNA indicated that the strain as lactacin B producing. The bio-interventions, lacto-pickles endowed with neutraceuticals showed antimicrobial activity against food borne pathogens Escherichia coli (MTCC443), Aeromonas hydrophila (MTCC1739), Klebsiella pneumonia (MTCC109), Listeria monocytogenes (MTCC657) and Staphylococcus aureus (MTCC3906).
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    ThesisItemOpen Access
    Identification and characterization of enzyme(s) causing browning of button mushroom, Agaricus bisporus (Lange) Sing.
    (Punjab Agricultural University, Ludhiana, 2020) Ravneet Kaur; Sodhi, H.S.
    Agaricus bisporus is the most acceptable mushroom in the world but being perishable it loses sheen as fresh produce. Present study has dealt with the enzymes related to browning of button mushroom, namely, tyrosinase (E.C 1.14.18.1), laccase (EC 1.10.3.2), peroxidase (EC 1.11.1.7), phenylalanine ammonia lyase (E.C. 4.3.1.5), lipoxygenase (EC 1.13.11.12) and catalase (EC 1.11.1.6). These were estimated in submerged state and the compost/casing (solid substrate). The enzyme activity of A. bisporus mycelium in broth was found to increase with time and maximum activity was that of peroxidase (7.41U/mg) and laccase (6.05U/mg). Mycelial run in compost indicated maximum specific activity of laccase enzyme (12.3 U/mg) while the activity of the other enzymes was significantly lower. During the pin head stage, there was an increase in the activity of tyrosinase, laccase, peroxidase, PAL, catalase, lipoxygenase enzymes as 17.67, 13.98, 14.67, 4.5, 2.33 and 17.75U/mg respectively. After harvesting during the first flush, tyrosinase activity was reduced up to 12.03 U/mg and remained at par during the second and third flush harvesting. A similar pattern was observed for laccase and PAL. Peroxidase activity showed a decline up to second flush and remained stable thereafter. Catalase activity reduced from the pin head stage to first flush and no significant change in the lipoxygenase activity was observed during reproductive phase of mushroom production. During the postharvest period, the activity of melanin synthesizing enzymes that is tyrosinase, laccase and peroxidase was found to increase with time during storage and maximum increase was that for tyrosinase leading to increase in browning index (BI) value. The activity of the lipoxygenase enzyme increased which led to increased electrolytic leakage and hence lipid peroxidation. During the storage period, there was a decrease in the activity of PAL while the activity of catalase was found to increase. Mushroom washing treatments (KMS, Citric acid and Salicylic acid) and fumigation (cinnamon oil and clove oil) along with packing treatments (polypropylene bags, carton trays with cling film cover and plastic trays with cling film cover) were studied up to 16d at 4 day intervals. The washing treatments using 0.2% KMS, 1% citric acid and 200µmole/L salicylic acid showed a significant decline in the browning associated enzymes and BI in all the packagings during 16d storage. In general mushrooms treated with 200µmole/L salicylic acid and packed in PT showed the best results with minimum BI value (23.09) and tyrosinase activity (27.72 U/mg) at 16th day of storage. Cinnamon oil and clove oil fumigation indicated a gradual decline in activity of melanogenic enzymes and BI with increasing oil concentration up to 40 ppm. Tyrosinase enzyme was purified and found active over pH range 6.4 to 7.2 showing 90% and 88% of the maximum activity at pH 7. The enzyme was stable at a temperature range 25-40°C with maximum activity at 35°C. The kinetic studies of enzyme showed that in case of catechol as substrate, the Km was found to be 0.71 mM with Vmax 2518 µmole/min/ml. In case of L-Dopa from Lineweaver-Burk plot, the Km value was found to be 0.87 mM with Vmax 1714 µmole/ml/min. The SDS-PAGE electrophoresis of the enzyme gave a single prominent band at 43 kDa. FTIR spectra of purified enzyme indicated a secondary structure that reflected the amide I and amide II bands. This study indicated that mushroom browning is a complex process triggered after the harvesting involving the enzymatic reactions. The study of the browning associated enzymes at the different stages of the cultivation and during the storage with different postharvest treatments showed that browning can be decelerated by the postharvest treatments which mainly act by restricting the tyrosinase and related enzymes.