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ThesisItem Restricted Development of lactic acid starter culture based fermented fruit and vegetable juices(Punjab Agricultural University, Ludhiana, 2021) Modi, Ritika; Sahota, ParampalThe 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.ThesisItem Embargo 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, ShivaniPleurotus 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.ThesisItem Restricted Evaluation of bacterial consortium to boost in-situ degradation of Sesbania sp. to improve soil fertility, plant growth and yield of maize crop(Punjab Agricultural University, 2023) Khipla, Neha; Sharma, PoonamPresent research work was aimed at evaluating bacterial consortium for boosting in-situ decomposition of Sesbania (green manure) to improve soil health, growth and yield of Kharif maize (var. PMH1). Three cellulolytic bacterial cultures viz. CD2, CD9 and CD14, identified through 16S rDNA sequence similarity were published at GenBank under strain name Lysinibacillus sp. Strain JNcd2 (OM721941.1), Glutamicibacter mishari Strain JNcd9 (OM723742.1) and Paenibacillus sp. Strain JNcd14 (OM727120.1), respectively. The bacterial strains were positive for various plant growth promoting traits such as ammonia production and excretion, phosphorus and zinc solubilization, starch hydrolysis, IAA and siderophore production. All the bacterial strains were tested positive for ligno-cellulolytic enzyme production in both qualitative and quantitative assays; and thereby, designated as Ligno-cellulolytic Bacterial Consortium (LBC). Primarily, the effect of LBC inoculation on decomposition and nutrient mineralization of Sesbania and Sesbania + rice straw was studied through 60 day laboratory incubation study during Kharif 2018. LBC inoculation increased weight loss rate of Sesbania by 17.7 % and of Sesbania + rice straw by 11.6 % over uninoculated control, till 16th day of decomposition. Total carbon content and C:N ratio of both the organic inputs was significantly lower in LBC inoculated treatment over uninoculated control, till 30th day of decomposition. Highest mean soil viable population of total bacteria and cellulolytic bacteria was recorded in Sesbania + LBC treatment, total fungi and nonsymbiotic diazotrophs in Sesbania + rice straw treatment; while, PSB and actinomycetes in Sesbania + rice straw + LBC treatment. Soil respiration, organic carbon, ammonical and nitrate nitrogen available phosphorus and potassium content was higher by 19.2 %, 6.8 % 15.2 %, 2.7 %, 1.6 % and 5.4 % in Sesbania + LBC treatment, and by 4.1 %, 0.8 %, 5.3 %, 3.4 %, 2.7 % and 4.9 % in Sesbania + rice straw + LBC treatment, respectively than uninoculated controls, at 60th day of decomposition. Field studies were conducted for two consecutive Kharif seasons (2019 and 2020) to study the effect of LBC inoculation with different sole inorganic and integrated fertilizer treatments on soil bio-chemical properties, plant growth, nutrient acquisition and yield of maize. Before sowing of maize, soil treatment with Sesbania + LBC showed highest fertility status over bare, than sole LBC and Sesbania treatment. In maize rhizosphere, soil viable microbial population (total bacteria, fungi, PSB and cellulolytic bacteria) and enzymatic activities (dehydrogenase, alkaline phosphatase, urease, cellulose and invertase) remained significantly higher in treatment with 100 % N + Sesbania + rice straw mulch (RSM) + LBC, during both experimental years. However, soil actinomycetes and non-symbiotic diazotrophs were highest in 75% N + Sesbania + RSM + LBC treatment and soil amylase activity in 100 % N + Sesbania + LBC treatment. Soil chemical properties viz. electrical conductivity, organic carbon, available P and K content, were highest in 100 % N + Sesbania + RSM + LBC treatment; soil available N content in 100 % N + Sesbania + LBC treatment; and soil pH varied numerically. During both experimental years; highest plant nutrient (NPK) uptake, growth and yield attributes, grain and stover yield of maize was recorded with treatment 100 % N + Sesbania + RSM + LBC. It was concluded that integrating LBC with organic and inorganic fertilizers could ensure efficient decomposition and nutrient mineralization in soil, and thus contribute to improved soil health, growth and productivity of maize.ThesisItem Restricted Impact Of Sulphur Oxidizing Bacteria On Soil Health And Yield Of Brassica Napus L. In Field Conditions(Punjab Agricultural University, 2023) Vishnu A L; Sharma, PoonamPresent research work was aimed at evaluating the impact of sulphur oxidizing bacteria (SOB) on soil health and yield of Brassica napus (var. GSC 7) in field conditions. Three SOB cultures viz. SOB 5, SOB 10 and SOB 38 were able to tolerate the recommended concentration of different pesticides (pendimethalin, clodinafop, isoproturon, bavistin and chlorpyriphos) excluding ridomil. SOB 38 tolerated highest concentration of clodinafop, isoproturon, bavistin, ridomil and chlorpyriphos with MIC of 0.80 ppm, 12 ppm, 0.30 ppm, 0.18 ppm and 0.40 ppm respectively; while SOB 10 showed highest tolerance to pendimethalin with MIC of 1.40 ppm. Results of disc diffusion method revealed that in all SOB cultures maximum inhibition zone was seen with MIC as compared to ½ MIC and recommended concentration of pesticides. Growth profiling of all three cultures revealed that SOB 38 showed highest OD540nm of 2.098, 2.444, 1.467 and 0.889 at recommended dose of clodinafop, isoproturon, bavistin and chlorpyriphos respectively. SOB 10 (OD540nm 2.244) showed best growth at recommended dose of pendimethalin. While, SOB 5 revealed highest OD540nm (1.689) at ½ MIC dose of ridomil. SOB 38 was found superior for functional activities including quantitative P solubilization (1.761µg/ml), IAA (38.43 µg/ml), siderophore (12.7 µg/ml), ammonia excretion (3.033 µg/ml) and sulphate ion production (0.124mM). Highest qualitative P and Zn solubilization was seen in SOB 5; while maximum siderophores were produced by SOB 10. All three SOB cultures viz. SOB 5, SOB 10 and SOB 38 were identified through 16S rRNA sequence similarity and were submitted at GenBank under strain names Enterobacter ludwigii strain Remi_9 (ON764276), Enterobacter hormaechei strain AUH-ENM30 (ON764272) and Bacillus sp. strain 5BM21Y12 (ON764271) respectively. Field studies were conducted for two consecutive Rabi seasons (2019-20 and 2020-21) to study the effect of potential SOB (SOB 38), inorganic sulphur and pesticides on soil bio-chemical properties, plant growth, nutrient acquisition and yield of Brassica napus. Soil viable microbial population viz. total bacteria, fungi, actinobacteria, diazotrophs, PSB and SOB; enzymatic activities viz. dehydrogenase, alkaline phosphatase, urease, and sulfite oxidase, were significantly higher in treatment with 100 % S + SOB 38 + pesticides, during both experimental years. Soil chemical properties viz. electrical conductivity, organic carbon, available N, P and K content, were highest in 100 % S + SOB 38 + pesticides treatment; while soil pH varied numerically. During both experimental years; highest plant nutrient (NPKS) uptake, growth, yield attributes, seed yield and total biomass in Brassica napus was recorded with treatment 100 % S + SOB 38 + pesticides. Total crude protein and oil content in seed of Brassica napus was insignificant between the treatments. Application of pesticide tolerant SOB 38 (Bacillus sp.) in integration with inorganic fertilizers (NPS) improved soil microbial dynamics and available nutrients which enhanced crude oil, protein, nutrient acquisition, growth and yield of Brassica napus.ThesisItem Embargo Consolidated bioprocess for microalgae cultivation and biogas production from fruit and vegetable wastes(Punjab Agricultural University, 2023) Saroj Bala; Gupta, UrmilaThe main objective of the present study was to formulate fruit and vegetable peels-based media for microalgae cultivation followed by biogas production. A total of five stress-tolerant microalgae strains, viz., Nostoc, Chlorella, Scenedesmus, Spirulina, and Oscillatoria available at the Biogas Laboratory, Department of Renewable Energy Engineering, College of Agricultural Engineering and Technology, PAU, Ludhiana, were used in the present study. Physical (grinding), chemical (choline chloride: citric acid) and biological (kinnow peels based bioenzymes) pretreatment of fruit and vegetable waste were generally used to maximize the extraction of macronutrients and micronutrients for the growth of microalgae. Microalgal strains were grown on fruit and vegetable wastes leachate supplemented standard media (5%, 10%, and 15% of fruit waste, vegetable waste, and mixture of fruit and vegetable wastes). These strains were screened for biomass production and other parameters (chlorophyll, protein, carbohydrate, and lipids). Among all the five strains, Oscillatoria sp. were found to produce maximum biomass and other parameters on algal cultivation media (ACM) as well as combination of 5% fruit and vegetable waste leachate supplemented with standard media. Oscillatoria sp. growth was optimized using response surface methodology (RSM) based on a central composite design (Statgraphics Centurian XVI.I). Characterization of microalgae biomass cultivated on fruit and vegetable waste medium and standard media (ACM) by GCMS, FTIR and thermogravimetric analysis. The optimized and most desirable cultural conditions were at 10 pH, 5% concentration of biologically pretreated fruit and vegetable leachate, 5% inoculum concentration, and 30 days of incubation period. The solid spent waste from physical, chemical and biologically pretreated fruit wastes, vegetable wastes and mixture of fruit cum vegetable wastes were evaluated for their biogas production potential. It was found that the highest biogas was produced by biologically pretreated fruit and vegetable spent waste (51.315 l/kg FVW, 172.86 l/kg TS, and 207 l/kg VS) as compared to control (39.737 l/kg FVW, 133.86 l/kg TS, and 160.32 l/kg VS) and % enhancement of biogas was 29.136%. Oscillatoria sancta PCC 7515 were evaluated for their biogas production potential alone and in co-digestion with fruit and vegetable wastes along with cow dung as substrate and cow dung slurry as inoculum. Upscaling studies conducted using 15 kg biologically pretreated fruit and vegetable wastes showed maximum biomass production of 4.45 g/L along with 13.89% increase in biogas production. Thus, consolidated bioprocess of fruit and vegetable wastes for microalgae cultivation and biogas production is technically feasible and financially sustainable.ThesisItem Embargo Bioprocessing of corn stover for bioethanol production(Punjab Agricultural University, Ludhiana, 2022) Mandeep Kaur; Kocher, Gurvinder SinghThe 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.ThesisItem Open 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, PoonamThe 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.ThesisItem Open Access Development of probiotic vegetable pickles(Punjab Agricultural University, Ludhiana, 2021) Kusam Lata; Sahota, Param PalFermented 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).ThesisItem Open 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.
