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Open Access
Investigation on biochemical basis of resistant starch with respect to physiochemical properties in cereals-millets, rice and wheat
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-12) Verma, Vivek Chandra; Agrawal, Sanjeev
The present investigation was carried out to assess the physio-chemical properties of starch attributing towards resistant starch in millets (Barnyard millet; VL Madira-207, Finger millet; VL Mandua-352 and Foxtail millet; DHFT-109-3), rice (PB-2 and PD-19) and wheat (PBW-343 and UP-262). The study emphasize on the factors which influences the resistant starch in cereals. In the present study starch isolated from different cereals were viewed under SEM to analyse the shape and size of starch granules. The size of starch granules were diverse range in cereals like millets (4.6-12.23 μm) rice (6.22-177.13 μm) and wheat (2.50-198.23 μm). Small and large spherical, irregular and polygonal shape of starch granules was observed. The FTIR bands at 1047 and 1022 cm-1 gives ordered and amorphous structure of starches and absorbance ratio at 1047/1022 gives indexes of the short-range order of double helices ranged from 0.94-1.24. Total starch (57-70%), amylase (31-45%) and amylose-amylopectin ratio (0.46-0.83) was determined in different cereals under observation. The amylose content was positively correlated with resistant starch at P < 0.01 while total starch had non-significant impact on resistant starch. The other nutrient of cereals like dietary fibre (2.66-6.98%), total soluble protein (9.33-16.29%) and soluble protein fraction like albumin (1.41-2.44%), globulins (1.25-2.23%) and prolamines (2.04-2.88%) was estimated to establish the correlation with resistant starch. These other nutrient of cereals showed positive correlation with resistant starch at P < .01. In vitro digestion of native starch, gelatinized starch and retrograded starch was done at time interval of 30, 60, 90, 120 min and it was found that the rate of enzymatic digestion follows the order; Gelatinized starch > Native starch > Retrograded starch. During endosperm developmental stages at S1, S2, S3, S4 (7, 14, 21, 28 DAA) the key enzymes for starch biosynthesis like AGPase, SSS and GBSS were estimated. The activity of starch biosynthetic enzymes follows the order AGPase > GBSS > SSS in all these varieties during endosperm development. The higher activity of AGPase, GBSS and SSS found in rice comprises higher accumulation of amylose and total starch during endosperm development. The accumulation of amylose and total starch in millets was found at par to the wheat varieties which is related to the activities pattern of enzymes. All three enzymes under study showed the positive correlation with each other during endosperm development. Accumulation of amylose showed positive correlation with AGPase and GBSS, while non-significant correlation with SSS. Total starch accumulation showed positive correlation with AGPase.
Open Access
Optimization of pretreatment parameters and nanoparticle based enzyme immobilization for lignocellulosic biomass saccharification
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-09) Singh, Archana; Verma, A.K.
Pretreatment is a prerequisite technology to facilitate the recovery of fermentable sugars from lignocellulosic biomass. Various lignocellulosic biomass viz. sugarcane leaves, rice straw, wheat straw, orange peel and pomegranate peel were used for pretreatment with ionic liquid EmimAc (1-ethyl-3-methylimidazolium acetate), aqueous ammonia and a combination of both followed by saccharification with immobilized cellulase and β-glucosidase (BGL) on magnetic nanoparticles. Magnetic nanoparticles (Fe3O4) and cellulase/BGL immobilized nanoparticles were characterized by TEM, SEM, FTIR and Zeta potential. Immobilization efficiency of cellulase and BGL were found to be 86.36% and 87.73%, immobilization yield were 83.38 and 85.18% respectively. The immobilized cellulase had optimum pH 5.0 and optimum temperature 50°C. The immobilized cellulase retained 90% of initial activity after 60 min incubation at 50°C while free cellulase had only 49% of its initial activity. Km value for free and immobilized cellulase was found to be 0.625 mM and 0.714 mM and the Vmax was 35.71 and 38.16 μmole/min/mg respectively. Immobilized cellulase retained up to 97% of residual activity on 6th day when stored at 4°C and 45% at 25°C whereas free cellulase had 86% of residual activity at 4°C and 20% at 25°C. Immobilized cellulase retained about 87% of residual activity after 10 cycle of reuse. The immobilized BGL had optimum pH 5.0 and optimum temperature 50°C. The immobilized BGL retained 91% of initial activity after 60 min incubation at 50°C while free BGL had only 51% of its initial activity. Km value for free and immobilized BGL was found to be 0.555 mM and 0.779 mM and the Vmax was 38.46 and 43.47 μmole/min/mg. Immobilized BGL retained up to 95% of residual activity on 6th day when stored at 4°C and 42% at 25°C whereas free BGL had 87% of residual activity at 4°C and 18% at 25°C. Immobilized BGL retained about 85% of residual activity after 10 cycle of reuse. The combined use of EmimAc and aqueous ammonia in 1:1 for pretreatment at 6 hrs with <2 mm size of biomass followed by saccharification with 3.2 U of immobilized cellulase showed a synergistic effect on biomass with release of 93% glucose. Combined pretreated biomass were analysed for saccharification by addition of 3.2 U BGL with 3.2 U cellulase and 97% of glucose released. Furthermore, EmimAc and immobilized cellulase were recycled for 6 times with 89% of efficiency; EmimAc, immobilized cellulase and immobilized BGL were recycled for 6 times with 90% of efficiency. Thus, compared to the other pretreatment method, this combine method is more efficient and cost effective. To identify the cheapest source for enzymes biogas slurry was analysed for different enzymes. These were cellulase (0.41), BGL (0.30), Xylanase (0.31), α-amylase (0.06), β-amylase (0.20), Dehydrogenase (0.37), and Protease (0.29) U/mL. Saccharification was done with crude enzyme isolated from biogas slurry and found 69% of glucose release in combined pretreated biomass.
Open Access
Evaluation of brown and yellow seeded quality germplasm of oilseed Brassica for their potential nutrients
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Sajwan, Neha; Punetha, Himanshu
Oilseed Brassica is one of the most important crop among the seven edible oilseed crops. India’s top Rapeseed & Mustard producing states is Rajasthan followed by Madhya Pradesh and Haryana. In northern India, mustard oil is popular for cooking purposes, in the preparation of pickles and also used in cosmetics due to its excellent health benefits. The defatted meal of oilseeds Brassica is an excellent source of many bioactive compounds, proteins, minerals, vitamins and antioxidants. It also comprises some anti-nutritional compounds such as glucosinolates and phytic acid. In the present investigation, 15 genotypes of oilseed Brassica were evaluated for their potential nutrients. The total phenolic content observed to be maximum in LES-56 (5.92 ± 0.132 mg/g), total flavonoid content found highest in RLC-6 (0.536±0.009 mg/g) and the ortho-dihydroxy phenol recorded highest in Kranti (0.412±0.007 mg/g). The highest antioxidant content was recorded in PM-29 (23.27 ± 0.202 mg/g) followed by NRCHB-101 (21.19 ± 0.583 mg/g) and LES-56 (20.54 ± 0.329 mg/g). The glucosinolates in Brassica genotypes in desired range (i.e., <30 μmol/g) was observed in PDZ9 (19.69±1.229μmol/g), PDZ-10 (20.35±1.847μmol/g) and RLC-6 (27.73±0.011μmol/g). The minimum phytic acid content was recorded in PDZ-9 (1.7± 0.173 mg/100g). The crude protein ranges from 23.35±0.710 % (in RLC-6) to 30.06±1.121 % (in LES-56). The methionine content was found to be maximum in LES-54 (2.398± 0.063 g/100g protein) and the highest tryptophan content was found in NPJ-203 (1.219± 0.029 g/100g protein). The highest content of nitrogen, phosphorus, and potassium was recorded in LES-56 as compared to Kranti. The maximum content of Copper was observed in PM-30 (9.2 mg kg-1), Zinc was found to be highest in RGN73 (149.5 mg kg-1) and Iron was observed to be highest in RH-749 (187.7 mg kg-1). Thus, the oilseed Brassica can be utilized in the preparation of functional food as a feasible food ingredient due to the presence of potential nutrients.
Open Access
Effect of micro/macro environment of rhizogenesis for optimization of withanolide contents in Withania somnifera
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Singh, Manali; Agrawal, Sanjeev
India is a varietal emporium of medicinal plants and is one of the richest countries in the world with regard to genetic resources of medicinal plants. Withania somnifera is a revered herb of Indian medicinal system. Owing to the presence of pharmacologically bioactive compounds. The roots and leaves of Ashwagandha contain various alkaloids viz. Withanolides and withaferin A. Among withanolides, withaferin A and withanolide A has been reported to be dominant metabolite distributed among various tissues of this plant in varying concentrations. Since the location and environmental factors play a key role in the biosynthesis of the bioactive metabolites so the agro-climatic conditions are conductive for introducing and domesticating new exotic plant varieties with enhanced and consistent withanolide A/withaferin A contents. Thus keeping in mind the pharmacological significance of the bioactive ingredients, the present study focuses on the different strategies employed for their enhanced production. MS medium supplemented with Kinetin 1.5 mg/l was found to have highest withaferin A content i.e. 0.93 mg/g (DW) while withanolide A content was highest in MS medium supplemented with Kinetin 1.5 mg/l i.e 0.348 mg/l (DW). Agrobacterium rhizogenes transformed Poshita recorded highest total withaferin A content i.e 5.969 mg/g (DW) and total withanolide content i.e 2.017 mg/g(DW). In aquaponics the total withaferin A content of 6 month grown seedling, was higher in Jawahar-20 i.e 2.904 mg/g (DW) while withanolide content was higher in Poshita i.e 4.019 mg/g (DW). Moreover use of different elicitors at very low concentrations were found to enhance the withaferin A /withanolide A content. Thus both the varieties i.e. Jawahar-20 and Poshita are the promising varieties of Withania somnifera, with higher withaferin A /withanolide A production. Thus implementation of tissue culture techniques, Hairy root culture, usage of elicitors, bio augmented soil and aquaponic system can offer promising approach for enhanced growth and sustainable production of pharmacologically bioactive ingredients in perspective of improving cultivation of Ashwagandha.
Open Access
Biochemical characterization of selected chemotypes of Withania somnifera
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Pant, Vinita; Agrawal, Sanjeev
Withania somnifera, commonly known as Ashwagandha, is a traditional herb of Indian origin having extensive therapeutic importance. Ashwagandha contain various bioactive compounds in which withanolides, especially withaferin A and withanolides A are of major therapeutic value. Conventional propagation of Ashwagandha faces major problems due to many diseases, poor seed viability and germination. Owing to its huge commercial application, micropropagation method has become a rapid and efficient method of its production. In present investigation four chemotypes (P4, P11, P20and J9), were taken for in vitro propagation and maintenance through tissue culture technique. The plant extracts were prepared of these four in vitro grown chemotypes and two field grown varieties (Jawahar-20 and Poshita) in two different solvents viz. methanol: water (80:20) and water for the efficient recovery of their bioactive compounds. The extracts were prepared for their investigation on biochemical parameters like total phenolic content (TPC), total flavonoid content (TFC), ortho-dihydroxy phenol (ODP) and total antioxidant activity (TAA). Biochemical evaluation of these two extracts showed better phytochemical recovery in aqueous methanolic extract (80%) with respect to water. The phytochemical content also varied with the chemotypes. Among the field grown varieties Poshita showed higher content of TPC, ODP content and TAA compared to Jawahar-20, however the TFC was higher in Jawahar-20. Similarly among the in vitro grown chemotypes P4 showed maximum content of TPC, ODP content and TAA and the TFC content was higher in J9 type. Further the variation was also analysed by phytochemical profiling through thin layer chromatography along with protein study using SDS-PAGE. The phytochemical content and protein composition varied with the variation in chemotypes as well as solvent composition used for extraction.
Open Access
Characterization and comparison of bioactive constituents in the in vitro regenerated and wild roots of Himalayan medicinal herb Valeriana jatamansi Jones
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Saini, Soniya; Kumar, Vandana A.
The medicinal herb Valeriana jatamansi Jones is a native of Northeastern Himalayan region belonging to family Valerianaceae and growing at altitudes of 1500-3000 m asl. The in vitro direct-, indirect rhizogenesis and plantlet regeneration was achieved from leaf explant in MS medium supplemented with ppm quantities of NAA (2.0)+BAP (2.0); IBA (1.0)+NAA (1.0)+BAP (0.1) and BAP (2.0)+NAA (0.2), respectively. Multiple shoots and plantlet regeneration was achieved from nodal explant in MS medium supplemented with ppm quantities of NAA(0.1)+TDZ(1.0) and IBA(1.0)+AgNO3(1.0), respectively. Phytochemical screening in crude and methanolic root extract by FTIR confirmed the presence of alkaloids, saponins, tannins, flavonoids, steroids and terpenes, and polyphenols. Methanol was the best solvent followed by dichloromethane and ethylacetate for extraction of phytochemicals from root and leaf of wild plant. The highest phytochemicals like phenol (351.0mg GAE g-1 extract), flavonoids (84.0mg QE g-1 extract), tannin (814.0mg TAE g-1 extract) and orthodihydroxy phenol (47.0mg CE g-1 extract) content and antioxidant activities like total antioxidant (935.4 AAE g- 1), FRAP (810.1 AAE g-1 extract), DPPH radical scavenging (IC 50=11.8μg ml-1), Superoxide radical scavenging (IC50=18.4μg ml-1), Hydroxyl radical scavenging (IC50=18.1μg ml-1) and Metal ion chelating (IC50=11.5μg ml-1) were found to be highest in methanolic extract of 6 months grown wild roots at 35ºC. The methanolic extracts of 6 months grown roots at 35ºC showed lowest MIC (62.50 μg) and maximum efficacy at par to gentamycin standard against 6 human pathogenic strains i.e., S. typhaemurium-52, S. typhaemurium-149, S. entiritidis-ATCC 13076, S. typhaemurium-ATCC 51812, E. coli-ATCC-25922 and E coli strain-13. Effect of maturity of roots (in vitro- and wild grown) and thermal stress indicated that the phytochemical, antioxidant, antioxidant enzymes and antimicrobial activities in 6 months grown in vitro roots were comparable to 3 months grown wild roots indicating adaptation towards higher temperature tolerance, since V. jatamansi was a temperate herb. Micronutrient analysis showed that Fe (0.254 ppm) was found to be significantly highest than Zn (0.137 ppm) in wild leaf and root. The third largest element in root was Mn (0.052 ppm) and Ca (0.118 ppm) in wild leaf because Mn facilitated the uptake of other nutrients like Fe and inorganic phosphate in root and Ca acted as binding agent in cell wall fusion in leaf. GC-MS analyses identified 38 constituents comprising 78.33% of total compositions of which 28% were contributed by patchouli alcohol confirming V. jatamansi as a patchouli chemotype. Valerenic acid content quantified by HPLC in the in vitro and wild roots was 8.6 mg g-1 and 25.09 mg g-1 dry weight, respectively.
Open Access
Micropropagation studies on endangered species Gentiana kurroo Royle using calcium supplemented growth medium
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Kohli, Manju; Kumar, Vandana A.
Gentiana kurroo containing high amounts of irridoids vis gentiopicroside, gentiomarin and gentiopicrin, is a critically endangered medicinal plant found at 1500 to 3500m altitude. Its micropropagation in four MS media i.e M1(GA3:BAP:Kin), M1C(M1+extra calcium pantothenate), M2(NAA:BAP) and M2C(M2+extra calcium pantothenate) was compared on the basis of number of leaves, nodes and shoot length(cm). Culture grown in M1C, M1, M2C & M2 media showed average leaves (19.5, 14.6, 25.1, & 14.8, respectively), nodes (4.5, 3, 5.5 &3.5, respectively) and shoot length (5.1, 2.6, 7.8 & 2.78cm, respectively) at 90 days of growth. Root formation was observed only in M1C (short, thick, slow growing) and M2C (long, thin, rapidly growing) media but roots grow earlier & better in M2C media. The decrease of electrical conductivity in leaf grown in M1C (38.2%) ,M2C (36.6%), M1(35.6%) & M2(33.3%) clearly indicated cell membrane stability due to extra calcium. The increase of chlorophyll A & B contents in M1C over M1 and M2C over M2 was 46%, 24.5% and 20%, 10%, respectively, suggested the role of calcium in improving photosynthesis and, therefore, size of cultures. Highest calcium content was observed in M1C (2.455μg/g), maximum gentiopicroside content was observed in leaf parts grown in M1C (0.32mg/g) and root parts grown in M2C (0.38 mg/g). Maximum protein content in leaf grown in M1C was 1723.81. Catalase activity increase in M1C over M1 & M2C over M2 was 63.3% & 67%, respectively. Superoxide dismutase activity decrease in M1C over M1 & M2C over M2 was 12% & 33%, respectively. Glutathione reductase activity decrease in M1C over M1 & M2C over M2 was 2.7% & 2%, respectively. Calcium was recognized to play critical role for holding cell wall of together and improving the overall culture growth.
Open Access
Biochemical and ecochemical assessment of healthy and declining Dalbergia sissoo plantation to recognize plant mortality reason
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-07) Pathak, Himanshu; Dubey, Ashutosh
Dalbergia sissoo is widely distributed tree in tropical and sub tropical regions in general and is a significant component of the alley cropping system of agroforestry in particular. The tree is reported to have positive pharmacological and ethno-medicinal properties. Equivalently, there are a good number of reports indicating high mortality of the tree due to varied factors ranging from edaphic, pathological, silvicultural, entomological as well as certain soil factors. However, conclusive scientific studies are not sufficient in this regard. Keeping in mind the above-stated problem a lab experiment was conducted in 2017-2018 for biochemical and ecochemical assessment of healthy and declining Dalbergia sissoo plantation (four year old) and to recognize plant mortality reasons. From both healthy as well as declining tree, soil samples (at 5, 10 and 15 cm depth; from near as well as one meter away from the tree trunk) and leaf samples were evaluated for different physical (Soil pH, EC and NPK content) and biochemical properties (proline, chlorophyll, ascorbic acid, phenol, flavonoid antioxidant, enzyme activity etc.). Soil pH was significantly higher in case of declining and organic matter content was significantly higher in case of healthy tree at almost all the depths of sampling with minor variations while Soil EC didn’t differ significantly among the two types of trees. N content declined and K content increased in case of declining tree, while P content didn’t differ significantly for the two stages of trees. Total protein content in soil was observed to be significantly higher in healthy tree as compared to the declining one at all the depths. Amongst the soil enzymes tested, specific activity of protease and beta glucosidase were observed to be marginally higher in case of healthy tree while urease activity was relatively greater in declining tree. Rhizospheric microbial activity at all sampling depths was significantly greater in case of healthy tree indicating good rhizosphere health in case of healthy tree as compared to the declining tree. It followed from the results that primary stress/ageing indicators; proline content as well as membrane injury index value were expectedly on a higher side in the declining tree while ascorbic acid, auxin and gibberellic acid i.e. growth promoters were observed to be greater in case of healthy tree. Concentration of micronutrients Fe, Ni and Zn was greater in case of declining trees. The result is in line with the fact that these micronutrients are the major cofactors of stress/ageing induced plant enzymes; the latter are upregulated in case of stress/ageing. In continuation with the above reasoning the amount of phenol, flavonoids and total antioxidants were observed to be significantly greater in case of declining tree. Total chlorophyll and total protein content in leaves were higher in case of green tree as compared to the declining one. The stress/ageing marker enzymes SOD (Superoxide dismutase), catalase and peroxidase were found significantly higher in case of the declining tree as compared to the healthy tree. An SDS-PAGE and GC-MS analysis of the samples from two types of trees indicated different but specific set of protein and metabolite fractions being active respectively, in healthy and declining trees.
Open Access
Evaluation of pyrolysis products from biomass for commercial applications
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-07) Lekhak, Brijesh; Verma, A.K.
Pyrolysis products (aqueous phase of bio-oil, bio-oil and biochar) of sugarcane leaves, wheat straw, rice husk and pine needles were used for wheat seeds germination. Biochemical analysis of wheat seeds grown in presence of 2 mg/ml wheat biochar showed increased GA3 (86.58%), _-amylase (249%), reducing sugar (23%) and total dehydrogenase activities (149%). While there was a decrease in _-amylase activity (23%) and starch (30%) during first 20 hours of imbibition of seeds. Bioinformatic analysis of GC-MS data of biochar extract, revealed that the beta-sitosterol had lower _G value with karrikins receptor of Arabidopsis. GC-MS analysis of pine needles bio-oil showed approximately 50% phenols. These phenols were extracted by liquid-liquid extraction method with 86.52% yield. Antioxidant assay of extracted phenolic compounds showed high DPPH, Total antioxidant and reducing power activity. MTT assay of the aqueous phase of pine needle bio-oil was found lowest IC50 value for E.coli. Pine needles bio-oil was used for resin formation to replace phenol to synthesize the bio-oil- phenol-formaldehyde (BPF) resins. FTIR analysis showed that 30% BPF resins could have wood adhesive activity. Insecticidal properties of the aqueous phase of bio-oil and pure bio-oil against the third instar larvae of H. armigera and anti-ovipositional activity against the adult H. armigera was screened and found that the H. armigera was tolerant against all types of the aqueous phase of bio-oil and pure bio-oil. Ovipositional activity maximum decreased by pine needles bio-oil (52%). While more number of eggs were laid on wheat straw bio-oil (57%) showed attractant nature like pheromones. Catalytic esterification of sugarcane leaves bio-oil with n-propanol produce 61.5% yield with calorific values increased by 11.06% making fuel more suitable to be used in engines.

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