Sameer S. Bhagyawant

Multivariate Analysis Based on Nutritional Value, Antinutritional Profile and Antioxidant Capacity of Forty Chickpea Genotypes Grown in India

Background: Chickpea (Cicer arietinum L.) is an important pulse crop with several potential health benefits. Providing an affordable alternative to animal protein, the chickpea seed is consumed as food in various platters. However, bioavailability of seed proteins is usually low. This seems due to the presence of antinutritional factors, such as phytates, trypsin inhibitors and tannins. Objectives: This study has been conducted to evaluate the multivariate analysis of nutritional and antinutritional aspects of 40 chickpea genotypes. Methods: seeds were maintained at 4°C with 40% relative humidity. Seeds were grinded in a grinder and the contents were passed through 80 μm sieve. Powdered seed samples were first defatted using chilled acetone and air dried. Nutritional and other phytochemical analysis were performed under ambient conditions of temperature and humidity. Results: The seeds exhibit an average nutritional content of total protein (≥ n110.38 mg-1 100 g), total free amino acids (≥ 292.28 mg-1 100 g) and nutritional minerals like Fe (≥ 0.66 mg-1 100 g) and Zn (≥ 0.59 mg-1 100 g). The multivariate analysis for all the chickpea genotypes studied, based on their principal components, show unique position according to their nutritional status. Moreover, hierarchical clustering agglomerative genotypes as basis for genotypes, grouped into two major clusters of MC-1 and MC-2. The study revealed that chickpea genotypes exhibit divergent nutritional and antinutritional properties. Conclusion: Based on the present study and evaluation, the genotype selection for future breeding programmes so as to develop nutritionally elite cultivar can be planned.

Vector-delivered artificial miRNA effectively inhibited replication of Chikungunya virus.

Abstract Chikungunya virus (CHIKV) has emerged as one of the most significant arboviral threats in many parts of the world. In spite of large scale morbidity, and long lasting polyarthralgia, no licensed vaccine or antivirals are available for the clinical management of CHIKV infection. In this study, a novel RNA interference based strategy has been adopted for effective inhibition of CHIKV. Four artificial microRNAs (amiRNAs) were designed to target different regions of CHIKV genome. These amiRNAs significantly inhibited CHIKV replication in Vero cells at both RNA and protein levels as assessed by qRT-PCR, immunoblotting and immunofluorescence techniques. Further inhibition of the infectious CHIKV up to 99.8% was demonstrated by plaque reduction assay. Concatemerization of amiRNA resulted in higher inhibition of CHIKV than individual amiRNAs. In addition, we studied the effect of combination of RNAi based therapy with other classical antivirals like chloroquine, ribavirin and mycophenolic acid, that helped in understanding the rational selection of RNAi based combination therapy. These findings provide a promising avenue for the development of novel amiRNA or combination based therapeutics against emerging CHIKV.

Phytochemical Evaluation of Moth Bean (Vigna aconitifolia L.) Seeds and Their Divergence

In the present study, phytochemical contents of 25 moth bean (Vigna aconitifolia) seed accessions were evaluated. This includes protease inhibitors, phytic acid, radical scavenging activity, and tannins. The studies revealed significant variation in the contents of theses phytochemicals. Presence of photochemical composition was correlated with seed storage proteins like albumin and globulin. Qualitative identification of total seed storage protein abundance across two related moth bean accessions using two-dimensional gel electrophoresis (2D-GE) was performed. Over 20 individual protein fractions were distributed over the gel as a series of spots in two moth bean accessions. Seed proteome accumulated spots of high intensity over a broad range of pI values of 3–10 in a molecular weight range of 11–170 kDa. In both seed accessions maximum protein spots are seen in the pI range of 6–8.

Characterization of Seed Storage Proteins from Chickpea Using 2D Electrophoresis Coupled with Mass Spectrometry

Proteomic analysis was employed to map the seed storage protein network in landrace and cultivated chickpea accessions. Protein extracts were separated by two-dimensional gel electrophoresis (2D-GE) across a broad range 3.0–10.0 immobilized pH gradient (IPG) strips. Comparative elucidation of differentially expressed proteins between two diverse geographically originated chickpea accessions was carried out using 2D-GE coupled with mass spectrometry. A total of 600 protein spots were detected in these accessions. In-gel protein expression patterns revealed three protein spots as upregulated and three other as downregulated. Using trypsin in-gel digestion, these differentially expressed proteins were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) which showed 45% amino acid homology of chickpea seed storage proteins with Arabidopsis thaliana.

Plant-Derived Enzymes: A Treasure for Food Biotechnology

Abstract Enzymes are highly specific biological catalysts involved in food biotechnology. Enzyme production and application in the food manufacturing industry are based on a profound understanding of the enzymes in traditional foods. Plant-derived enzymes include amylase, invertase, papain, bromelain, ficin, lipoxygenase, etc. These enzymes have played an important part in food production, for example, syrups, bakery products, alcoholic beverages, dairy products, etc. Besides the use of plants as a factory for enzyme production in the food industry, they can also serve as raw material (enhancer) for the enhancement of microbial enzyme activity employed in the food industry. New approaches such as direct genetic modification are now in progress that might significantly contribute to the improvement of the nutritional value of plant-derived foods or the quality of food using enzymes derived from plant sources. Earlier enzymes were isolated from living cells (plants and animals), which led to their large-scale commercial production and wider application in the food industry. Today, microorganisms are the most important source of commercial enzymes. However, the safety of the source organism is the primary consideration in assessing an enzyme product. Food animals and edible plants have a history of safe use as sources of enzymes for the food industry.

Effect of Decortication and Heat pretreatment on Oil content extracted from Safflower Seeds variety PBNS-12

Sustainable development has improved the quality of life associate with requirement, production and processing of agricultural belongings among other things. Processing aim is to improve quality and concentration of products with their nutritional value influencing its cost also. Safflower oil is a value added product of safflower seeds which have biotechnological importance in the field of food and pharmaceutical industry. The presence of antinutritional factors (ANFs) like tannin, phytate and phenolic glucosides reduce the qualitative value of safflower seed oil. Different processing methodologies like boiling (temperature based pretreatment), decortications have been employed to reduce level of ANFs in safflower seeds. In our study, combinatorial effects of boiling and decortications have been employed to improve oil content in safflower seeds by reducing level of tannin. The boiling pretreatment was done with two sets of seed samples i.e. corticated and decorticated seeds. Each set was divided into four portions i.e. B10, B20, B30 and C. Samples B10, B20 and B30 were heated for 10 min, 20 min and 30 min respectively at 800C, while sample C served as the control for the experiment. The results showed an enhancement in percentage oil content by 1.16 fold and reduction in tannin content by 0.78 fold from B10 to B30 in corticated and decorticated set of seeds after boiling treatment. The combinatorial pretreatment methods using decortication with boiling treatment increase two fold oil content and 1.12 fold reduction in tannin content than control corticated

Development of nsP2 protease based cell free high throughput screening assay for evaluation of inhibitors against emerging Chikungunya virus

Chikungunya virus has emerged as one of the most important global arboviral threats over the last decade. Inspite of large scale morbidity, with long lasting polyarthralgia, so far no licensed vaccine or antiviral is available. CHIKV nsP2 protease is crucial for processing of viral nonstructural polypeptide precursor to release enzymes required for viral replication, thus making it a promising drug target. In this study, high cell density cultivation (HCDC) of Escherichia coli in batch process was carried out to produce rCHIKV nsP2pro in a cost-effective manner. The purified nsP2pro and fluorogenic peptide substrate have been adapted for fluorescence resonance energy transfer (FRET) based high throughput screening (HTS) assay with Z’ value and CV of 0.67 ± 0.054 and <10% respectively. We used this cell free HTS system to screen panel of metal ions and its conjugate which revealed zinc acetate as a potential candidate, which was further found to inhibit CHIKV in Vero cells. Scale-up process has not been previously reported for any of the arboviral nonstructural enzymes. The successful scale-up method for viral protease together with a HTS assay could lead to the development of industrial level large-scale screening platform for identification of protease inhibitors against emerging and re-emerging viruses.

Chickpea Lectin Inhibits Human Breast Cancer Cell Proliferation and Induces Apoptosis Through Cell Cycle Arrest

BACKGROUND Breast cancer demands safe adjuvant to overcome the side effects of standard drug tamoxifen. Diet derived bioactive compounds are reported to exhibit modulation of cancer growth leading to cell death. Chickpea is a protein rich edible legume with several bioactive compounds that includes lectin as well. Characterization of chickpea lectin for its effect against cancer cells has been investigated in this study. METHOD Cicer arietinum L. lectin (CAL) agglutinating trypsin-treated rabbit blood cells was purified employing DEAE-cellulose and SP-sephadex ion exchange chromatography. The lectin was characterized for its biological activity vis-à-vis antiproliferative and apoptotic effects through cell cycle arrest in MCF-7 human breast cancer cells. RESULT There is a significant inhibition of the survival of breast cancer cells due to chickpea lectin in a dose dependent manner for 24 hr. Lectin treated cells revealed distinct features of apoptosis. Flow cytometric analysis at 80 µg/ml of lectin induced S and G2 phase cell cycle arrest. CAL induced apoptosis in MCF-7 cells associated with lactate dehydrogenase leakage, cell cycle arrest and reactive oxygen species generation. CONCLUSION Our studies show that chickpea lectin exerted anticancer activity and could be exploited as an essential source for medicine leading to the treatment of breast cancer.

Cloning, expression and purification of virB10 protein of Brucella melitensis and evaluation of its role as a serological marker for Brucella infection in experimental and natural host

Brucellosis is a zoonotic disease caused by various species of the genus Brucella. The control of this disease mainly depends on its accurate and early diagnosis. Culture methods employed for diagnosis are time consuming and require well equipped biosafety level 3 laboratories and hence serological tests are favored alternative for brucellosis diagnosis. At present serological diagnosis is based on LPS (lipopolysaccharide) which is less specific as it shows cross reactivity with other gram-negative bacteria. There is a need to develop serological diagnostic assay based on purified recombinant antigen of Brucella. T4SS (Type IV Secretion System) is an important virulent factor of Brucella and required for infection suggesting their expression in vivo and can be targeted as serological marker for infection. To test this concept, the present study is designed to clone, express and purify virB10 gene of Brucella T4SS under denaturing conditions and to evaluate its use as serological marker of Brucella infection. The immunoreactivity of this recombinant antigen was checked with antisera collected after experimental infection in Balb/C mice with B. melitensis 16M, BR31 (human clinical isolate) and Y. enterocolitica O:9. The recombinant protein was also tested against a panel of 46 bovine sera samples collected from Leh, India. Antibody response against VirB10 was detected in experimental and natural host suggesting that it can be explored as potential target for serodiagnosis of Brucella infection.

High level expression and immunochemical characterization of botulinum neurotoxin type F light chain

Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger. This concern was addressed by purifying the recombinant catalytically active light chain of BoNT/F. BoNT/F-LC gene was amplified from the genomic DNA using specifically designed primers and expressed in Escherichia coli. Expression and purification profile were optimized under different conditions for biologically active light chain production. Specific polyclonal antibodies generated against type F illustrates in vivo neutralization in mice and rabbit. These antibodies play key role in conceiving the development of high throughput SPR based detection system which is a highly precise label free technique for protein interaction analysis. The presented work is first of its kind, signifying the production of highly stable and active rBoNT/F-LC and its immunochemical characterization. The study aids in paving the path towards developing a persistent detection system as well as in presenting comprehended scheme for in vitro small molecule therapeutics analysis.