New withanolides from leaves along with alkyl glucoside from roots and stem as well as variations in withanolide contents in leaves of accessions of Withania somnifera, Ashwagandha- the Indian ginseng$.

Based on the major components in the leaves, the ashwagandha has been found to exist in several chemotypic forms in India. From the leaves of various accessions of Withania somnifera, which were maintained in our institute, three new steroids namely, 4-acetoxy-20ß-hydroxy-1-oxo-witha-2,5,24-trienolide (7), 24,25-dihydro-14α-hydroxy withanolide D (9), 5α,6ß,17α,27-tetrahydroxy-1-oxo-witha-2,24-dienolide (12) together with thirteen known withanolides were identified by spectroscopic methods. From the roots and stem of one accession and leaves of another, a new alkyl ester glucoside (4) has also been isolated. The new withanolides 7, 9 and 12 have been tentatively named as withanolide 135 A, withanolide 135B and withanolide 108, respectively.

Enzymatic processing of Citrus reticulata (Kinnow) pomace using naringinase and its valorization through preparation of nutritionally enriched pasta.

Citrus fruits are consumed either as whole fruits or as juice after processing. Processing of fruits yields a significant number of by-products in the form of pulp, peel and seeds, which are often discarded and major cause of environmental concern. Bitterness in the waste residue of citrus products is one of the leading hindrance in its valorization and supplementation in other food products. Aim of this study was to reduce the bitterness of Citrus reticulata (kinnow) pomace using enzymatic method and its supplementation in production of nutritionally rich pasta. Under optimized conditions (1U/mg enzyme naringinase concentration, temperature 50 °C, at pH 4.5 and treatment time 4 h), the maximum reduction (65.95%) of naringin (bitterness causing compound) was observed coupled with increase (60.13%) in naringenin (non-bitter compound). The debittered kinnow pomace has been further characterized for physio-chemical changes and morphological changes before and after treatment. The debittered kinnow pomace was then supplemented for the preparation of antioxidant and nutrient enriched pasta.

Comparative study of various processes used for removal of bitterness from kinnow pomace and kinnow pulp residue.

The current study was focused on new approaches for debittering of by-products like kinnow pomace and kinnow pulp residue by using various food grade mild chemical methods, such as alkali treatment, acid treatment, and solventogenesis. Whereas in the studied various chemical treatments, the solventogenesis method with acetone resulted in maximum extraction of naringin and limonene from kinnow pomace and pulp residue and showed high acceptability for food product development. The acetone treatment was further optimized by RSM for the maximum extraction of naringin and limonene. Under optimized conditions, the maximum amount of naringin and limonene extracted were found to be 8.955, 2.122 mg/g from kinnow pomace and 9.971, 3.838 mg/g from pulp residue, respectively. This process can not only result in the effective utilization of agro-industrial by-product but also provide a sustainable solution to the environmental pollution caused by kinnow juice industry.

Alginate-pectin co-encapsulation of dextransucrase and dextranase for oligosaccharide production from sucrose feedstocks.

The genes for dextransucrase and dextranase were cloned from the genomic regions of Leuconostoc mesenteroides MTCC 10508 and Streptococcus mutans MTCC 497, respectively. Heterologous expression of genes was performed in Escherichia coli. The purified enzyme fractions were entrapped in the alginate-pectin beads. A high immobilization yield of dextransucrase (~ 96%), and dextranase (~ 85%) was achieved. Alginate-pectin immobilization did not affect the optimum temperature and pH of the enzymes; rather, the thermal tolerance and storage stability of the enzymes was improved. The repetitive batch experiments suggested substantially good operational stability of the co-immobilized enzyme system. The synergistic catalytic reactions of alginate-pectin co-entrapped enzyme system were able to produce 7-10 g L-1 oligosaccharides of a high degree of polymerization (DP 3-9) from sucrose (~ 20 g L-1) containing feedstocks, e.g., table sugar and cane molasses. The alginate-pectin-based co-immobilized enzyme system is a useful catalytic tool to bioprocess the agro-industrial bio-resource for the production of prebiotic biomolecules.

Value additon of kinnow industry byproducts for the preparation of fiber enriched extruded products.

In the present study dietary fiber enriched vermicelli from wheat flour supplemented with debittered kinnow industry by-products (pulp residue and pomace) has been developed. Functional, cooking and textural properties of both supplemented and unsupplemented vermicelli were evaluated. Vermicelli containing 15% debittered kinnow pulp residue and pomace showed minimum cooking loss (18.5, 20.0%) but higher swelling index (2.06, 1.87), water absorption capacity (153, 202 g/100 g) and optimal cooking time (9.34, 9.02 min). Firmness and fracturability of vermicelli supplemented debittered pulp residue (10.0 and 21.5) and pomace (16.7 and 16.1) was higher as compared to control sample (6.1 and 2.1) respectively. Further, redness, firmness, TPC, DPPH activity and water absorption capacity of vermicelli got increased with addition of debittered kinnow pulp and pomace. The utilization of debittered kinnow pulp and pomace in vermicelli can provide dual benefit like production of healthy food products along with solving the problem of solid waste disposal of kinnow industry byproducts.

Interspecies comparative features of trichomes in Ocimum reveal insights for biosynthesis of specialized essential oil metabolites.

Ocimum species commonly referred to as "Tulsi" are well-known for their distinct medicinal and aromatic properties. The characteristic aroma of Ocimum species and cultivars is attributed to their specific combination of volatile phytochemicals mainly belonging to terpenoid and/or phenylpropanoid classes in their essential oils. The essential oil constituents are synthesized and sequestered in specialized epidermal secretory structures called as glandular trichomes. In this comparative study, inter- and intra-species diversity in structural attributes and profiles of expression of selected genes related to terpenoid and phenylpropanoid biosynthetic pathways have been investigated. This is performed to seek relationship of variations in the yield and phytochemical composition of the essential oils. Microscopic analysis of trichomes of O. basilicum, O. gratissimum, O. kilimandscharicum, and O. tenuiflorum (green and purple cultivars) revealed substantial variations in density, size, and relative proportions of peltate and capitate trichomes among them. The essential oil yield has been observed to be controlled by the population, dominance, and size of peltate and capitate glandular trichomes. The essential oil sequestration in leaf is controlled by the dominance of peltate glandular trichome size over its number and is also affected by the capitate glandular trichome size/number with variations in leaf area albeit at lower proportions. Comprehension and comparison of results of GC-MS analysis of essential oils showed that most of the Ocimum (O. basilicum, O. tenuiflorum, and O. gratissimum) species produce phenylpropanoids (eugenol, methyl chavicol) as major volatiles except O. kilimandscharicum, which is discrete in being monoterpenoid-rich species. Among the phenylpropanoid-enriched Ocimum (O. basilicum, O. gratissimum, O. tenuiflorum purple, O. tenuiflorum green) as well, terpenoids were important constituents in imparting characteristic aroma. Further, comparative abundance of transcripts of key genes of phenylpropanoid (PAL, C4H, 4CL, CAD, COMT, and ES) and terpenoid (DXS and HMGR) biosynthetic pathways was evaluated vis-à-vis volatile oil constituents. Transcript abundance demonstrated that richness of their essential oils with specific constituent(s) of a chemical group/subgroup was manifested by the predominant upregulation of phenylpropanoid/terpenoid pathway genes. The study provides trichomes as well as biosynthetic pathway-based knowledge for genetic improvement in Ocimum species for essential oil yield and quality.

Efficient and economic process for the production of bacterial cellulose from isolated strain of Acetobacter pasteurianus of RSV-4 bacterium.

In the present investigation, several residues from agro-forestry industries such as rice straw acid hydrolysate, corn cob acid hydrolysate, tomato juice, cane molasses and orange pulp were evaluated as the economical source for the production of bacterial cellulose. The bacterial cellulose attained the significant yield of 7.8 g/L using tomato juice, followed by 3.6 g/L using cane molasses and 2.8 g/L using orange pulp after 7 days of incubation. Furthermore, the optimum pH and temperature of fermentation for maximum production of bacterial cellulose was 4.5 and 30 ±â€¯1 °C. The identified bacterium Acetobacter pasteurianus RSV-4 has been deposited at repository under the accession number MTCC 25117. The produced bacterial cellulose was characterized through FTIR, SEM, TGA and DSC and found to be of very good quality. The bacterial cellulose produced by identified strain on these various agro-waste residues could be a cost effective technology for commercial its production.

Metagenomic analysis of geothermal water reservoir sites exploring carbohydrate-related thermozymes.

Hot springs are geothermally heated underground water that create a natural habitat for diverse thermophilic microorganisms. The study presents a comprehensive investigation of microbial diversity and functional potential of four thermal water reservoirs of 55 to 98 °C temperature range, located in Tattapani geothermal field of Chhattisgarh, India, by using culture-independent metagenome sequencing approach. The MG-RAST taxonomic profiling of metagenome samples revealed the predominance of bacterial domains (94.8 to 98.2%), followed by archaea (1.1 to 4.8%), eukaryota (0.1 to 0.5%), and viruses (0.04 to 0.09%). The quality filtered reads (42.1 to 68.1 million) were assembled into 66 to 330 thousand non-redundant contigs (>200 bp length) in the four metagenome samples. The functional annotation using CAZy database identified a total of 4083 putative genes with functional domains involved in catalysis of carbohydrate degradation or modification or synthesis of glycosidic bonds. The study detected many novel biocatalysts associated with hydrolysis of lignocellulosic biomass polymers- cellulose, hemicellulose, lignin, and pectin. Metagenome assembly and catalytic functions of two metagenomic contigs, encoding ß-glucosidase, and xylanase, were experimentally validated. The findings emphasized these geothermal water reservoir sites as the repository of biocatalyst-encoding genes of carbohydrate-related and lignocellulosic biomass processing significance.

Tomato processing byproduct valorization in bread and muffin: improvement in physicochemical properties and shelf life stability.

Tomato processing industry generates huge waste like tomato skin, seed, and pulp which creates environmental issues. Since tomato pomace contains bioactive compounds and pigments, present study was conducted to investigate the effect of tomato pomace addition on physicochemical characteristics and shelf-life stability of the developed bread and muffin. Refined flour was partially substituted with 35 and 40% tomato pomace in bread and muffin, respectively. Tomato pomace addition in bread and muffin was observed to significantly (p < 0.05) increase the dietary fiber, vitamin C, antioxidant activity and minerals (Na, K, Mg, Ca, Fe). The color parameters for bread and muffins were quantified in terms of L* (lightness), a* (redness/greenness) and b* (yellowness/blueness). There was an increase in a* and b*, while L* values decreased. Tomato based bread and muffin were found to possess softer texture as compared to control products. Microbial study has depicted the enhanced shelf-life of tomato based bread and muffin. Shelf life of preservative added tomato based bread was 8 days and muffins were 12 days. Tomato pomace could be a very useful commodity for incorporation into bread and muffin to have a complete nutritive food product.

De novo sequencing, assembly and characterisation of Aloe vera transcriptome and analysis of expression profiles of genes related to saponin and anthraquinone metabolism.

BACKGROUND: Aloe vera is a perennial, succulent, drought-resistant plant that exhibits many pharmacological characteristics such as wound healing ability against skin burns, anti-ulcer, anti-inflammatory, anti-tumor, anti-viral, anti-hypercholesterolemic, anti-hyperglycemic, anti-asthmatic and much more. Despite great medicinal worth, little genomic information is available on Aloe vera. This study is an initiative to explore the full-scale functional genomics of Aloe vera by generating whole transcriptome sequence database, using Illumina HiSeq technology and its progressive annotation specifically with respect to the metabolic specificity of the plant. RESULTS: Transcriptome sequencing of root and leaf tissue of Aloe vera was performed using Illumina paired-end sequencing technology. De novo assembly of high quality paired-end reads, resulted into 1,61,733 and 2,21,792 transcripts with mean length of 709 and 714 nucleotides for root and leaf respectively. The non-redundant transcripts were clustered using CD-HIT-EST, yielding a total of 1,13,063 and 1,41,310 unigenes for root and leaf respectively. A total of 6114 and 6527 CDS for root and leaf tissue were enriched into 24 different biological pathway categories using KEGG pathway database. DGE profile prepared by calculating FPKM values was analyzed for differential expression of specific gene encoding enzymes involved in secondary metabolite biosynthesis. Sixteen putative genes related to saponin, lignin, anthraquinone, and carotenoid biosynthesis were selected for quantitative expression by real-time PCR. DGE as well as qRT PCR expression analysis represented up-regulation of secondary metabolic genes in root as compared to leaf. Furthermore maximum number of genes was found to be up-regulated after the induction of methyl jasmonate, which stipulates the association of secondary metabolite synthesis with the plant's defense mechanism during stress. Various transcription factors including bHLH, NAC, MYB were identified by searching predicted CDS against PlantTFdb. CONCLUSIONS: This is the first transcriptome database of Aloe vera and can be potentially utilized to characterize the genes involved in the biosynthesis of important secondary metabolites, metabolic regulation, signal transduction mechanism, understanding function of a particular gene in the biology and physiology of plant of this species as well as other species of Aloe genus.

An integrated bio-process for production of functional biomolecules utilizing raw and by-products from dairy and sugarcane industries.

The study investigated an integrated bioprocessing of raw and by-products from sugarcane and dairy industries for production of non-digestible prebiotic and functional ingredients. The low-priced feedstock, whey, molasses, table sugar, jaggery, etc., were subjected to transglucosylation reactions catalyzed by dextransucrase from Leuconostoc mesenteroides MTCC 10508. HPLC analysis approximated production of about 11-14 g L-1 trisaccharide i.e. 2-α-D-glucopyranosyl-lactose (4-galactosyl-kojibiose) from the feedstock prepared from table sugar, jaggery, cane molasses and liquid whey, containing about 30 g L-1 sucrose and lactose each. The trisaccharide was hydrolysed into the prebiotic disaccharide, kojibiose, by employing recombinant ß-galactosidase from Escherichia coli. The enzyme ß-galactosidase achieved about 90% conversion of 2-α-D-glucopyranosyl-lactose into kojibiose. The D-fructose generated by catalytic reactions of dextransucrase was targeted for catalytic transformation into rare sugar, D-allulose (or D-psicose), by treating the samples with Smt3-D-psicose 3-epimerase. The catalytic reactions resulted in the conversion of ~ 25% D-fructose to D-allulose. These bioactive compounds are known to exert a plethora of benefits to human health, and therefore, are preferred ingredients for making functional foods.

Transcriptome mining and in silico structural and functional analysis of ascorbic acid and tartaric acid biosynthesis pathway enzymes in rose-scanted geranium.

Rose-scented geranium (Pelargonium sp.) is widely known as aromatic and medicinal herb, accumulating specialized metabolites of high economic importance, such as essential oils, ascorbic acid, and tartaric acid. Ascorbic acid and tartaric acid are multifunctional metabolites of human value to be used as vital antioxidants and flavor enhancing agents in food products. No information is available related to the structural and functional properties of the enzymes involved in ascorbic acid and tartaric acid biosynthesis in rose-scented geranium. In the present study, transcriptome mining was done to identify full-length genes, followed by their bioinformatic and molecular modeling investigations and understanding of in silico structural and functional properties of these enzymes. Evolutionary conserved domains were identified in the pathway enzymes. In silico physicochemical characterization of the catalytic enzymes revealed isoelectric point (pI), instability index, aliphatic index, and grand average hydropathy (GRAVY) values of the enzymes. Secondary structural prediction revealed abundant proportion of alpha helix and random coil confirmations in the pathway enzymes. Three-dimensional homology models were developed for these enzymes. The predicted structures showed significant structural similarity with their respective templates in root mean square deviation analysis. Ramachandran plot analysis of the modeled enzymes revealed that more than 84% of the amino acid residues were within the favored regions. Further, functionally important residues were identified corresponding to catalytic sites located in the enzymes. To, our best knowledge, this is the first report which provides a foundation on functional annotation and structural determination of ascorbic acid and tartaric acid pathway enzymes in rose-scanted geranium.

Development of a thermo-stable and recyclable magnetic nanobiocatalyst for bioprocessing of fruit processing residues and D-allulose synthesis.

The aim of the study was to covalently immobilize Smt3-D-psicose 3-epimerase onto functionalized iron oxide magnetic nanoparticles. After immobilization, Km of the immobilized enzyme increased, however, Vmax was nearly the same as that of its free form, indicating that immobilization has no detrimental effects on its catalytic output. The covalent immobilization caused a reduction in the deactivation rate constant (kd) values leading to 4-5 fold enhancement in its half-life at 50-65°C, indicating significant thermal stability of the iron-enzyme nanobioconjugate. The immobilized enzyme showed excellent storage stability by losing only 20% activity even after 60days of storage at 4°C. The immobilized enzyme retained up to 90% of its initial activity even after 10 consecutive cycles of catalyzing D-fructose epimerization reactions. Thus, after immobilization the enzyme exhibited remarkable improvements in thermal tolerance, storage stability and recycling efficiency, useful for development of industrially exploitable process for D-allulose production.

Improved levulinic acid production from agri-residue biomass in biphasic solvent system through synergistic catalytic effect of acid and products.

In this study, levulinic acid (LA) was produced from rice straw biomass in co-solvent biphasic reactor system consisting of hydrochloric acid and dichloromethane organic solvent. The modified protocol achieved a 15% wt LA yield through the synergistic effect of acid and acidic products (auto-catalysis) and the designed system allowed facile recovery of LA to the organic phase. Further purification of the resulting extractant was achieved through traditional column chromatography, which yielded a high purity LA product while recovering ∼85% wt. Upon charcoal treatment of the resultant fraction generated an industrial grade target molecule of ∼99% purity with ∼95% wt recovery. The system allows the solvent to be easily recovered, in excess of 90%, which was shown to be able to be recycled up to 5 runs without significant loss of final product concentrations. Overall, this system points to a method to significantly reduce manufacturing cost during large-scale LA preparation.

Processing of wet Kinnow mandarin (Citrus reticulata) fruit waste into novel Brønsted acidic ionic liquids and their application in hydrolysis of sucrose.

In citrus processing industries, where up to 60% of the whole fruit is discarded, generates citrus waste (peel, seeds, membrane, and pulp) on a massive scale. The waste does not currently have high-value applications, instead the majority is disposed of or pelletized for animal feed. Therefore, a concise and efficacious protocol for processing of Kinnow mandarin (Citrus reticulata) fruit waste to numerous novel Brønsted acidic ionic liquids (3a-k) have been developed. BAILs were characterized using spectroscopic techniques (FT-IR and NMR). Water immiscibility of ILs 3a, 3g and 3h, a property never observed with sulfonic acid ILs, allowed the catalytic application of BAIL 3a in hydrolysis of sucrose/table sugar, giving a mixture of d-glucose and d-fructose in excellent yields. The ionic liquid could be recycled for >3 times without significant loss of activity.

Efficient detoxification of corn cob hydrolysate with ion-exchange resins for enhanced xylitol production by Candida tropicalis MTCC 6192.

The present study demonstrates utilization of secondary agricultural wastes for xylitol production. The highest xylan-to-xylose (70%) conversion was achieved using dilute nitric acid as catalyst followed by resin treatment. Results show that resin treatment efficiently removed nitrate salt (70%), phenolic content and 5-HMF (70%). Highest xylitol yield (85%) was achieved during fermentation using Candida tropicalis MTCC 6192 from the neutralized hemicellulosic hydrolysate medium. Good recovery (>15%) was achieved from corncob with 85% xylose to xylitol conversion during fermentation. This two-step process for transformation of agri-waste to xylitol is much simpler and it could possibly be considered for up scaling after process optimization parameters.

Effect of atmospheric cold plasma (ACP) with its extended storage on the inactivation of Escherichia coli inoculated on tomato.

The study investigated the efficacy of atmospheric cold plasma (ACP) treatment on inactivation of E. coli load during extended storage period of 48h at both the temperatures of refrigeration (4°C) as well as room (25°C). The tomato samples were spot inoculated with E. coli and exposed to ACP at 15 and 60kV for 5, 10, 15, and 30min followed by their storage at 4°C and 25°C. The highest log reduction of 6logCFUmL-1 was achieved in population of E. coli after 15min of ACP treatment at 60kV, which was sustained up to storage duration of 48h at both the temperatures. Furthermore, significant reduction in E. coli was found at plasma treatment of 60kV in comparison to 15kV. The inactivation of E. coli was significantly (p<0.01) affected by combination of treatment higher voltage at extended treatment time, however, treatment time with prolonged storage of sealed ACP treated tomato was observed as a key factor in reduction of E. coli. In addition, investigation of E. coli exposed tomato surface was done using scanning electron microscopy that clearly showed the breakdown of cell cover of E. coli as a consequence of ACP. The study predicts the promising potential of the technique in sanitization of vegetables that are eaten raw like tomato.

De novo transcriptome analysis of rose-scented geranium provides insights into the metabolic specificity of terpene and tartaric acid biosynthesis.

BACKGROUND: Rose-scented geranium (Pelargonium sp.) is a perennial herb that produces a high value essential oil of fragrant significance due to the characteristic compositional blend of rose-oxide and acyclic monoterpenoids in foliage. Recently, the plant has also been shown to produce tartaric acid in leaf tissues. Rose-scented geranium represents top-tier cash crop in terms of economic returns and significance of the plant and plant products. However, there has hardly been any study on its metabolism and functional genomics, nor any genomic expression dataset resource is available in public domain. Therefore, to begin the gains in molecular understanding of specialized metabolic pathways of the plant, de novo sequencing of rose-scented geranium leaf transcriptome, transcript assembly, annotation, expression profiling as well as their validation were carried out. RESULTS: De novo transcriptome analysis resulted a total of 78,943 unique contigs (average length: 623 bp, and N50 length: 752 bp) from 15.44 million high quality raw reads. In silico functional annotation led to the identification of several putative genes representing terpene, ascorbic acid and tartaric acid biosynthetic pathways, hormone metabolism, and transcription factors. Additionally, a total of 6,040 simple sequence repeat (SSR) motifs were identified in 6.8% of the expressed transcripts. The highest frequency of SSR was of tri-nucleotides (50%). Further, transcriptome assembly was validated for randomly selected putative genes by standard PCR-based approach. In silico expression profile of assembled contigs were validated by real-time PCR analysis of selected transcripts. CONCLUSION: Being the first report on transcriptome analysis of rose-scented geranium the data sets and the leads and directions reflected in this investigation will serve as a foundation for pursuing and understanding molecular aspects of its biology, and specialized metabolic pathways, metabolic engineering, genetic diversity as well as molecular breeding.

A novel approach of integrated bioprocessing of cane molasses for production of prebiotic and functional bioproducts.

In this work, the sugar industry by-product cane molasses was investigated as feedstock for acceptor reactions by dextransucrase from Leuconostoc mesenteroides MTCC 10508, leading to the biosynthesis of oligosaccharides. The starch industry corn fiber residue was used as a source for acceptor molecules, maltose, in the reaction. Production of approximately 124g oligosaccharides (DP3-DP6) per kg of fresh molasses was achieved. Further, cane molasses based medium was demonstrated as a sole carbon source for L. mesenteroides growth and dextransucrase production. d-Fructose released by dextransucrase activity as processing by-product was transformed into the functional monosaccharide with zero caloric value, d-psicose, by inducing its epimerization. Quantitative analysis approximated 37g d-psicose per kg of fresh molasses. Thus, the study established a novel approach of integrated bioprocessing of cane molasses into prebiotic and functional food additives.

Thermo-chemical pretreatment of rice straw for further processing for levulinic acid production.

A variety of pretreatment protocols for rice straw fiber reconstruction were evaluated under mild conditions (upto 0.2%wt. and 121°C) with the object of improving polymer susceptibility to chemical attack while preserving carbohydrate sugars for levulinic acid (LA) production. Each of the protocols tested significantly enhanced pretreatment recoveries of carbohydrate sugars and lignin, and a NaOH protocol showed the most promise, with enhanced carbohydrate preservation (upto 20% relative to the other protocols) and more effective lignin dissolution (upto 60%). Consequently, post-pretreatment fibers were evaluated for LA preparation using an existing co-solvent system consisting of HCl and THF, in addition supplementation of DMSO was attempted, in order to improve final product recovery. In contrast to pretreatment response, H2SO4 protocol fibers yielded highest LA conc. (21%wt. with 36% carbohydrate conversion efficiency) under the modest reaction conditions. Apparent spectroscopic analysis witnessed for fiber destruction and delocalization of inherent constituents during pretreatment protocols.