Enhancing bioflavor production by solid-state fermentation using Kluyveromyces marxianus and l-phenylalanine.

This study includes the utilization of sweet lemon peel (SLP) and sugarcane bagasse (SB) in solid-state fermentation using Kluyveromyces marxianus for bioflavor compounds production adopting response surface methodology. The major flavor compounds, 2-phenylethanol (2-PE) and 2-phenylethyl acetate (2-PEA) were quantified using gas chromatography-mass spectrometry with and without adding any supplements. Quantification of flavor compounds indicated that without adding any accessory in the substrate, the concentration of 2-PE using SLP and SB was 0.15 ± 0.003 mg/g and 0.14 ± 0.002 mg/g, respectively. Whereas 2-PEA concentration using SLP and SB was observed as 0.01 ± 0.008 mg/g and 0.02 ± 0.001 mg/g, respectively. The addition of l-phenylalanine (l-phe) in the substrates showed 30%-75% enhancement in the production of 2-PE and 2-PEA. The present study indicates that the K. marxianus is a potential microbial cell factory for the production of 2-PE and 2-PEA with the addition of synthetic l-phe having a plethora of applications in food and pharmaceutical industries.

Dual Inhibition of DPP-4 and Cholinesterase Enzymes by the Phytoconstituents of the Ethanolic Extract of Prosopis cineraria Pods: Therapeutic Implications for the Treatment of Diabetes-associated Neurological Impairments.

BACKGROUND: Insulin resistance causes decreased uptake of glucose which promotes the susceptibility of type 2 associated neurological impairments. METHODS: The study was aimed to evaluate the inhibition potential of the ethanolic extract of Prosopis cineraria (EPC) pods against DPP-4 and cholinesterase enzymes by in-vitro, in-vivo and in-silico assessments. The present study consists of in vivo studies on a diabetes-induced rat model by HOMA (Homeostasis model assessment) and related parameters, in vitro studies through the DPP-4 enzyme assay and cholinesterase assays using Ellman's reaction. The in-silico studies were conducted by the molecular docking of Cinerin C with targeted enzymes. The phytochemical characterization of the extract was demonstrated through LCMS studies. The antioxidant studies on the extract were performed by FRAP and TEAC assays. RESULTS: The extract showed 64.8% maximum inhibition of DPP-4, 34.91% inhibition of AChE and 74.35% inhibition of BuChE. The antioxidant capacity of the extract was observed to be 847.81±16.25µM Fe2+ equivalent in the FRAP assay and 0.40 ± 0.08 mmol/l of Trolox equivalent in the TEAC assay. The in vivo study showed competent glycaemic control against significant HOMA IR (1.5), HOMA % ß (26.5) and HOMA % S (68.8) as well as pancreatic cell mass proliferation. The insilico analysis also revealed positive interactions of Cinerin C with targeted enzymes (DPP4 and cholinesterase). CONCLUSION: It can be concluded that the phytoconstituents of Prosopis cineraria pod extract can be significantly considered in neuropharmacology to resolve insulin resistance-induced neurological complications as it showed inhibition against DPP-4, AChE and BuChE target enzymes.

Endophytic Fungi-Alternative Sources of Cytotoxic Compounds: A Review.

Cancer is a major cause of death worldwide, with an increasing number of cases being reported annually. The elevated rate of mortality necessitates a global challenge to explore newer sources of anticancer drugs. Recent advancements in cancer treatment involve the discovery and development of new and improved chemotherapeutics derived from natural or synthetic sources. Natural sources offer the potential of finding new structural classes with unique bioactivities for cancer therapy. Endophytic fungi represent a rich source of bioactive metabolites that can be manipulated to produce desirable novel analogs for chemotherapy. This review offers a current and integrative account of clinically used anticancer drugs such as taxol, podophyllotoxin, camptothecin, and vinca alkaloids in terms of their mechanism of action, isolation from endophytic fungi and their characterization, yield obtained, and fungal strain improvement strategies. It also covers recent literature on endophytic fungal metabolites from terrestrial, mangrove, and marine sources as potential anticancer agents and emphasizes the findings for cytotoxic bioactive compounds tested against specific cancer cell lines.

Production of Potent Antimicrobial Compounds from Streptomyces cyaneofuscatus Associated with Fresh Water Sediment.

The genus Streptomyces under phylum actinobacteria has been recognized as a prolific source for the production of bioactive secondary metabolites. An actinobacterial strain designated as DST103 isolated from a wetland fresh water sediment of Tamdil Lake, Mizoram, Northeast, India was identified as Streptomyces cyaneofuscatus (KY287599) using 16SrRNA gene sequencing which shares 99.87% sequence similarity with Streptomyces cyaneofuscatus NRRL B-2570 T . The strain showed broad spectrum antimicrobial activities against Gram negative bacteria (Escherichia coli MTCC 739 and Pseudomonas aeruginosa MTCC 2453), Gram positive bacteria (Micrococcus luteus NCIM 2170 and Staphylococcus aureus MTCC 96) and yeast pathogen Candida albicans MTCC 3017). The methanolic extract of the strain DST103 exhibited highest antimicrobial activity against E. coli (IC50 = 2.10 µg/mL) and minimum activity against S. aureus (IC50 = 43.63 µg/mL). Five antibiotics [trimethoprim (18 µg/g), fluconazole (6 µg/g), ketoconazole (18 µg/g), nalidixic acid (135 µg/g), and rifampicin (56 µg/g)] were detected and quantified using ultra-performance liquid chromatography (UPLC-ESI-MS/MS). Further, biosynthetic potential genes [polyketide synthases type II, non-ribosomal peptide synthetases, and aminodeoxyisochorismate synthase (phzE)] were also detected in strain DST103 which may possibly be responsible for the production of antimicrobial compounds. Additionally, gas chromatography-mass spectrometry analysis showed the presence of four volatile compounds which might be responsible for their diverse biological activity. The present study revealed the presence of bioactive compounds in strain DST103, which may be a promising resource for the discovery of novel bioactive metabolites against wide range of pathogens.

Rhizospheric Bacterial Community of Endemic Rhododendron arboreum Sm. Ssp. delavayi along Eastern Himalayan Slope in Tawang.

Information on rhizosphere microbiome of endemic plants from high mountain ecosystems against those of cultivated plantations is inadequate. Comparative bacterial profiles of endemic medicinal plant Rhododendron arboreum Sm. subsp. delavayi rhizosphere pertaining to four altitudinal zonation Pankang Thang (PTSO), Nagula, Y-junction and Bum La (Indo-China border; in triplicates each) along cold adapted Eastern slope of Himalayan Tawang region, India is described here. Significant differences in DGGE profile between below ground bulk vs. rhizospheric community profile associated with the plant was identified. Tagged 16S amplicon sequencing from PTSO (3912 m) to Bum La (4509 m), revealed that soil pH, total nitrogen (TN), organic matter (OM) significantly influenced the underlying bacterial community structure at different altitudes. The relative abundance of Acidobacteria was inversely related to pH, as opposed to TN which was positively correlated to Acidobacteria and Proteobacteria abundance. TN was also the significant predictor for less abundant taxonomic groups Chloroflexi, Gemmatimonadetes, and Nitrospirae. Bum La soil harbored less bacterial diversity compared to other sites at lower altitudes. The most abundant phyla at 3% genetic difference were Acidobacteria, Actinobacteria, and Proteobacteria amongst others. Analysis of similarity indicated greater similarity within lower altitudinal than higher altitudinal group (ANOSIM, R = 0.287, p = 0.02). Constraining the ordination with the edaphic factor explained 83.13% of variation. Unique phylotypes of Bradyrhizobium and uncultured Rhizobiales were found in significant proportions at the four regions. With over 1% relative abundance Actinobacteria (42.6%), Acidobacteria (24.02%), Proteobacteria (16.00%), AD3 (9.23%), WPS-2 (5.1%), and Chloroflexi (1.48%) dominated the core microbiome.

Detection of antibiotic-resistant bacteria endowed with antimicrobial activity from a freshwater lake and their phylogenetic affiliation.

Antimicrobial resistance poses a serious challenge to global public health. In this study, fifty bacterial strains were isolated from the sediments of a freshwater lake and were screened for antibiotic resistance. Out of fifty isolates, thirty-three isolates showed resistance against at least two of the selected antibiotics. Analysis of 16S rDNA sequencing revealed that the isolates belonged to ten different genera, namely Staphylococcus(n = 8), Bacillus(n = 7), Lysinibacillus(n = 4), Achromobacter(n=3), bacterium(n = 3), Methylobacterium(n = 2), Bosea(n = 2), Aneurinibacillus(n = 2), Azospirillum(n = 1), Novosphingobium(n = 1). Enterobacterial repetitive intergenic consensus (ERIC) and BOX-PCR markers were used to study the genetic relatedness among the antibiotic resistant isolates. Further, the isolates were screened for their antimicrobial activity against bacterial pathogens viz., Staphylococcus aureus(MTCC-96), Pseudomonas aeruginosa(MTCC-2453) and Escherichia coli(MTCC-739), and pathogenic fungi viz., Fusarium proliferatum (MTCC-286), Fusarium oxysporum (CABI-293942) and Fusarium oxy. ciceri (MTCC-2791). In addition, biosynthetic genes (polyketide synthase II (PKS-II) and non-ribosomal peptide synthetase (NRPS)) were detected in six and seven isolates, respectively. This is the first report for the multifunctional analysis of the bacterial isolates from a wetland with biosynthetic potential, which could serve as potential source of useful biologically active metabolites.

A Novel Triculture System (CC3) for Simultaneous Enzyme Production and Hydrolysis of Common Grasses through Submerged Fermentation.

The perennial grasses are considered as a rich source of lignocellulosic biomass, making it a second generation alternative energy source and can diminish the use of fossil fuels. In this work, four perennial grasses Saccharum arundinaceum, Panicum antidotale, Thysanolaena latifolia, and Neyraudia reynaudiana were selected to verify their potential as a substrate to produce hydrolytic enzymes and to evaluate them as second generation energy biomass. Here, cellulase and hemi-cellulase producing three endophytic bacteria (Burkholderia cepacia BPS-GB3, Alcaligenes faecalis BPS-GB5 and Enterobacter hormaechei BPS-GB8) recovered from N. reynaudiana and S. arundinaceum were selected to develop a triculture (CC3) consortium. During 12 days of submerged cultivation, a 55-70% loss in dry weight was observed and the maximum activity of ß-glucosidase (5.36-12.34 IU) and Xylanase (4.33 to 10.91 IU) were observed on 2nd and 6th day respectively, whereas FPase (0.26 to 0.53 IU) and CMCase (2.31 to 4.65 IU) showed maximum activity on 4th day. Around 15-30% more enzyme activity was produced in CC3 as compared to monoculture (CC1) and coculture (CC2) treatments, suggested synergetic interaction among the selected three bacterial strains. Further, the biomass was assessed using Fourier-transform infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM). The FTIR analysis provides important insights into the reduction of cellulose and hemicellulose moieties in CC3 treated biomass and SEM studies shed light into the disruption of surface structure leading to access of cellulose or hemicelluloses microtubules. The hydrolytic potential of the CC3 system was further enhanced due to reduction in lignin as evidenced by 1-4% lignin reduction in biomass compositional analysis. Additionally, laccase gene was detected from A. faecalis and E. hormaechei which further shows the laccase production potential of the isolates. To our knowledge, first time we develop an effective endophytic endogenous bacterial triculture system having potential for the production of extracellular enzymes utilizing S. arundinaceum and N. reynaudiana as lignocellulosic feedstock.

Evaluation of Phenolic Content Variability along with Antioxidant, Antimicrobial, and Cytotoxic Potential of Selected Traditional Medicinal Plants from India.

Plants have been used since ancient times as an important source of biologically active substances. The aim of the present study was to investigate the phytochemical constituents (flavonoids and phenolics), antioxidant potential, cytotoxicity against HepG2 (human hepato carcinoma) cancer cell lines, and the antimicrobial activity of the methanol extract of selected traditional medicinal plants collected from Mizoram, India. A number of phenolic compounds were detected using HPLC-DAD-ESI-TOF-MS, mainly Luteolin, Kaempferol, Myricetin, Gallic Acid, Quercetin and Rutin, some of which have been described for the first time in the selected plants. The total phenolic and flavonoid contents showed high variation ranging from 4.44 to 181.91 µg of Gallic Acid equivalent per milligram DW (GAE/mg DW) and 3.17 to 102.2 µg of Quercetin/mg, respectively. The antioxidant capacity was determined by DPPH (IC50 values ranges from 34.22 to 131.4 µg/mL), ABTS (IC50 values ranges from 24.08 to 513.4 µg/mL), and reducing power assays. Antimicrobial activity was assayed against gram positive (Staphylococcus aureus), gram negative (Escherichia coli, Pseudomonas aeruginosa), and yeast (Candida albicans) demonstrating that the methanol extracts of some plants were efficacious antimicrobial agents. Additionally, cytotoxicity was assessed on human hepato carcinoma (HepG2) cancer cell lines and found that the extracts of Albizia lebbeck, Dillenia indica, and Bombax ceiba significantly decreased the cell viability at low concentrations with IC50 values of 24.03, 25.09, and 29.66 µg/mL, respectively. This is the first report of detection of phenolic compounds along with antimicrobial, antioxidant and cytotoxic potential of selected medicinal plants from India, which indicates that these plants might be valuable source for human and animal health.

Distribution and Identification of Endophytic Streptomyces Species from Schima wallichii as Potential Biocontrol Agents against Fungal Plant Pathogens.

The prospective of endophytic microorganisms allied with medicinal plants is disproportionally large compared to those in other biomes. The use of antagonistic microorganisms to control devastating fungal pathogens is an attractive and eco-friendly substitute for chemical pesticides. Many species of actinomycetes, especially the genus Streptomyces, are well known as biocontrol agents. We investigated the culturable community composition and biological control ability of endophytic Streptomyces sp. associated with an ethanobotanical plant Schima wallichi. A total of 22 actinobacterial strains were isolated from different organs of selected medicinal plants and screened for their biocontrol ability against seven fungal phytopathogens. Seven isolates showed significant inhibition activity against most of the selected pathogens. Their identification based on 16S rRNA gene sequence analysis, strongly indicated that all strains belonged to the genus Streptomyces. An endophytic strain BPSAC70 isolated from root tissues showed highest percentage of inhibition (98.3 %) against Fusarium culmorum with significant activity against other tested fungal pathogens. Phylogenetic analysis based on 16S rRNA gene sequences revealed that all seven strains shared 100 % similarity with the genus Streptomyces. In addition, the isolates were subjected to the amplification of antimicrobial genes encoding polyketide synthase type I (PKS-I) and nonribosomal peptide synthetase (NRPS) and found to be present in most of the potent strains. Our results identified some potential endophytic Streptomyces species having antagonistic activity against multiple fungal phytopathogens that could be used as an effective biocontrol agent against pathogenic fungi.

Antimicrobial biosynthetic potential and genetic diversity of endophytic actinomycetes associated with medicinal plants.

Endophytic actinomycetes are one of the primary groups that share symbiotic relationships with medicinal plants and are key reservoir of biologically active compounds. In this study, six selective medicinal plants were targeted for the first time for endophytic actinomycetes isolation from Gibbon Wild Life Sanctuary, Assam, India, during winter and summer and 76 isolates were obtained. The isolates were found to be prevalent in roots followed by stem and leaves. 16S rRNA gene sequence analysis revealed 16 genera, including rare genera, Verrucosispora, Isoptericola and Kytococcus, which have never been previously reported as endophytic. The genus Streptomyces (66%) was dominant in both seasons. Shannon's diversity index showed that Azadirachta indica (1.49), Rauwolfia serpentina (1.43) and Emblica officinalis (1.24) were relatively good habitat for endophytic actinomycetes. Antimicrobial strains showed prevalence of polyketide synthase (PKS) type-II (85%) followed by PKS type-I (14%) encoded in the genomes. Expression studies showed 12-fold upregulation of PKSII gene in seventh day of incubation for Streptomyces antibioticus (EAAG90). Our results emphasize that the actinomycetes assemblages within plant tissue exhibited biosynthetic systems encoding for important biologically active compounds.

Isolation, abundance and phylogenetic affiliation of endophytic actinomycetes associated with medicinal plants and screening for their in vitro antimicrobial biosynthetic potential.

Microorganisms associated with medicinal plants are of interest as the producers of important bioactive compounds. To date, the diversity of culturable endophytic actinomycetes associated with medicinal plants is in its initial phase of exploration. In this study, 42 endophytic actinomycetes were isolated from different organs of seven selected medicinal plants. The highest number of isolates (n = 22, 52.3%) of actinomycetes was isolated from roots, followed by stems (n = 9, 21.4%), leaves (n = 6, 14.2%), flowers (n = 3, 7.1%), and petioles (n = 2, 4.7%). The genus Streptomyces was the most dominant among the isolates (66.6%) in both the locations (Dampa TRF and Phawngpuii NP, Mizoram, India). From a total of 42 isolates, 22 isolates were selected for further studies based on their ability to inhibit one of the tested human bacterial or fungal pathogen. Selected isolates were identified based on 16S rRNA gene analysis and subsequently the isolates were grouped to four different genera; Streptomyces, Brevibacterium, Microbacterium, and Leifsonia. Antibiotic sensitivity assay was performed to understand the responsible antimicrobials present in the isolates showing the antimicrobial activities and revealed that the isolates were mostly resistant to penicillin G and ampicillin. Further, antimicrobial properties and antibiotic sensitivity assay in combination with the results of amplification of biosynthetic genes polyketide synthase (PKS-I) and non-ribosomal peptide synthetase (NRPS) showed that the endophytic actinomycetes associated with the selected medicinal plants have broad-spectrum antimicrobial activity. This is the first report of the isolation of Brevibacterium sp., Microbacterium sp., and Leifsonia xyli from endophytic environments of medicinal plants, Mirabilis jalapa and Clerodendrum colebrookianum. Our results emphasize that endophytic actinomycetes associated with medicinal plants are an unexplored resource for the discovery of biologically active compounds.