Characterization of Diesel Degrading Indigenous Bacterial Strains, Acinetobacter pittii and Pseudomonas aeruginosa, Isolated from Oil Contaminated Soils.

In this study, 13 diesel degrading bacteria were isolated from the oil contaminated soils and the promising strains identified as Acinetobacter pittii ED1 and Pseudomonas aeruginosa BN were evaluated for their diesel degrading capabilities. These strains degraded the diesel optimally at 30 °C, pH 7.0 and 1% diesel concentration. Both the strains produced biofilm at 1% diesel concentration indicating their ability to tolerate diesel induced abiotic stress. Gravimetric analysis of the spent medium after 7 days of incubation showed that A. pittii ED1 and P. aeruginosa BN degraded 68.61% and 76% diesel, respectively, while biodegradation reached more than 90% after 21 days. Fourier Transform Infrared (FTIR) analysis of the degraded diesel showed 1636.67 cm-1 (C=C stretch, N-H bond) peak corresponding to alkenes and primary amines, while GC-TOF-MS analysis showed decline in hydrocarbon intensities after 7 days of incubation. The present study revealed that newly isolated A. pittii ED1 and P. aeruginosa BN were able to degrade diesel hydrocarbons (C11-C18, and C19-C24) efficiently and have potential for bioremediation of the oil-contaminated sites. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01317-3.

Production, properties, and applications of fructosyltransferase: a current appraisal.

BACKGROUND: Fructosyltransferases (FTases) are drawing increasing attention due to their application in prebiotic fructooligosaccharide (FOS) generation. FTases have been reported to occur in a variety of microorganisms but are predominantly found in filamentous fungi. These are employed at the industrial scale for generating FOS which make the key ingredient in functional food supplements and nutraceuticals due to their bifidogenic and various other health-promoting properties. SCOPE AND APPROACH: This review is aimed to discuss recent developments made in the area of FTase production, characterization, and application in order to present a comprehensive account of their present status to the reader. Structural features, catalytic mechanisms, and FTase improvement strategies have also been discussed in order to provide insight into these aspects. KEY FINDINGS AND CONCLUSIONS: Although FTases occur in several plants and microorganisms, fungal FTases are being exploited commercially for industrial-scale FOS generation. Several fungal FTases have been characterized and heterologously expressed. However, considerable scope exists for improved production and application of FTases for cost-effective production of prebiotic FOS.HIGHLIGHTSFructosyltrasferase (FTase) is a key enzyme in fructo-oligosaccharide (FOS) generationDevelopments in the production, properties, and functional aspects of FTasesMolecular modification and immobilization strategies for improved FOS generationFructosyltransferases are innovation hotspots in the food and nutraceutical industries.

Investigation of the internal structure and dynamics of cellulose by 13C-NMR relaxometry and 2DPASS-MAS-NMR measurements.

Internal structure and dynamics of commercial and natural cellulose were studied by measuring chemical shift anisotropy (CSA) parameters, and spin-lattice relaxation rate (1/T1) at each and every chemically different carbon nuclear site. CSA parameters were measured by 13C two-dimensional phase adjusted spinning sideband (2DPASS) cross-polarization magic angle spinning (CP-MAS) NMR experiment. Site specific spin-lattice relaxation time was measured by Torchia-CP method. Anisotropy parameters of C4 and C6 regions are higher than C1 and C235 regions and asymmetry of C4 line is lower than any other carbon site. The higher values of CSA parameters of C4 and C6 nuclei arise due to the rotation of O4-C4, C1-O4, O5-C5-C6-O6 and C4-C5-C6-O6 bonds at torsion angles ψ, Φ, χ and χ' respectively and the influence of interchain and intrachain hydrogen bondings. Two distinct peaks are also observed for C4 and C6 resonance line position-one peak arises primarily due to the nuclei in amorphous region and another one arises due to the same nuclei resides in paracrystalline region. The spin-lattice relaxation time and the CSA parameters are different at these two distinct peak positions of C4 and C6 line. Molecular correlation time of each and every chemically different carbon site was calculated with the help of CSA parameters and spin-lattice relaxation time. The molecular correlation time of the amorphous region is one order of magnitude less than the crystalline region. The distinction between amorphous and paracrystalline regions of cellulose is more vividly portrayed by determining spin-lattice relaxation time, CSA parameters, and molecular correlation time at each and every chemically different carbon site. This type of study correlating the structure and dynamics of cellulose will illuminate the path of inventing biomimetic materials.

A comprehensive and comparative study of the internal structure and dynamics of natural ß-keratin and regeneratedß-keratin by solid state NMR spectroscopy.

Structure and dynamics of natural and regenerated chicken feather ß-keratin were investigated by 13C cross-polarization (CP) magic angle spinning (MAS) solid state nuclear magnetic resonance (SSNMR) spectral analysis, 13C and 1H spin-lattice relaxation time measurements, and 13C two dimensional phase adjusted spinning sidebands (2DPASS) MAS SSNMR measurements. Chemical shift anisotropy (CSA) parameters of both natural and regenerated chicken feather ß-keratin were extracted by using 2DPASS MAS SSNMR experiment. The beauty of 2DPASS MAS SSNMR experiment is it can correlate the isotropic and anisotropic dimension with the help of shearing transformation and two dimensional Fourier Transformation. Molecular correlation time at each and every magnetically inequivalent carbon site of both natural and regenerated chicken feather ß-keratin were also determined. The change in molecular dynamics of structural protein after pretreatment was monitored by 2DPASS MAS SSNMR and 13C relaxation measurement. This type of comprehensive study will provide the information about the interrelation between the structure and dynamics of structural protein and will also shed light in the way of developing methods for conversion of animal by-products to novel product.

Biotechnological potential of microbial inulinases: Recent perspective.

Among microbial enzymes, inulinases or fructo-furanosylhydrolases have received considerable attention in the past decade, and as a result, a variety of applications based on enzymatic hydrolysis of inulin have been documented. Inulinases are employed for generation of fructose and inulo-oligosaccharides (IOS) in a single-step reaction with specificity. The high fructose syrup can be biotransformed into value-added products such as ethanol, single cell protein, while IOS are indicated in nutraceutical industry as prebiotic. Myriad microorganisms produce inulinases, and a number of exo- and endo-inulinases have been characterized and expressed in heterologous hosts. Initially, predominated by Aspergilli, Penicillia, and some yeasts (Kluyveromyces spp.), the list of prominent inulinase producers has gradually expanded and now includes extremophilic prokaryotes and marine-derived microorganisms producing robust inulinases. The present paper summarizes important developments about microbial inulinases and their applications made in the last decade.

Production of inulinase, fructosyltransferase and sucrase from fungi on low-value inulin-rich substrates and their use in generation of fructose and fructo-oligosaccharides.

Owing to applications in the food and nutraceutical industries, inulinases, fructosyltransferases and sucrases have gained considerable attention in recent times. Twenty-five fungal strains were screened for production of these enzymes on three different media formulated using inulin-rich plant extracts prepared from asparagus root, dahlia tuber and dandelion root extract. Culture filtrates of the fungi were examined for hydrolytic activities. Fungi belonging to genus Aspergillus, A. niger GNCC 2655 (11.3 U/ml), A. awamori MTCC 2879 (8.2 U/ml), A. niger ATCC 26011 (7.9 U/ml) secreted high titers of inulinase followed by Penicillium sp. NFCCI 2768 (2.6 U/ml) and Penicillium citrinum MTCC 1256 (1.1 U/ml). High sucrase activity was noticed in A. niger GNCC 2613 (113 U/ml) and A. awamori MTCC 2879 (107.8 U/ml). Analysis of end products of inulinase action by HPLC revealed that most of the enzymes were exo-inulinases liberating fructose exclusively from inulin. Five fungi, P. citrinum MTCC 1256, Penicillium rugulosum MTCC 3487, Penicillium sp. NFCCI 2768, A. fumigatus GNCC 1351 and A. niger ATCC 26011 however, produced a mixture of endo- and exo-inulinases liberating oligosaccharides (GF3 and GF2) along with fructose. High inulinase/sucrase yielding strains were evaluated for extracellular and intracellular hydrolytic and transfructosylating activities and intracellular enzyme profiles were found to be considerably different in terms of titers and end products.

Isolation and identification of actinomycetes for production of novel extracellular glutaminase free L-asparaginase.

Over the recent years glutaminase free L-asparaginase has gained more importance due to better therapeutic properties for treatment of acute lymphoblastic leukemia. Actinomycetes are known for L-asparaginase activity. In the current study, 80 actinomycetes were isolated from various soil habitats by serial dilution technique. Presence of L-asparaginase was investigated in a total of 240 actinomycetes by tubed agar method using modified M-9 medium. A total of 165 actinomycetes were found positive for L-asparaginase activity. Among these, 57 actinomycetes producing larger zones of L-asparagine hydrolysis were further screened for their capacity to produce glutaminase-free L-asparaginase. Four L-glutaminase-free actinomycetes were found to be potential L-asparaginase producers. These actinomycetes were identified as Streptomyces cyaneus (SAP 1287, CFS 1560), S. exfoliates (CFS 1557) and S. phaeochromogenes (GS 1573) on the basis of morphological and biochemical identification studies. Maximum L-asparaginase activity (19.2 Uml(-1)) was observed in culture filtrate of S. phaeochromogenes under submerged fermentation. Results indicate that S. phaeochromogenes could be a potential source of glutaminase free L-asparaginase for commercial purpose. To the best of our knowledge, this is the first report on production of glutaminase free L-asparaginase from S. cyaneus, S. exfoliatus and S. phaeochromogenes.

Prebiotics, Probiotics and Postbiotics: The Changing Paradigm of Functional Foods.

The rampant use of antibiotics has led to the emergence of multidrug resistance and is often coupled with gut dysbiosis. To circumvent the harmful impact of antibiotics, probiotics have emerged as an effective intervention. However, while the new probiotics are being added to the list, more recently, the nature and role of their counterparts, viz. prebiotics, postbiotics and parabiotics have also drawn considerable attention. As such, intricate relationships among these gut-biotics vis-à-vis their role in imparting health benefits is to be delineated in a holistic manner. Prebiotic dietary fibers are selectively fermented by probiotics and promote their colonization in the gut. The proliferation of probiotics leads to production of fermentation by-products (postbiotics) which affect the growth of enteropathogens by lowering the pH and producing inhibitory bacteriocins. After completing life-cycle, their dead remnants (parabiotics e.g. exopolysaccharides and cell wall glycoproteins) also inhibit adhesion and biofilm formation of pathogens on the gut epithelium. These beneficial effects are not just endemic to gut but a systemic response is witnessed at different gut-organ axes. Thus, to decipher the role of probiotics, it is imperative to unravel the interdependence between these components. This review elaborates on the recent advancements on various aspects of these gut-biotics and the mechanism of potential attributes like anti-oxidant, anti-inflammatory, anti-neoplastic, anti-lipidemic and anti-hyperglycemic benefits.

Optimized production and characterization of endo-ß-mannanase by Aspergillus niger for generation of prebiotic mannooligosaccharides from guar gum.

Optimized production of Aspergillus niger ATCC 26011 endo-ß-mannanase (ManAn) on copra meal resulted in 2.46-fold increase (10,028 U/gds). Purified ManAn (47 kDa) showed high affinity towards guar gum (GG) as compared to konjac gum and locust bean gum with Km 2.67, 3.25 and 4.07 mg/mL, respectively. ManAn efficiently hydrolyzed GG and liberated mannooligosaccharides (MOS). Changes occurring in the rheological and compositional aspects of GG studied using Differential scanning calorimetry (DSC), Thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) revealed increased thermal stability and crystallinity of the partially hydrolyzed guar gum (PHGG). Parametric optimization of the time and temperature dependent hydrolysis of GG (1% w/v) with 100 U/mL of ManAn at 60 °C and pH: 5.0 resulted in 12.126 mg/mL of mannotetraose (M4) in 5 min. Enhanced growth of probiotics Lactobacilli and production of short chain fatty acids (SCFA) that inhibited enteropathogens, confirmed the prebiotic potential of PHGG and M4.

Role of Synbiotics (Prebiotics and Probiotics) as Dietary Supplements in Type 2 Diabetes Mellitus Induced Health Complications.

Diabetes is a metabolic disorder whose prevalence has become a worrying condition in recent decades. Chronic diabetes can result in serious health conditions such as impaired kidney function, stroke, blindness, and myocardial infarction. Despite a variety of currently available treatments, cases of diabetes and its complications are on the rise. This review article provides a comprehensive account of the ameliorative effect of prebiotics and probiotics individually or in combination i.e. synbiotics on health complications induced by Type 2 Diabetes Mellitus (T2DM). Recent advances in the field underscore encouraging outcomes suggesting the consumption of synbiotics leads to favorable changes in the gut microbiota. These changes result in the production of bioactive metabolites such as short-chain fatty acids (crucial for lowering blood sugar levels), reducing inflammation, preventing insulin resistance, and encouraging the release of glucagon-like peptide-1 in the host. Notably, novel strategies supplementing synbiotics to support gut microbiota are gaining attraction as pivotal interventions in mitigating T2DM-induced health complications. Thus, by nurturing a symbiotic relationship between prebiotics and probiotics i.e. synbiotics, these interventions hold promise in reshaping the microbial landscape of the gut thereby offering a multifaceted approach to managing T2DM and its associated morbidities. Supporting the potential of synbiotics underscores a paradigm shift toward holistic and targeted interventions in diabetes management, offering prospects for improved outcomes and enhanced quality of life for affected individuals. Nevertheless, more research needs to be done to better understand the single and multispecies pre/pro and synbiotics in the prevention and management of T2DM-induced health complications.

Impact of Dietary Probiotics on the Immune and Reproductive Physiology of Pubertal Male Japanese Quail (Coturnix coturnix japonica) Administered at the Onset of Pre-Puberty.

Fertility in males is dependent on the proper production of sperms involving the synchronization of numerous factors like oxidative stress, inflammatory processes, and hormonal regulation. Inflammation associated with oxidative stress is also known to impair sperm function. Nutritional factors like probiotics and prebiotics have the potential benefits to modulate these factors which may enhance male fertility. In the present study, immature male Japanese quail at the beginning of 3rd week were administered with Lactobacillus rhamnosus (L), Bifidobacterium longum (B), and mannan-oligosaccharides (M) through dietary supplementation in individual groups as well as in combinations like LB and MLB. Markers of oxidative stress including SOD and catalase were examined by native PAGE; inflammatory biomarkers (IL-1ß, IL-10, and NFκB), apoptotic markers (caspase 3 and caspase 7), steroidal hormones, and their receptors estrogen receptor alpha (ERα) and beta (ERß) were assessed in testis. The study reveals that dietary supplementation of 1% L, B, and M in combination significantly and positively increases the overall growth of immature male quail specifically testicular weight and gonadosomatic index (GSI). Furthermore, significant improvement in testicular cell size; increased steroidal hormones like testosterone, FSH, and LH levels; increase in SOD, catalase enzymes; decrease in apoptotic factors Caspase 3, Caspase 7 and immune system strength observed indicated by a decrease in expression of IL-1ß, NFκB; and increase of IL-10 in testis when LBM was used in combination. These variations are attributed to the increase in testicular estrogen receptors alpha and beta, facilitated by the neuroendocrine gonadal axis, ultimately leading to improved male fertility. It can be concluded that the dietary supplementation in combination with L, B, and M enhances male fertility in immature quail by increased expression of estrogen receptors via gut microbiota modulation. It also sheds light on the potential use of these nutritional factors in avian species as therapeutic interventions to overcome low fertility problems in quail thereby benefitting the poultry industry.

Synthesis and characterization of keratinase laden green synthesized silver nanoparticles for valorization of feather keratin.

This study focuses on the efficient and cost-effective synthesis of silver nanoparticles (AgNPs) using plant extracts, which have versatile and non-toxic applications. The research objectives include synthesizing AgNPs from readily available plant extracts, optimizing their production and multi scale characterization, along with exploring their use for enzyme immobilization and mitigation of poultry feather waste. Among the plant extracts tested, the flower extract of Hibiscus rosa-sinensis (HF) showed the most potential for AgNP synthesis. The synthesis of HF-mediated AgNPs was optimized using response surface methodology (RSM) for efficient and environment friendly production. Additionally, the keratinase enzyme obtained from Bacillus sp. NCIM 5802 was covalently linked to AgNPs, forming a keratinase nanocomplex (KNC) whose biochemical properties were evaluated. The KNC demonstrated optimal activity at pH 10.0 and 60 °C and it displayed remarkable stability in the presence of various inhibitors, metal ions, surfactants, and detergents. Spectroscopic techniques such as FTIR, UV-visible, and X-ray diffraction (XRD) analysis were employed to investigate the formation of biogenic HF-AgNPs and KNC, confirming the presence of capping and stabilizing agents. The morphological characteristics of the synthesized AgNPs and KNC were determined using transmission electron microscopy (TEM) and particle size analysis. The study highlighted the antimicrobial, dye scavenging, and antioxidant properties of biogenic AgNPs and KNC, demonstrating their potential for various applications. Overall, this research showcases the effectiveness of plant extract-driven green synthesis of AgNPs and the successful development of keratinase-laden nanocomplexes, opening possibilities for their use in immobilizing industrial and commercial enzymes.

Withaferin-A attenuates diabetes mellitus induced male reproductive dysfunction mediated by ERα in brain and testes of Swiss albino mice.

Diabetes mellitus (DM) is a chronic metabolic disease, characterized by persistent hyperglycemia resulting from diminished insulin secretion or insulin resistance. The present study evaluated the ameliorative effects of Withaferin-A (WA) on DM-induced reproductive dysfunction in mice. For the same, mice were intraperitoneally injected with Streptozotocin (STZ), (40 mg/kg/day) for 5 consecutive days to induce DM. Mice were then treated with WA (8 mg/kg/day) in normal and diabetic conditions (STZ + WA). Next, blood glucose levels, oral glucose tolerance, intraperitoneal insulin tolerance, oxidative stress and reproductive parameters were estimated. For reproductive performance, immunofluorescent localization of gonadotropin-releasing hormone (GnRH-I) and estrogen receptor alpha (ERα) in the preoptic area and paraventricular nucleus region of hypothalamus and ERα in testes was performed. STZ-induced diabetes triggered reproductive dysfunctions as mediated by low GnRH-I and ERα in the brain and ERα in the testes along with declined testosterone and estradiol levels. Treatment with WA significantly reduced the blood glucose levels and enhanced glucose clearance accompanied by reduced oxidative stress in the brain, pancreas and testes as indicated by the low levels of H2O2 and MDA in diabetic mice treated with WA (STZ + WA). This study reports, for the first time, that WA can efficiently ameliorate DM-induced reproductive dysfunctions by enhancing endogenous testosterone, estrogen and increased GnRH-I and ERα in the brain and ERα in the testes of DM-induced male mice.

Production of inulinase from Kluyveromyces marxianus using dahlia tuber extract.

Various carbon sources were evaluated for production of inulinase by yeast, Kluyveromyces marxianus MTCC 3995. Highest inulinase activity was observed with Dahlia extract (25.3 nkat mL(-1)) as carbon source. The enzyme activity was 1.4 folds higher than that observed in media containing pure chicory inulin (17.8 nkat mL(-1)). The yeast showed good growth on a simple medium containing dahlia extract (20% w/v) and yeast extract (2%w/v) as carbon and nitrogen source respectively, in 96 h. at 28°C and 120 rpm. Lowest inulinase yield (4.8 nkat mL(-1)) was seen in the medium containing glucose as C-source. Although varied inulinase levels were noticed on different C- sources, Inulinase: Sucrase (I/S) ratios were noticed to be similar. Among various protein sources tested, yeast extract was found to be the best source followed by beef extract (17.9 nkat mL(-1)) and peptone (13.8 nkat mL(-1)). The enzyme was optimally active at pH (4.0) and 50°C. TLC analysis of end product revealed that inulinase hydrolyzed inulin exclusively into fructose. Results suggest that the dahlia extract induced exoinulinase synthesis in Kluyveromyces marxianus and can be utilized as a potential substrate for inulinase production.

Recent Developments in Industrial Mycozymes: A Current Appraisal.

Fungi, being natural decomposers, are the most potent, ubiquitous and versatile sources of industrial enzymes. About 60% of market share of industrial enzymes is sourced from filamentous fungi and yeasts. Mycozymes (myco-fungus; zymes-enzymes) are playing a pivotal role in several industrial applications and a number of potential applications are in the offing. The field of mycozyme production, while maintaining the old traditional methods, has also witnessed a sea change due to advents in recombinant DNA technology, optimisation protocols, fermentation technology and systems biology. Consolidated bioprocessing of abundant lignocellulosic biomass and complex polysaccharides is being explored at an unprecedented pace and a number of mycozymes of diverse fungal origins are being explored using suitable platforms. The present review attempts to revisit the current status of various mycozymes, screening and production strategies and applications thereof.

Purification of Aspergillus tamarii mycelial fructosyltransferase (m-FTase), optimized FOS production, and evaluation of its anticancer potential.

In the present study, generation of prebiotic fructooligosaccharides (FOS) using Aspergillus tamarii FTase was optimized by applying response surface methodology. Optimal FOS (251 g L-1 ) was generated at 28.4°C, pH 7.0 and 50% (w/v) sucrose leading to 1.97-fold yield enhancement. The m-FTase was purified using ultrafiltration followed by HiTrap Q HP anion exchange chromatography resulting in 2.15-fold purified FTase with 12.76 U mg-1 specific activity. Purified FTase (75 kDa) had Km and Vmax values of 1049.717 mM and 2.094 µmol min-1  mg-1 , respectively. FOS incorporation led to upregulation of caspase 3, caspase 9, and Bax genes suggesting mitochondrial apoptosis activation in cancer cells. The study describes characteristics of purified FTase from A. tamarii, production optimization of FOS and unravels the role of FOS in anticancer activity against HT-29 cells. PRACTICAL APPLICATION: This study provides detailed insights of kinetic and thermodynamic characteristics of purified FTase, a prebiotic FOS-generating enzyme. Moreover, the role of the apoptotic genes involved in anticancer activity, and the prebiotic potential of FOS is also investigated. These findings are important in the context of FOS applications, and the optimized production strategies make it useful for industrial application.

Molecular insight into Aspergillus oryzae ß-mannanase interacting with mannotriose revealed by molecular dynamic simulation study.

Fungal ß-mannanases hydrolyze ß-1, 4-glycosidic bonds of mannans and find application in the generation of mannose and prebiotic mannooligosaccharides (MOS). Previously, a MOS generating ß-mannanase from Aspergillus oryzae MTCC 1846 (ßManAo) was characterized and its structural and functional properties were unraveled through homology modeling and molecular dynamics in this study. The ßManAo model was validated with 92.9% and 6.5% of the residues found to be distributed in the most favorable and allowed regions of the Ramachandran plot. Glu244 was found to play a key role in the interaction with mannotriose, indicating conserved amino acids for the catalytic reaction. A detailed metadynamic analysis of the principal components revealed the presence of an α8-helix in the C-terminus which was very flexible in nature and energy landscapes suggested high conformation sub-states and the complex dynamic behavior of the protein. The binding of the M3 substrate stabilized the ß-mannanase and resulted in a reduction in the intermediate conformational sub-states evident from the free energy landscapes. The active site of the ß-mannanase is mostly hydrophilic in nature which is accordance with our results, where the major contribution in the binding energy of the substrate with the active site is from electrostatic interactions. Define Secondary Structure of Proteins (DSSP) analysis revealed a major transition of the protein from helix to ß-turn for binding with the mannotriose. The molecular dynamics of the ßManAo-mannotriose model, and the role and interactions of catalytic residues with ligand were also described. The substrate binding pocket of ßManAo was found to be highly dynamic and showed large, concerted movements. The outcomes of the present study can be exploited in further understanding the structural properties and functional dynamics of ßManAo.

Parametrically optimized feather degradation by Bacillus velezensis NCIM 5802 and delineation of keratin hydrolysis by multi-scale analysis for poultry waste management.

Enormous amounts of keratinaceous waste make a significant and unexploited protein reserve that can be utilized through bioconversion into high-value products using microbial keratinases. This study was intended to assess the keratinase production from a newly isolated B. velezensis NCIM 5802 that can proficiently hydrolyze chicken feathers. Incubation parameters used to produce keratinase enzyme were optimized through the Response Surface Methodology (RSM) with chicken feathers as substrate. Optimization elevated the keratinase production and feather degradation by 4.92-folds (109.7 U/mL) and 2.5 folds (95.8%), respectively. Time-course profile revealed a direct correlation among bacterial growth, feather degradation, keratinase production and amino acid generation. Biochemical properties of the keratinase were evaluated, where it showed optimal activity at 60 °C and pH 10.0. The keratinase was inhibited by EDTA and PMSF, indicating it to be a serine-metalloprotease. Zymography revealed the presence of four distinct keratinases (Mr ~ 100, 62.5, 36.5 and 25 kDa) indicating its multiple forms. NMR and mass spectroscopic studies confirmed the presence of 18 free amino acids in the feather hydrolysates. Changes in feather keratin brought about by the keratinase action were studied by X-ray diffraction (XRD) and spectroscopic (FTIR, Raman) analyses, which showed a decrease in the total crystallinity index (TCI) (1.00-0.63) and confirmed the degradation of its crystalline domain. Scanning electron microscopy (SEM) revealed the sequential structural changes occurring in the feather keratin during degradation. Present study explored the use of keratinolytic potential of the newly isolated B. velezensis NCIM 5802 in chicken feather degradation and also, unraveled the underlying keratin hydrolysis mechanism through various analyses.

Production of mannooligosaccharides from various mannans and evaluation of their prebiotic potential.

Aspergillus quadrilineatus endo-ß-mannanase effectively degraded konjac glucomannan (66.09% w/v), copra meal (38.99% w/v) and locust bean galactomannan (20.94% w/v). High performance liquid chromatography (HPLC) analysis of KG hydrolysate indicated its mannooligosaccharides (MOS) content (656.38 mg/g) with high amounts of DP 5 oligosaccharide. Multi-scale characterization of mannan hydrolysate was done using FTIR and 13C NMR which revealed α and ß form of galactose or glucose in MOS, respectively. CM and LBG hydrolysates (1 mg/mL) have shown cytotoxic effect and reduced cell viability of Caco-2 cells by 45% and 62%, respectively. MOS DP (1-4) derived from LBG supported better Lactobacilli biofilm formation as compared to KG hydrolysate containing high DP MOS (5-7). Lactobacilli effectively fermented MOS to generate acetate and propionate as main short chain fatty acids. Lactobacilli produced leucine, isoleucine and valine as branched chain amino acids when grown on LBG hydrolysate.

Production and characterization of keratinase by Ochrobactrum intermedium for feather keratin utilization.

A newly isolated bacterium producing 55.5 U/mL keratinase on feather meal minimal medium was identified as Ochrobactrum intermedium. Optimization of process parameters by one-variable-at-a-time (OVAT) approach (substrate concentration 0.5% w/v, inoculum size 5% w/v, pH 7.0, 200 rpm for 96 h at 40 °C) resulted in 2.1-fold increase in keratinase secretion (117 U/mL). Keratinase was optimally active at pH 9.0 and 40 °C and was stable at pH 9.0 and 60 °C for 120 min. Calcium ions enhanced keratinase activity (158%) significantly, while it was strongly inhibited by both PMSF and EDTA, indicating it to be a metallo-serine protease. Keratinase degraded native chicken feathers efficiently resulting in 97.9% weight loss along with release of 745.5 µg/mL soluble proteins and 4196.69 µg/mL amino acids. Feather hydrolysate generated by NKIS 1 exhibited significant anti-oxidant and free-radical scavenging activity (90.46%). The present study revealed that O. intermedium NKIS 1 has potential applications in the biodegradation of chicken feathers and the value-addition of poultry waste.