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.

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.

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.

Heterologous expression and molecular modelling of L-asparaginase from Bacillus subtilis ETMC-2.

Bacterial L-asparaginase is the key therapeutic enzyme in cancer therapy and is also witnessing demand as a food processing aid. In this study, L-asparaginase of newly isolated Bacillus subtilis ETMC-2 was cloned and over-expressed in Escherichia coli as an active soluble protein using ligation independent cloning strategy. The molecular mass was estimated to be 40 kDa and was optimally active at 50 °C. Zymography revealed that the enzyme was active in homo-tetramer state (~160 KDa). The encoded protein after BLASTp analysis on NCBI showed 99.73% similarity with L-ASNase that of Bacillus sp. Physico-chemical properties were predicted using Protparam leading to categorization of the enzyme as a stable protein with an instability index (II) of 19.02. The calculated aliphatic index (85.44) indicated the high thermal stability of the protein with GRAVY value of -0.317. Protein-Ligand docking revealed that the residues Thr89, Thr121, and Asp122 were fundamental in protein-ligand complexation. After homology modelling, model validation was performed using Ramachandran plot, VERIFY3D, and RMSD. The paper describes cloning, heterologous expression, catalytic characteristics and physico-chemical properties of the type II B. subtilis L-ASNase.

Prebiotic mannooligosaccharides: Synthesis, characterization and bioactive properties.

Functional oligosaccharides are non-digestible food ingredients that confer numerous health benefits. Among these, mannooligosaccharides (MOS) are emerging prebiotics that have characteristic potential bio-active properties. Microbial mannanases can be used to break down mannan rich agro-residues to yield MOS. Various applications of MOS as health promoting functional food ingredient may open up newer opportunities in food and feed industry. Enzymatic hydrolysis is the widely preferred method over chemical hydrolysis for MOS production. Presently, commercial MOS is being derived from yeast cell wall mannan and is widely used as prebiotic in feed supplements for poultry and aquaculture. Apart from stimulating the growth of probiotic microflora, MOS impart anticancer and immunomodulatory effects by inducing different gene markers in colon cells. This review summarizes recent developments and future prospects of enzymatic synthesis of MOS from various mannans, their structural characteristics and their potential health benefits.

Characterization of biogenically synthesized silver nanoparticles for therapeutic applications and enzyme nanocomplex generation.

The present study describes green synthesis of silver nanoparticles (AgNPs) and inulin hydrolyzing enzyme nanocomplexes (ENC) using Azadirachta indica (Ai) and Punica granatum (Pg) leaf extracts. Surface topology and physico-chemical characteristics of AgNPs were studied using surface plasmon resonance (SPR), FTIR, SEM, AFM and EDX analyses. Particle size analysis using dynamic light scattering and AFM studies revealed that Ai-AgNPs (76.4 nm) were spherical in shape having central bigger nano-regime with smaller surroundings while Pg-AgNPs (72.1 nm) and ENCs (Inulinase-Pg-AgNPs ~ 145 nm) were spherical particles having smooth surfaces. Pg-AgNPs exhibited significant photocatalysis of a thiazine dye, methylene blue. Both Ai- and Pg-AgNPs showed selective antibacterial action by inhibiting pathogenic Bacillus cereus, while the probiotic Lactobacillus strains remained unaffected. Ai-AgNPs showed potential anti-biofilm effect (30% viability) on B. cereus biofilms. Pg-AgNPs showed anti-cancer effect against human colon cancer cell lines (Caco-2) resulting in 40% cell death in 48 h. Enzymes (inulinase, L-asparaginase and glucose oxidase) were successfully immobilized onto nanoparticles together with the biogenic synthesis of AgNPs and recyclability of the Inulinase-Pg-AgNPs complex was demonstrated. The study elaborates characteristics of green synthesized nanoparticles and their potential applications as anti-cancer, antibacterial and antioxidant nano drugs that could be used in food and nutraceutical industries. Enzyme immobilization on AgNPs without any toxic cross-linker opens up newer possibilites in enzyme-nanocomplex research.

Characteristics and bioactive properties of mannooligosaccharides derived from agro-waste mannans.

Mannooligosaccharides (MOS) were derived using Aspergillus oryzae ß-mannanase (ManAo) from different mannan-rich agro-wastes, palm kernel cake (PKC), guar gum and copra meal (CM). Guar gum (GG) released higher amount of MOS (56.31% w/w) from which purification of mannobiose (0.68 mg) and mannotriose (1.26 mg) was demonstrated using size-exclusion chromatography. FTIR analysis of mannan hydrolysates showed characteristic peaks in 1200-900 cm-1 region indicating the presence of MOS. 1H &13C NMR spectra showed presence of anomeric sugar forms of MOS in different mannan hydrolysates. MOS from locust bean gum and guar gum had both α- and ß-anomers while PKC and CM had only α-anomer. Growth promotional activities of different MOS were demonstrated using two probiotic Lactobacilli. Besides, enzymatically derived MOS also showed metal (Fe2+) chelating and anti-oxidant activities, wherein best anti-glycating agent was evaluated as MOS from PKC. PKC derived MOS showed highest cytotoxicity (74.19%) against human colon adenocarcinoma cell line (Caco-2). This study demonstrated the prebiotic potential of agro-waste derived MOS and possibility of their utilization as a functional food ingredient.

Development and catalytic characterization of L-asparaginase nano-bioconjugates.

The present paper describes efficient immobilization of L-glutaminase free L-asparaginase for developing a new therapeutic system for anticancer therapy. L-asparaginase (L-ASNase) was covalently immobilized on the functionalized aluminum oxide nanoparticles (AONP) and titanium oxide nanoparticles (TONP). The nano-bioconjugates (AONP-ASNase and TONP-ASNase) were characterized by scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FTIR) and UV-Vis spectral analysis that revealed the successful immobilization. The nano-bioconjugates were optimally active at pH 7.0 and 40 °C. TONP-ASNase activity was enhanced in the presence of NH4+ (160%) and Mn2+ (165%) while AONP-ASNase bioconjugates showed increased relative activity with ethyl acetate (142%) and toluene (160%). The nano-bioconjugates displayed excellent reusability and maintained >90% average activity after nine successive cycles. Maximum cytotoxicity (61%) was noticed with AONP-ASNase (10 µg/ml) against human leukemia MOLT-4 cells. Regarding kinetic values, AONP-ASNase showed better affinity (Km 1.9 µmol) to L-asparagine as compared to free L-ASNase. After 23 days storage at 37 °C, bioconjugates retained 40% residual activity while free L-ASNase was completely deactivated. Thermodynamic characterization revealed higher conversion rate of the E-S complex in case of nano-bioconjugates.

Catalytic characteristics and application of l-asparaginase immobilized on aluminum oxide pellets.

l-asparaginase from Escherichia coli (l-ASNase) was covalently immobilized on aluminum oxide pellets (AlOPs) using a cross-linking agent, glutaraldehyde. Maximum immobilization yield (85.0%) was obtained after optimizing immobilization parameters using response surface methodology (RSM). Both free and immobilized l-ASNase (AlOP-ASNase) were optimally active at 37°C and pH7.5. However, the bioconjugate exhibited enhanced activity and stability at different pH and temperatures. It had higher affinity (low Km) and was comparatively more stable in presence of some solvents (ethyl acetate, acetone, acetonitrile), metal ions (Ag+, Zn2+) and ß-mercaptoethanol. AlOP-ASNase was reused in a glass column reactor for l-asparagine hydrolysis upto nine successive cycles without any loss in activity. The AlOP-ASNase was effective in lowering l-asparagine level in blanched potato chips indicating its potential use in mitigating acrylamide formation in starchy foods. This cost-effective enzyme preparation had shelf-life of more than 30days and can be effectively used in starch based food industries.

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.

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.