Assessing the viability of carbamoylethyl pullulan-g-stearic acid based smart polymeric micelles for tumor targeting of raloxifene.

The present investigation entails the synthesis of smart pullulan polymeric micelles for evaluating its tumor targeting potential. For this purpose, two step polymerization synthesis reactions were conducted. In the first step, carbamoylethylation occurs by reaction of the free alcoholic moieties at 6th position of glucopyranose unit of pullulan with acrylamide in presence of alkali to obtain carbamoylethyl pullulan (CmP). In the second step, CmP undergoes graft polymerization with stearic acid (SA) to obtain CmP-g-stearic acid diblock co-polymer (CmP-g-SA) as evident from FTIR and NMR analysis. The XpRD spectra showed crystalline nature that was further confirmed by SEM indicating rough and poly-porous morphology. The QbD based optimized formulations of raloxifene HCl (RLX) loaded polymeric micelles (RLX PMs) exhibited pH-dependent release profile with added advantage of 1.2 times reduction in percentage hemolysis giving substantial compatibility with erythrocytes. In vivo pharmacokinetic performance of RLX PMs suggested enhanced mean residence time and volume of distribution. Besides, the biodistribution study of RLX PMs manifested enhanced entry of RLX in mammary carcinoma tissues as compared to normal tissues suggested that CmP-g-SA based micelles enhanced the anti-tumor activity of RLX. Overall, the findings pointed toward the biocompatibility of CmP-g-SA as a potential carrier system for the delivery of RLX.

Nutritional Enhancement of Chicken Feather Waste by Bacillus aerius NSMk2.

Keratinous waste is the bulk by-product of various livestock industries. Slow natural degradation of keratin and less efficient chemical hydrolysis imposes challenge for the search of alternative recycling methods. Keratin degrading microbes hydrolyse keratin to soluble peptides and amino acids. Bacillus aerius NSMk2 showed great potential for hydrolysis of chicken feather waste. Bacillus aerius NSMk2 cells grown in phosphate buffer supplemented with chicken feathers showed high disulfide reductase activity and release of sulfhydryl groups. The release of proteins and amino acids were statistically optimized at varied pH (4.0-11.0), temperature (30.0-45.0 °C) and agitation (100-250 rpm), and maximum release was recorded at pH 7.5, temperature 37 °C and shaking (175 rpm). FTIR and SEM showed sulfitolysis and extensive keratinolysis of feathers resulting in complete hydrolysis of white chicken feathers after 84 h. MALDI-TOF mass spectrometry confirmed the release of low molecular weight peptides in the range of 399 to 3289.4 m/z. The present study demonstrates management of otherwise hard-to-degrade keratinous waste and simultaneous nutritional enhancement of waste chicken feathers to value-added hydrolysate that can be used in livestock feed formulations or biofertilizer in agro-industry.

Investigating the potential of carboxymethyl pullulan for protecting the rabbit eye from systematically induced precorneal tear film damage.

The present investigation was aimed to develop a rabbit model for protecting the rabbit eye from systematically induced precorneal tear film (PTF) damage, evaluation of carboxymethyl pullulan for its protective action against PTF damage and its curative potential. For the same, pullulan was modified by carboxymethylation and structural modification was confirmed by spectral attributes. Further, the carboxymethyl pullulan (CMP) solutions (0.1-2.0%, w/v) were evaluated for their physical properties and its concentration 1.5% (w/v) was found to fit the criterion to prepare an eye solution. The safety and non-toxicity of CMP (1.5%, w/v) eye solution was confirmed by HET-CAM method and rabbit eye irritation test. Further, a systematic rabbit eye model was developed that mimic PTF damage in day to day life. Therefore, three levels of PTF damage were developed equating symptoms of damage due to high temperature (level I) or long term mobile use (level II) or heavy air pollution (level III). Thus, a representative model with benzalkonium chloride (BAC, 0.1% v/v, 0.2% v/v and 0.3% v/v), administered two drops twice a day for two days to develop level I, level II and level III eye damage. The CMP (1.5%, w/v) eye solution possessed a protective potential against level I and II PTF damage. The rabbit eyes remained unharmed and comparable with the normal control during the complete experimental period. Additionally, CMP (1.5%, w/v) eye solution has shown early fast recovery (8 days) from PTF damage induced by instillation of PTF damage agent (BAC). Carboxymethyl pullulan eye protective solution has normalized the tear film stability in rabbit eye model. It is established from the present work that, carboxymethyl pullulan has protective action against precorneal tear film damage and it potentiates the early recovery too.

Solid-State Fermentation of Carrot Pomace for the Production of Inulinase by Penicillium oxalicum BGPUP-4.

Inulinases are an important class of industrial enzymes which are used for the production of high-fructose syrup and fructooligosaccharides. Inulin, a polyfructan, is generally employed for the production of inulinase, which is a very expensive substrate. A number of agroindustrial residues have been used for cost-effective production of inulinases. In the present study, carrot pomace was selected as a substrate for the production of inulinase by Penicillium oxalicum BGPUP-4 in solid-state fermentation. Carrot pomace is one of the good substrates for bioprocesses, because it is rich in soluble and insoluble carbohydrates. A central composite rotatable design (CCRD) used in response surface methodology was employed for the optimal production of inulinase from carrot pomace. Using CCRD, 15 runs were practiced to optimize the range of three independent variables: moisture content (70-90%), incubation time (4-6 days) and pH (5.0-7.0) for inulinase production. Carrot pomace supplemented with 0.5% inulin as an inducer, 0.2% NH4H2PO4, 0.2% NaNO3, 0.2% KH2PO4, 0.05% MgSO4·7H2O and 0.001% FeSO4·7H2O was used for the production of inulinase in solid-state fermentation at 30 °C. Inulinase production (322.10 IU per g of dry substrate) was obtained under the optimized conditions, i.e. moisture content of 90%, incubation time 4 days and pH=7.0. The corresponding inulinase/invertase (I/S) ratio (3.38) was also high, which indicates the inulolytic nature of the enzyme. Multiple correlation coefficients R for inulinase production and I/S ratio were 0.9995 and 0.9947, respectively. The R value very close to one indicates an excellent correlation between experimental and predicted results.

Algal lectins as promising biomolecules for biomedical research.

Lectins are natural bioactive ubiquitous proteins or glycoproteins of non-immune response that bind reversibly to glycans of glycoproteins, glycolipids and polysaccharides possessing at least one non-catalytic domain causing agglutination. Some of them consist of several carbohydrate-binding domains which endow them with the properties of cell agglutination or precipitation of glycoconjugates. Lectins are rampant in nature from plants, animals and microorganisms. Among microorganisms, algae are the potent source of lectins with unique properties specifically from red algae. The demand of peculiar and neoteric biologically active substances has intensified the developments on isolation and biomedical applications of new algal lectins. Comprehensively, algal lectins are used in biomedical research for antiviral, antinociceptive, anti-inflammatory, anti-tumor activities, etc. and in pharmaceutics for the fabrication of cost-effective protein expression systems and nutraceutics. In this review, an attempt has been made to collate the information on various biomedical applications of algal lectins.

Multifunctional iron bound lactoferrin and nanomedicinal approaches to enhance its bioactive functions.

Lactoferrin (Lf), an iron-binding protein from the transferrin family has been reported to have numerous functions. Even though Lf was first isolated from milk, it is also found in most exocrine secretions and in the secondary granules of neutrophils. Antimicrobial and anti-inflammatory activity reports on lactoferrin identified its significance in host defense against infection and extreme inflammation. Anticarcinogenic reports on lactoferrin make this protein even more valuable. This review is focused on the structural configuration of iron-containing and iron-free forms of lactoferrin obtained from different sources such as goat, camel and bovine. Apart for emphasizing on the specific beneficial properties of lactoferrin from each of these sources, the general antimicrobial, immunomodulatory and anticancer activities of lactoferrin are discussed here. Implementation of nanomedicinial strategies that enhance the bioactive function of lactoferrin are also discussed, along with information on lactoferrin in clinical trials.

Microbial lectins and their prospective mitogenic potential.

The binding of mitogenic lectins to the glycoconjugates on cell surface receptor triggers multitude of reactions involving different signal transduction pathways which ultimately results in cell proliferation. Since 1960 after the chance discovery of mitogenic property of lectins by Nowell, it has attracted more attention. The property of stimulation of mature lymphocytes aided the immunological investigations to study and analyse the mechanism of cell stimulation and division. Plant lectins are extensively studied for mitogenic activity and their widespread applications are also reported. During the past two decades, the study of biological properties of microbial lectins has increased tremendously. Their biological activities including mitogenic potential are equally applicable as that of plant lectins. The review mainly focuses on mitogenic potential of microbial lectins. It will provide a comprehensive view regarding distribution of this remarkable property among microbes including algae, fungi and bacteria, mechanisms and models of mitogenic stimulation, and also assays being employed to detect the mitogenic activity.

Pullulan in pharmaceutical and cosmeceutical formulations: A review.

Pullulan, an α-glucan polysaccharide, is colorless, odorless, non-toxic, non-carcinogenic, highly biocompatible, edible and biodegradable in nature. The long chains of glucopyranose rings in pullulan structure are linked together by α-(1 â†’ 4) and α-(1 â†’ 6) glycosidic linkages. The occurrence of both glycosidic linkages in the pullulan structure contributes to its distinctive properties. The unique structure of pullulan makes it a potent candidate for both pharmaceutical and cosmeceutical applications. In pharmaceuticals, it can be used as a drug carrier and in various dosage formulations. It has been widely used in drug targeting, implants, ocular dosage forms, topical formulations, oral dosage forms, and oral liquid formulations, etc. Pullulan can be used as a potential carrier of active ingredients and their site-specific delivery to skin layers for cosmeceutical applications. It has been extensively used in cosmeceutical formulations like creams, shampoo, lotions, sunscreen, facial packs, etc. The current review highlights applications of pullulan in pharmaceutical and cosmeceutical applications.

Downstream processing and structural confirmation of pullulan - A comprehensive review.

Pullulan is a microbial polymer, commercially produced from Aureobasidium pullulans. Downstream processing of pullulan involves a multi-stage process which should be efficient, safe and reproducible. In liquid-liquid separations, firstly cell free extract is separated. Cell biomass can be separated after fermentation either by centrifugation or filtration. Due to practically insolubility of pullulan in organic solvents, ethanol and isopropanol are the most commonly used organic solvents for its recovery. Pullulan can also be purified by chromatographic techniques, but these are not cost effective for the purification of pullulan. Efficient aqueous two-phase system can be used for the purification of pullulan. The current review describes the methods and perspectives used for solid-liquid separation, liquid-liquid separations and finishing steps for the recovery of pullulan. Techniques used to determine the structural attributes of pullulan have also been highlighted.

Current trends of lectins from microfungi.

Lectins are widespread in nature and have been isolated from plants, animals, microorganisms, and viruses. Although several lectins have been reported from microfungi, many more genera still remain unexplored and their physiological role is also uncertain. Microfungal lectins show wide disparity regarding their specificity to erythrocytes. Only a few lectins display specificity to particular human blood types. In addition, they also show agglutination to various animal erythrocytes. Many lectins from microfungi exhibit stringent specificity to animal glycoproteins, while a few have much more simplified sugar binding properties. The role of few microfungal lectins in host-parasite interactions, as storage proteins, and in growth and morphogenesis has been proposed. The current review focuses on an overview of lectins from microfungi, their specificity towards erythrocytes and carbohydrates, physicochemical characteristics, and their possible role and applications.

Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus MAK2 in a stirred tank reactor.

Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca(+)² caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5-l reactor in 34 h. An increase in sugar concentration (15 g l⁻¹) in the fermentation medium further increased naringinase production (8.9 IU ml⁻¹) in the bioreactor. Thus, availability of naringinase renders it attractive for potential biotechnological applications in citrus processing industry.

Pullulan in biomedical research and development - A review.

Pullulan is an imperative microbial exo-polymer commercially produced by yeast like fungus Aureobasidium pullulans. Its structure contains maltosyl repeating units which comprises two α-(1 â†’ 4) linked glucopyranose rings attached to one glucopyranose ring through α-(1 â†’ 6) glycosidic bond. The co-existence of α-(1 â†’ 6) and α-(1 â†’ 4) glycosidic linkages endows distinctive physico-chemical properties to pullulan. It is highly biocompatible, non-toxic and non-carcinogenic in nature. It is extremely resistant to any mutagenicity or immunogenicity. The unique properties of pullulan make it a potent candidate for biomedical applications viz. drug delivery, gene delivery, tissue engineering, molecular chaperon, plasma expander, vaccination, etc. This review highlights the potential of pullulan in biomedical research and development.

Mushroom lectins: current status and future perspectives.

Lectins are nonimmune proteins or glycoproteins that bind specifically to cell surface carbohydrates, culminating in cell agglutination. These are known to play key roles in host defense system and also in metastasis. Many new sources have been explored for the occurrence of lectins during the last few years. Numerous novel lectins with unique specificities and exploitable properties have been discovered. Mushrooms have attracted a number of researchers in food and pharmaceuticals. Many species have long been used in traditional Chinese medicines or functional foods in Japan and other Asian countries. A number of bioactive constituents have been isolated from mushrooms including polysaccharides, polysaccharopeptides, polysaccharide-protein complexes, proteases, ribonucleases, ribosome inactivating proteins, antifungal proteins, immunomodulatory proteins, enzymes, lectins, etc. Mushroom lectins are endowed with mitogenic, antiproliferative, antitumor, antiviral, and immune stimulating potential. In this review, an attempt has been made to collate the information on mushroom lectins, their blood group and sugar specificities, with an emphasis on their biomedical potential and future perspectives.

Further screening of Aspergillus species for occurrence of lectins and their partial characterization.

Fifteen species of Aspergillus were screened for occurrence of lectins. Nine of them (A. sydowii, A. candidus, A. allahabadi, A. terricola, A. ficuum, A. sparsus, A. carneus, A. pulvinus and A. aculeatus) were found to possess lectin activity. None of the species elaborated lectin in culture supernatant. All the lectins agglutinated rat, pig and rabbit erythrocytes. A. sydowii, A. candidus, A. allahabadi, A. terricola, A. ficuum, A. sparsus, A. carneus and A. aculeatus lectins agglutinated all human type erythrocytes equally, while A. pulvinus lectin specifically agglutinated human type A and O erythrocytes. Neuraminidase and protease treatment to erythrocytes substantially augmented lectin titres manyfold. Lectins showed specificity to mucin and asialofetuin and all of them were specific to L-arabinose except that of A. carneus. Lectins from A. sydowii, A. ficuum, A. sparsus and A. carneus displayed remarkable specificities to D-xylose. Maximum lectin activity was expressed by 11 day old cultures of A. sydowii (titre 32), A. ficuum (titre 64) and A. sparsus (titre 1024). Lectins from A. aculeatus, A. candidus and A. terricola were expressed by 7-10 days, 6-9 days and 5-11 days old cultures, respectively. A. allahabadi cultures exhibited maximum lectin activity (titre 32) after 8-10 days of cultivation. A. carneus and A. pulvinus expressed optimal titres of 32 and 8, respectively on the 9th day.

Functionalization of multiwalled carbon nanotubes for enzyme immobilization.

A simple strategy for enzyme immobilization onto homo- and hetero-functionalized multiwalled carbon nanotubes (MWCNTs) is described. Homo-functionalization of MWCNTs can be carried out using an amino-silane compound, i.e., 3-aminopropyl-triethoxysilane (APTES). It is an important silane coupling agent, which promotes interfacial behavior of nanomaterials. Whereas, hetero-functionalization of MWCNTs can be performed using APTES and cross-linking agent glutaraldehyde (GA). Key parameters for the immobilization of an enzyme on MWCNTs are selection of an efficient functionalizing agent, sonication of MWCNTs, enzyme load and enzyme coupling time. The optimal level of these factors is very important to obtain high specific activity and immobilization efficiency of the developed immobilized biocatalyst. Both homo- and hetero-functionalized MWCNTs can be used to develop an efficient immobilized biocatalyst having good operational stability. However, hetero-functionalized MWCNTs are more potent candidate for enzyme immobilization. The present methodology provides very efficient approach for enzyme immobilization on a versatile support to achieve high enzyme selectivity and operational stability for various industrial applications.

Purification and characterisation of a xylose-specific mitogenic lectin from Fusarium sambucinum.

A xylose-specific intracellular lectin, showing hemagglutination only with rabbit erythrocytes was purified from mycelium of Fusarium sambucinum which was designated as FSL. An array of anion exchange chromatography on Q-Sepharose and gel-exclusion chromatography on Sephadex G-100 resulted in 84.21% yield and 53.99-fold purification of lectin with specific activity of 169.53 titre/mg. Molecular weight of FSL determined by SDS-PAGE was 70.7 kDa, which was further confirmed by gel-exclusion chromatography. Native-PAGE analysis of FSL showed its monomeric nature. FSL was observed to be a glycoprotein containing 2.9% carbohydrate. Hapten inhibition profile of FSL displayed its strong affinity towards D-xylose (MIC 1.562 mM), L-fucose (MIC 6.25 mM), D-mannose (MIC 3.125 mM), fetuin (MIC 15.62 µg/mL), asialofetuin (MIC 125 µg/mL) and BSM (MIC 3.125 µg/mL). Affinity of FSL towards xylose is rare. FSL was found stable over a pH range 6.0-7.5 and upto 40 °C temperature. Hemagglutination activity of FSL remained unaffected by divalent ions. Lectin concentration of 5 µg/mL was found sufficient to stimulate proliferation of murine spleen cells and its concentration 75 µg/mL exhibited highest mitogenic potential. FSL exhibited maximum mitogenic stimulatory index of 14.35. The purification, characterisation and mitogenicity of F. sambucinum lectin has been reported first time.

Mushroom lectins in biomedical research and development.

Lectins are unique biorecognition proteins which recognize and interact with various cell surface carbohydrates/glycoproteins. These ubiquitous molecules are involved in various cell-cell interactions and can be exploited to analyze cell surface associated interactions and biological functions. Amongst fungi, lectins have been extensively explored from mushrooms as compared to microfungi or yeasts. Lectins from basidiomycetes have diverse features in terms of their physico-chemical characteristics and carbohydrate specificity. A plethora of lectins from genera Aleuria, Agrocybe, Boletus, Pleurotus, Russula, Schizophyllum, Volvariella, etc. exhibit potential applications in biomedical field. The current review summarizes the potential sources and characteristics of mushroom lectins. Potential involvement of mushroom lectin as anticancer, mitogenic, antiviral, antimicrobial, antioxidant and therapeutic agents has been discussed.

Carbamoylethyl locust bean gum: Synthesis, characterization and evaluation of its film forming potential.

The synthesis of carbamoylethyl locust bean gum (CLBG) was optimized using Plackett-Burman design. The generated model showed high significance (p < 0.05) to all the response variables which justifies the authenticity of the designed model. The optimal conditions i.e. acrylamide (5.12 mM), sodium hydroxide (3.00 mM), reaction temperature (50.97 °C) and reaction time (2.00 h) supported maximum -CONH2 content (5.44%), -COOH content (3.04%), degree of substitution (0.85) and product yield (7.25%, w/w). Carbamoylethylation of locust bean gum (LBG) involved substitution of its hydroxyl (-OH) moieties with amide group (-CH2CH2CONH2). FTIR and NMR spectroscopy confirmed the addition of amide group to CLBG. Scanning electron microscopy assured the slight rough surface of CLBG particles. Differential scanning calorimetry showed that carbamoylethylation of LBG lowered its melting temperature range (205.60-272.45 °C). However, the amorphous nature, non-Newtonian flow and shear-thinning behaviour of pure LBG were retained in CLBG. Further, CLBG films prepared with glycerol (1%, w/w, plasticizer) showed partially smooth surface and have clear transversal cross-sections. CLBG-glycerol films were highly water resistant and almost transparent. Further, CLBG-glycerol films showed good tensile strength (18.55 ± 0.02 MPa) and higher percentage elongation (6.11 ± 0.01%). Water vapor transmission rate of CLBG-glycerol film was quite lower (0.211 ± 0.001 g.mm/h.m2.kPa) which verified its higher resistance towards water.

Screening of Penicillium species for occurrence of lectins and their characterization.

Out of 15 Penicillium species screened for lectin activities, P. griseofulvum and P. thomii were found to possess mycelial lectin activity. None of the species displayed extracellular or cell surface-bound lectin activity. Both species agglutinated rabbit erythrocytes. P. griseofulvum lectin showed specificity to human type O erythrocytes. While P. thomii lectin specifically agglutinated human type A erythrocytes. Highest lectin activities from P. thomii and P. griseofulvum were expressed after 8 and 7 days of growth, respectively. Lectins from both the species displayed a high binding affinity to chondroitin-6-sulphate, mucin, asialofetuin, D-sucrose, and D-trehalose. Ammonium sulphate at 50% saturation yielded 80% of the total lectin activity. Dialysis and ultrafiltration of the precipitates resulted in 1.79 and 3.46 fold purification of P. griseofulvum and P. thomii lectins, respectively. Both lectins showed pH optima between 7.0-8.0 and were stable near the neutral pH after 2 h. P. thomii lectin exhibited optimal activity at 35-40 degrees C, and P. griseofulvum lectin at 30-40 degrees C. P. thomii lectin showed a complete loss of activity above 40 degrees C, P. griseofulvum lectin was stable at or below 35 degrees C.

Optimization of culture conditions and characterization of a new lectin from Aspergillus niger.

Lectin activity was determined on solidified medium containing agar and in broth cultures of Aspergillus niger. The fungus was found to express 16 times higher activity in broth cultures, when grown in a medium adjusted to pH 5.5 at 30°C under stationary condition. Lectin activity was found to be expressed by 6-day-old mycelial cultures with maximum activity being expressed on 9th day of incubation. The crude lectin (total titer 1280) was found to be precipitated at 50% saturation of ammonium sulphate with 2.4-fold purifi cation and 83% yield in the precipitate. The partially purifi ed lectin was found to agglutinate all human, rat, mice and pig erythrocytes. It was found to have a strong binding affinity to mucin, asialofetuin and inulin.