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.

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.

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.

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.

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.

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.

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.

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.

Investigating aqueous phase separation of pullulan from Aureobasidium pullulans and its characterization.

In the present investigation, polyethylene glycol (PEG 6000) was used for downstream processing of pullulan from Aureobasidium pullulans by aqueous phase separation (APS) technique. The cell-free broth was processed with PEG solution (10-35%, w/w) and pullulan formed a clear separate phase with all concentrations of PEG i.e. pullulan in lower phase and PEG solution along with impurities in upper phase. Maximum pullulan recovery from cell-free broth was obtained by PEG 25% (w/w). The sample handling in APS technique is quite easier due to lesser volume of PEG used in comparison to organic solvent precipitation i.e. minimum required ratio of cell-free broth to PEG for maximum pullulan yield was 0.5:1 (PEG:cell-free broth). Additionally, APS technique was found to be temperature independent. Further, structural attributes of pullulan recovered by APS was confirmed by FTIR, NMR and TLC. This is the first report on downstream processing of pullulan using PEG based aqueous solution.

Structural aspects and biomedical applications of microfungal lectins.

Lectins are unique biorecognition molecules that identify cell surface carbohydrates/glycoproteins and play significant role in various interactions. They are ubiquitous glycan-specific proteins/glycoproteins of non-immune origin. Amongst microfungi, lectins have been widely reported from aspergilli, penicilli, Fusarium sp., etc., however a plethora of genera still remains unexplored. Microfungal lectins have wide diversity in their haemagglutination and carbohydrate specificity. They also exhibit great variations in their structural organization which influences lectin-glycan interactions. The present review summarizes the sources, characteristics and structural diversity of microfungal lectins. Prospective biomedical applications of microfungal lectins as anticancer, mitogenic, immunomodulatory, antioxidant and therapeutic agents have been discussed extensively.

Pullulan production from agro-industrial waste and its applications in food industry: A review.

Pullulan is a microbial exopolysaccharide produced from Aureobasidium pullulans by submerged fermentation of a medium supplemented with carbon, nitrogen and other essential nutrients. These nutrients are expensive which increase the cost of pullulan production. The requirement of alternative cost-effective substrates for pullulan production is a prerequisite. Agro-based industries generate a large volume of solid/liquid waste and its accumulation generates a severe environmental impact. These wastes are composed of carbohydrates, proteins and other constituents, and can be used as substrates for the development of low-cost processes for the production of various microbial products. This could be a good environmental friendly waste management system. Pullulan production from agro-industrial wastes can be carried out by both submerged and solid-state fermentation by A. pullulans. Owing to its unique properties, pullulan has wide applications in many food-based industries. This review highlights pullulan production from agro-industrial wastes and potential applications of pullulan in various food industries.

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.

Purification and characterization of a heterodimeric mycelial lectin from Penicillium proteolyticum with potent mitogenic activity.

In the present study, Penicillium proteolyticum lectin was purified by DEAE-Sepharose chromatography followed by Sephadex G-100 gel filtration chromatography. A sequence of ion-exchange and gel filtration chromatography resulted in 52.30 fold purified lectin with a high yield of 84.21%. The purified P. proteolyticum lectin is a glycoprotein with 2.83% linked carbohydrates. A single band in Native-PAGE, whereas two bands (25.1 kDa and 22.9 kDa) in SDS-PAGE confirmed the heterodimeric nature of the purified lectin. The apparent molecular weight (48 kDa) of P. proteolyticum lectin was further confirmed by gel filtration analysis. Heterodimeric P. proteolyticum lectin was stable within a pH range of 6.5-7.5 and upto a temperature of 30 °C. The lectin activity was strongly inhibited by complex glycoproteins and denaturants. Metal ions are not required for agglutination activity of purified lectin. The lectin showed significant mitogenic response towards mice splenocytes. It exhibited highest mitogenic activity at a concentration of 50 µg/mL. This is a first report on characteristics of purified P. proteolyticum lectin having potent mitogenic potential.

Fructose production from inulin using fungal inulinase immobilized on 3-aminopropyl-triethoxysilane functionalized multiwalled carbon nanotubes.

The main objective of the present work was to modify multiwalled carbon nanotubes (MWCNTs) using 3-aminopropyl-triethoxysilane (APTES) to generate amino-terminated surfaces for inulinase immobilization, which can be further used for fructose production. CCRD of response surface methodology was used for optimization of inulinase immobilization on MWCNTs. At optimized parameters (APTES concentration 4%; sonication time 4 h; enzyme coupling time 1.5 h and enzyme load 15 IU), maximal inulinase activity and immobilization yield was 60.7% and 74.4%, respectively. Immobilized inulinase showed same pH optima of free enzyme, while an elevation in temperature optima to 60 °C was observed after its immobilization. Immobilized inulinase also shown enhancement in pH stability and thermostability. Overall, 4.54-fold rise in half-life of inulinase was detected after immobilization at 60 °C. Km and Vmax of inulinase decreased after immobilization. Immobilized inulinase preserved 28% of its residual activity after 10 consecutive batch cycles of inulin hydrolysis for the production of fructose.

Purification and characterization of two isoforms of exoinulinase from Penicillium oxalicum BGPUP-4 for the preparation of high fructose syrup from inulin.

Two exoinulinases, Exo-I and Exo-II from the culture broth of Penicillium oxalicum BGPUP-4 was purified using three-step purification method i.e., isopropanol precipitation, Q-Sepharose and Sephadex G-100 column chromatography. The molecular weight of Exo-I and Exo-II was determined to be 64.85 kDa and 32.54 kDa, respectively using MALDI-TOF. Exo-I and Exo-II showed high specificity for inulin and their respective Vmax/Km ratio was 3.74 and 7.20. Besides, both the inulinases also displayed specificity for lactose, sucrose and raffinose. Exo-I and Exo-II were stable at a pH range of 4.0-8.0 with pH optima 5.0. Optimal temperature for both the inulinases was 55 °C, and both the isoforms retained approximately 50% of their activity up to 70 °C. Ag+, Hg2+, Ba2+, Cu2+ and Ca2+ ions shown stimulatory effect on inulinases activity, while Fe2+, Mn2+, Co2+and EDTA completely inhibited enzyme activity. Purified enzyme was successfully used for the preparation of high fructose syrup from inulin.

Carbamoylethyl pullulan: QbD based synthesis, characterization and corneal wound healing potential.

Corneal injuries are the major problem arises these days due to excessive use of mobile phone, TV, environment pollution, etc. Thus, a need is to evaluate materials having good corneal wound healing potential. To achieve this goal, pullulan was derivatized using carbamoylethylation and 24 factorial design has been found a good approach for the optimization of synthesis process of carbamoylethyl pullulan (CMEP). The design expert software applied for this purpose suggested significance of the generated model. ATR-FTIR, NMR and XRD spectral attributes validated the structure of CMEP. Interestingly, derivatization of pullulan to CMEP has increased its ΔH from 171.552 J/g to 203.5 J/g and decreased the endothermic transition from 323.44 °C to 248.67 °C. Further, rheological behavior suggested enhanced viscosity after carbamoylethylation indicating increase in H-bonding that further participate in the enhancement of corneal adhesive strength. The CMEP possessed high corneal wound healing property that was associated with its high corneal adhesive strength and oxygen neutralizing capacity. Thus, the findings suggested overwhelming influence of CMEP as a corneal wound healing agent.

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.

Purification and characterization of a mitogenic lectin from Penicillium duclauxii.

Lectins are proteins/glycoproteins of non-immune origin which interact specifically and non-covalently with carbohydrate moieties on the cell surface. In this study, a lectin was purified from Penicillium duclauxii by ion-exchange chromatography on DEAE-Sepharose and gel filtration chromatography on a Sephadex G-100 column. An overall recovery of 94.11% and 60-fold purification was achieved. The purified lectin had a molecular weight of 54.9 kDa and was found to be heterogeneous as revealed by double band of sub-units with molecular mass of 21.13 kDa and 33.26 kDa, under reducing conditions. It is a glycoprotein with carbohydrate content of 3.95%. Lectin induced haemagglutination of erythrocytes was inhibited strongly by glycoproteins such as bovine submaxillary mucin, porcine stomach mucin and fetuin. The maximum haemagglutinating activity of P. duclauxii lectin was maintained after incubation at a temperature and pH range of 20-35 °C and 6.0-8.0, respectively. The haemagglutinating activity of P. duclauxii lectin was unaffected by EDTA and various metal ions. The purified P. duclauxii lectin exhibited maximum mitogenic activity towards mouse splenocytes at a concentration of 75 µg/mL. This manuscript reports a novel lectin from P. duclauxii with potent mitogenic activity towards mouse splenocytes.

Sequential statistical optimization of lactose-based medium and process variables for inulinase production from Penicillium oxalicum BGPUP-4.

A statistical tool of response surface methodology was used sequentially to optimise lactose-based medium and process variables for inulinase production from Penicillium oxalicum BGPUP-4. Two-level CCRD with four variables, for each design, was used for the optimization study. The independent variables: lactose 1-3%, NH4H2PO4 0.2-0.5%, NaNO3 0.2-0.5%, pH 5.0-7.0 (design-1); temperature 25-35 °C, incubation time 4.0-6.0 days, inoculum size 1.0-3.0 mycelial agar discs and agitation 100-200 rpm (design-2) were selected for the present investigation. The optimised medium variables (lactose 3.70%, NH4H2PO4 0.35%, NaNO3 0.35% and pH 6.0) produced 44.44 (IU/ml) and 0.38 (g dry wt./50 ml) of inulinase and biomass yield, respectively. Thereafter, the optimization of process conditions (temperature 25 °C, incubation time 5 days, inoculum size 2 mycelial agar discs and agitation 150 rpm), increased the inulinase production 50.45 (IU/ml) and biomass yield 0.26 (g dry wt./50 ml). The good agreement between experimental and predicted values in both the designs, the coefficient of determination (R2 ) greater than 0.90 and very close to 1.0 shows an appropriate fitness of the polynomial quadratic models.