Pullulan Research Articles

Valorisation of chestnut spiny burs and roasted hazelnut skins extracts as bioactive additives for packaging films

The exploitation of agri-food by-products is a pertinent strategy in the context of the implementation of the circular economy concept in the agri-food industry. Therefore, the present work aimed to produce bioactive pullulan-based films loaded with polyphenolic-rich extracts from chestnut spiny burs (CSB, Castanea sativa Miller.) and roasted hazelnut skins (RHS, Corylus avellana L.). The hydroalcoholic extracts obtained from the CSB and RHS by-products were separately combined (1 %, 5 %, and 10 % w/w) with the filmogenic pullulan (PL) exopolysaccharide to fabricate flexible and bioactive films via simple solvent casting technique. The films present thermal stability up to 200 °C, good mechanical performance with Young’s modulus values higher than 2.6 GPa, and UV-light barrier properties. Furthermore, these flexible PL-based films exhibit a minimum antioxidant activity of ca. 94 % (DPPH scavenging activity) for the film composed of only 1 % of hydroalcoholic extracts (w/w relative to PL, i.e. 192 μg of extract per cm2 of film). Both sets of films were tested for antibacterial activity towards Staphylococcus aureus (a Gram-positive bacterium) and the results demonstrate antibacterial effectiveness with the increasing content of both hydroalcoholic extracts, reaching a maximum of 4–log CFU mL–1 reduction for the PL/RHS_10 film after 6 h. All the data evidences the potential applicability of these PL-based films containing hydroalcoholic extracts from agri-food by-products for the development of sustainable films for active food packaging.

Pullulan microneedle patches for the efficient transdermal administration of insulin envisioning diabetes treatment

The present study reports the fabrication of dissolvable microneedle (MN) patches using pullulan (PL), a water-soluble polysaccharide with excellent film-forming ability, for the transdermal administration of insulin, envisioning the non-invasive treatment of diabetes. PL MNs patches were successfully prepared by micromoulding and revealed good thermal stability (Tdmax = 294 °C) and mechanical properties (>0.15 N needle−1), penetrating skin up to 381 μm depth, as revealed by in vitro skin tests. After application into human abdominal skin in vitro, the MNs dissolved within 2 h releasing up to 87% of insulin. When stored at 4, 20 and 40 °C for 4 weeks, insulin was able to retain its secondary structure, as shown by circular dichroism spectropolarimetry. The prepared PL MNs were non-cytotoxic towards human keratinocytes, being suitable for skin application. These findings suggest that PL MNs have potential to deliver insulin transdermally, thus avoiding its subcutaneous administration.

Fabrication and characterization of pullulan-based nanocomposites reinforced with montmorillonite and tempo cellulose nanofibril

Nanocomposite film of pullulan (PULL), tempo cellulose nanofibrils (TOCNs) and, montmorillonite clay (MMT) were prepared using a solution casting method with aqueous solutions. X-ray diffraction data revealed that exfoliated MMT nanoplatelets are distributed within the PULL/TOCNs/MMT film structure. Fourier-transform infrared results revealed that there might be interactions among the TOCNs, MMT and PULL matrix led to improved tensile strength, thermal stability, water barrier properties, and decrease moisture susceptibility while maintained reasonable transparency and biodegradability of the ternary PULL nanocomposites. These excellent properties of the nanocomposites clearly indicate towards a new strategy for developing high-performance PULL-based nanocomposites by using two different types of fillers with various geometric shapes and aspect ratio. This kind of ternary nanocomposite film can be broadly used in food packaging and protection as a green and biodegradable film.

Pullulan as a potential enhancer of Lactobacillus and Bifidobacterium viability in synbiotic low fat yoghurt and its sensory quality

Yoghurt acts as a perfect delivery system for probiotic bacteria and some functional food ingredients, thereby enhancing their viability. Pullulan (PUL), a natural exopolysaccharide produced by yeast Aureobasidium pullulans is a low-calorie food ingredient and dietary fibre with potential prebiotic properties. The main aim of the present study was to investigate the effect of 0.5%–2.0% PUL addition on pH, sensory quality and viability of the yoghurt starter and probiotic bacteria in low-fat yoghurt during 28 days of storage. PUL addition enhanced the viability of Bifidobacterium and Lactobacillus, while it did not affect S. thermophilus count. The protective effect of PUL increased with concentration and was the highest for 1.5 and 2.0% PUL supplementation. Incorporation of PUL at 1.5% and 2.0% prolonged survival of B. animalis subsp. Lactis at a concentration above the therapeutic level (<10 6 cfu/mL) for the entire storage period. All the products showed a decrease in pH during the storage period. Further, the sensory analysis revealed that PUL incorporation improved thickness, smoothness, and viscosity of low-fat yoghurts, but decreased the intensity of milk taste and aroma, contributing to a decrease in overall acceptability.

Preparation and characterization of a novel porous whey protein concentrate/pullulan gel induced by heating for Cu2+ absorption

Whey protein concentrate (WPC) and pullulan (PUL) were used to prepare a hydrogel (WPC/PUL) as a novel Cu2+ adsorbent. The scanning electron microscopy, rheology, zeta-potential, sulfhydryl groups, surface hydrophobicity, infrared spectroscopy and thermal gravimetric analysis measurements were used to characterize WPC/PUL. The results showed the mechanism of gel formation may depend on the combination of electrostatic interaction, disulfide bond, hydrophobic interaction and hydrogen bond. The drying kinetic test was taken, and the drying process of WPC/PUL followed Page Model and the activation energy was 56.73 kJ/mol. Furthermore, WPC/PUL was used as a Cu2+ adsorbent and the absorption can reach more than 98% of the equilibrium adsorption (Qe) after shaking in Cu2+ solution for 10 min and Qe based on the pseudo-second-order kinetic model was 81.66 mg/g. Because the materials were safe and non-toxic, WPC/PUL could be used as a Cu2+ adsorbent during the food industry production process in the future.

Effects of κ-carrageenan on pullulan’s rheological and texture properties as well as pullulan hard capsule performances

κ-Carrageenan (κ-Ca) is often used to facilitate gelling of aqueous solutions of polysaccharides. However, studies on its effects on pullulan’s rheological and texture properties and pullulan (PUL) hard capsule performances have rarely been reported. Herein, effects of κ-Ca on PUL solutions, hydrogels, films and hard capsules were investigated. It was found that the gelling temperature of 15 % (w/w) PUL solutions with 0.07 % KCl increased from 34 ℃ to 42 ℃ as the concentration of κ-Ca increased from 0.6 % to 1.2 %, and the gelling temperature rose from 25 ℃ to 37 ℃ by adding a small amount of KCl (0.07 %) for 15 % PUL solutions with 0.9 % κ-Ca. As the κ-Ca concentration increased, hardness, fracturability and adhesiveness rose for PUL gels and tensile stress increased for PUL films. PUL capsules could be easily prepared by the aid of κ-Ca, and performances of capsules could be adjusted by changing the amount of κ-Ca.

Exploring the role of pullulan in the process of potato starch film formation

It is well known that mixing pullulan (PUL) with starch can overcome the poor physical properties of pure starch based film. However, the mechanism of improvement of physical properties of starch film with PUL is yet to be clearly understood. The objective of this study is to investigate the effect of PUL in potato starch (PS) condensed matter solution and illustrate the mechanism improvement of physical properties of PS film by mixing with PUL. The moisture content of PS-PUL film decreased by 18.61 % because PS-PUL film chain-structure was denser than that of PS film. Based on the analysis of condensed matter solution, PUL could reduce the speed of PS chain movement and increase the flexibility of PS rigidity chains. Thus, tensile strength of PS-PUL film decreased by 20.34 % and elongation at break of PS-PUL film increased by 23.45 %. An increase in bending endurance by 49.51 % also illustrates that PUL improved the chain flexibility in the process of composite film formation. The present research provides a novel research route to understand the mechanism of hydrophilic colloids affecting the physical properties of starch film at molecular level. Also, the work provides a theoretical basis for the establishment of a correlation system between the microstructure and macroscopic properties.

Pullulan-based dissolving microneedle arrays for enhanced transdermal delivery of small and large biomolecules

One specific technological advance in transdermal drug delivery is the development of dissolving microneedles (DMNs), which efficiently deliver therapeutics through a rapid dissolution of polymers after penetration into the skin. However, there is a limited range of water soluble, biodegradable polymers that can be used to manufacture DMN. Here, we report for the first time, the preparation and characterisation of a DMN system from the carbohydrate biopolymer, pullulan (PL). PL gels, of varying concentration, were studied for viscosity, film formation properties, and subsequently, microneedle formation. Model molecules and protein/peptide were loaded into PL DMN and characterised. The stability of model biomolecules, such as FITC-BSA and insulin, following DMN manufacture were assessed using circular dichroism. Ex-vivo porcine skin permeation studies using Franz diffusion cell apparatus for Flu-Na and FITC-BSA loaded PL-DMN were conducted. This study demonstrates that PL DMNs may serve as a promising tool for efficient transdermal drug delivery.

Release kinetics and antimicrobial properties of the potassium sorbate-loaded edible films made from pullulan, gelatin and their blends

In this study, the effect of increasing concentrations (0, 0.5, 1, 2% w/w) of potassium sorbate (KS) on the release and antimicrobial properties of edible films based on pullulan (PUL), gelatin (GEL) and their blends (75/25, 50/50, and 25/75) was investigated. The 75/25 and 50/50 polymer mixtures separated into GEL-rich microspheres. The biggest microscopic domains were formed at equal amounts of the polymers. As verified by Fourier-transform infrared spectroscopy, the blending did not cause any chemical changes within the functional groups of PUL and GEL. Release of KS from the PUL- and GEL-rich carriers was primarily due to dissolution and swelling of the polymer matrix, respectively. Consequently, basing on time required for 50% release, the GEL100 film offered at least 2.5 times slower release of KS compared to the PUL100 carrier. The gradual replacement of PUL with GEL tended to decrease KS release rate. The large microspheres of the PUL50/GEL50 films gave release profile similar to the blend system with higher GEL content. The KS diffusion mechanism was found to be Quasi-Fickian. The Weibull models were suitable for the efficient simulation of KS release from the films. At the lowest KS concentration (0.5%), the PUL-rich film-forming solutions (FFSs) tended to have the weakest antibacterial activity, likely due to their high pH values, as well as PUL-KS hydrogen bonding interactions. The FFSs with high levels of KS (2%) exhibited strong inhibitory effects on A. alternata, B. cinerea, P. notatum, S. cerevisiae, and K. apiculata.

Effect of pullulan on oil absorption and structural organization of native maize starch during frying

The oil-absorption behavior of native maize starch (NMS) during frying was investigated in the presence or absence of pullulan (PUL) using a LF-NMR method. The morphology, long-range order, short-range order, and thermal properties of fried NMS-PUL mixtures were further evaluated using SEM, XRD, ATR-FTIR, and DSC, respectively. Pullulan addition significantly reduced the oil content of the fried starch samples: 0.395, 0.310, 0.274, and 0.257 g/g in NMS with addition of 0, 1, 3, and 5% PUL, respectively (p < 0.05). SEM analysis showed that the intact granular morphology of the starch granules was preserved upon addition of pullulan. The XRD, FTIR, and DSC results showed that pullulan protected both the short-range double helices and long-range crystalline structure of the granules during frying. As a result, fried NMS-PUL mixtures were denser than fried NMS, thus inhibiting oil absorption during frying. These results may have important implications for creating healthier reduced-fat fried food products.

Preparation, properties, and structural characterization of β-glucan/pullulan blend films

This study investigates the physico-mechanical and structural properties of β-glucan (BG)/pullulan (PUL) composite edible films successfully prepared with 0–0.3 g of BG. Results demonstrated that BG addition significantly increases the elongation at break (p < 0.05), tensile strength, and water dissolution time of the resulting films. The transparency of the 0.2PUL:0.1BG film and the oxygen barrier property of the 0.15PUL:0.15BG film decreased remarkably compared with those of the plain films (0.3PUL:0BG and 0PUL:0.3BG) and other composite films (p < 0.05). FTIR indicated hydrogen bonding interactions between PUL and BG molecules, and microstructural observations showed that aggregated BG is homogeneously dispersed in the PUL continuous matrix. Among the films tested, the thermal stability of the 0.15PUL:0.15BG film was the best. A PUL:BG mixing ratio of 0.15:0.15 is thus suggested to provide the best film properties. This research offers an alternative method to improve PUL-based edible films.

Effect of pullulan on molecular chain conformations in the process of starch retrogradation condensed matter

Starch retrogradation is an inevitable process in the natural state caused by motion of starch molecular chains. However, traditional starch retrogradation research methods cannot explain this process from the aspect of chain motion. Therefore, the objective of this study was to investigate the effect of pullulan (PUL) on potato starch (PS) retrogradation and analyze this using condensed matter theory. The entanglement concentration (ce) was determined by Ubbelohde viscometer. The ce values of PS and PS-PUL were 0.22 and 0.32 g/dL, respectively. Small-angle X-ray Scattering (SAXS) was used to obtain the radius of gyration (Rg) values and investigate conformation evolution of PS and PS-PUL in different solution concentrations ranging from extremely dilute to concentrated. The Rg values of PS increased by 38.7% upon transition from single chain (0.001 g/dL) to an entanglement state (0.22 g/dL) and the percent reduction of Rg was only 3.3% from entanglement state (0.22 g/dL) to stabilization (1 g/dL). The percent increase of Rg values of PS-PUL reached to 34.7% from extremely dilute (0.001 g/dL) to concentrated (1 g/dL) solutions. The trend of Rg indicated that ce played a major role in starch retrogradation. Dynamic light scattering (DLS) was used to acquire the hydrodynamic radius (Rh) which was a significant parameter to calculate form factor (ρ) by the ratio: ρ = Rg/Rh. The ρ values of PS-PUL were generally lower than those for PS. This result illustrated that PUL delayed the process of starch retrogradation by increasing flexibility of PS molecular chain. Furthermore, the ρ value of PS-PUL was higher than PS at 0.22 g/dL (ce of PS). This result illustrated that entanglement could decreased flexibility of molecular chain. The present research provides a novel research route for exploring the influence of hydrocolloids on starch retrogradation from the viewpoint of chain motion.

Chitosan/Glycosaminoglycan Scaffolds: The Role of Silver Nanoparticles to Control Microbial Infections in Wound Healing

Cutaneous wounds represent a major issue in medical care, with approximately 300 million chronic and 100 million traumatic wound patients worldwide, and microbial infections slow the healing process. The aim of this work was to develop electrospun scaffolds loaded with silver nanoparticles (AgNPs) to enhance cutaneous healing, preventing wound infections. AgNPs were directly added to polymeric blends based on chitosan (CH) and pullulan (PUL) with hyaluronic acid (HA) or chondroitin sulfate (CS) to be electrospun obtaining nanofibrous scaffolds. Moreover, a scaffold based on CH and PUL and loaded with AgNPs was prepared as a comparison. The scaffolds were characterized by chemico–physical properties, enzymatic degradation, biocompatibility, and antimicrobial properties. All the scaffolds were based on nanofibers (diameters about 500 nm) and the presence of AgNPs was evidenced by TEM and did not modify their morphology. The scaffold degradation was proven by means of lysozyme. Moreover, the AgNPs loaded scaffolds were characterized by a good propensity to promote fibroblast proliferation, avoiding the toxic effect of silver. Furthermore, scaffolds preserved AgNP antimicrobial properties, although silver was entrapped into nanofibers. Chitosan/chondroitin sulfate scaffold loaded with AgNPs demonstrated promotion of fibroblast proliferation and to possess antimicrobial properties, thus representing an interesting tool for the treatment of chronic wounds.

Platelet lysate loaded electrospun scaffolds: Effect of nanofiber types on wound healing

In healthy individuals, wound healing is a highly efficient process. However, interruptions of normal healing give rise to chronic wounds, characterized by inflammation with impaired angiogenesis and re-epithelialization. The aim of this work was the design and the development of electrospun nanofibrous scaffolds based on sodium alginate (SA) and pullulan (PUL) and loaded with human platelet lysate (PL) intended for skin reparation, to take the advantage of nanofibrous scaffolds (with improved physical structure) and of SA as biopolymer.Two preparation approaches have been used to load PL in the scaffolds: as component of the PUL/SA matrix, to be electrospun, or as coating component, to cover the previously prepared electrospun PUL based membranes. A preformulation study to assess pullulan entanglement concentration and alginate or citric acid critical concentration, to obtain electrospun nanofibers, has been performed. The preparation process allowed to obtain insoluble systems starting from aqueous solutions and these were able to act as scaffolds for tissue engineering with suitable mechanical properties and PL release.PL loading in PUL/SA matrix nanofibers did not substantially modify the nanofiber morphology before crosslinking, while the crosslinking process, in presence of PL, determined less sharp nanofibers probably due to an increase in hydrophilicity caused by PL proteins. On the contrary, the coated nanofibers showed an increase in diameters due to PL loading.The two different approaches affected the fiber dimension and scaffold elasticity, especially for PL loaded systems. Anyhow, these differences were not crucial for fibroblast adhesion and proliferation which were mainly influenced by PL loading.In particular, fibroblasts presented different conformation and orientation mainly due to the presence of PL. This caused a cell random orientation compatible to a fibroblast-to-myofibroblast transition that could enhance wound healing.

Effect on Viability of Microencapsulated Lactobacillus rhamnosus with the Whey Protein-pullulan Gels in Simulated Gastrointestinal Conditions and Properties of Gels.

Lactobacillus rhamnosus GG (LGG) has low resistance to low pH and bile salt in the gastrointestinal juice. In this study, the gel made from whey protein concentrate (WPC) and pullulan (PUL) was used as the wall material to prepare the microencapsulation for LGG protection. The gelation process was optimized and the properties of gel were also determined. The results showed the optimal gel was made from 10% WPC and 8.0% PUL at pH 7.5, which could get the best protective effect; the viable counts of LGG were 6.61 Log CFU/g after exposure to simulated gastric juice (SGJ) and 9.40 Log CFU/g to simulated intestinal juice (SIJ) for 4 h. Sodium dodecyl sulphite polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that the WPC-PUL gel had low solubility in SGJ, but dissolved well in SIJ, which suggested that the gel can protect LGG under SGJ condition and release probiotics in the SIJ. Moreover, when the gel has highest hardness and water-holding capacity, the viable counts of LGG were not the best, suggesting the relationship between the protection and the properties of the gel was non-linear.

Diatom shell incorporated PHBV/PCL-pullulan co-electrospun scaffold for bone tissue engineering

Tissue engineering can benefit from wide variety of materials produced by microorganisms. Natural origin materials often possess good biocompatibility, biodegradability with sustainable production by microorganisms. A phytoplankton, diatom, produces an amorphous silica shell that can be obtained by a cost efficient production process. Diatom shells (DS) are promising for bone tissue engineering since silicon enhances bone regeneration. Biocompatible and biodegradable biopolymers with microorganism origin can be combined with DS to produce tissue engineering constructs. In this study, a novel multifunctional 3D fibrous scaffold for bone tissue engineering was produced by co-electrospinning system; antibiotic loaded poly(hydroxybutyrate-co-hydroxyvalerate)/poly(ε-caprolactone) (PHBV/PCL) fibers and DS incorporated pullulan (PUL) fibers. Controlled release of cefuroxime axetil (CA) from DS and scaffolds were investigated upon loading CA into DS or PHBV/PCL fibers. Purified DS were characterized with ESCA, SEM, and EDX analyses while scaffolds were evaluated in terms of morphology, porosity, degradation, calcium deposition, water retention and mechanical properties. In vitro studies showed that scaffolds bearing DS have improved human osteosarcoma (Saos-2) cell viability. Developed co-electrospun scaffold showed higher osteocompatibility with better cell spreading and cell distribution. Results showed that DS loaded, co-electrospun scaffold having both hydrophobic and hydrophilic characteristics can be a promising biomaterial for bone tissue engineering.

Intranasal administration of pullulan-based nanoparticles for enhanced delivery of adriamycin into the brain: in vitro and in vivo evaluation.

Intranasal (i.n.) administration is an efficient route for enhancing drug delivery to the brain, bypassing the blood-brain barrier (BBB) and eliminating systemic side effects. The purpose of this study was to investigate the nose-to-brain delivery efficiency of adriamycin (ADM) loaded in cholesterol-modified pullulan self-assembled nanoparticles (CHSP-SAN) via i.n. administration. The prepared nanodrugs (ADM-CHSP-SAN) were characterized as uniform size (112.8±1.02 nm), high drug loading capacity (7.65±0.58 %), and sustained release. CHSP-SAN showed good biocompatibility and low toxicity on HBMEC and C6 cells. The enhanced delivery of ADM across the BBB with CHSP-SAN was demonstrated by the reduced half maximal inhibitory concentration (IC50) value and the increased apoptosis proportion of C6 cells. The pharmacokinetics of ADM-CHSP-SAN was accessed by cerebral microdialysis technique. The pharmacokinetic results showed higher peak concentration (Cmax), area under the curve (AUC0-12h) and shorter peak time (Tmax) after i.n. administration that after intravenous (i.v.) administration. The i.n. administration of CHSP-SAN greatly increased ADM availability in cerebral tissue compared to that of ADM solution. Collectively, CHSP-SAN strikingly increased ADM transport across the BBB and improved its availability in brain via i.n. administration.

Intrinsic Viscosities of Polymer Blends: Sensitive Probes of Specific Interactions between the Counterions of Polyelectrolytes and Uncharged Macromolecules

In joint dilute aqueous solutions of pullulan (PUL) and poly(sodium 4-styrenesulfonate) (PSS-Na) the Na+ ions of the polyelectrolyte interact so favorably with the monomeric units of PUL that isolated coils containing both types of macromolecules are formed upon dilution. When water is replaced against a 1 M solution of NaCl, this effect dies out because of the large surplus of Na+ ions. On the other hand, if the water contains increasing amounts of the respective counterpolymer instead of NaCl, the formation of mixed isolated coils is fostered, where a further diminution of the intrinsic viscosities is caused by the lower the solvent quality. The molar mass of PSS-Na plays an important role for the viscosities of the solutions in pure water. The reason lies in the different extent of the electrostatic self-shielding as a function of the polyelectrolyte concentration.

Tapioca starch-pullulan interaction during gelation and retrogradation

The interaction between pullulan (PU) and tapioca starch (TS) during gelatinization and retrogradation was studied in this paper. TS and PU were prepared into a TS/PU composite system at the ratios of 10/0, 9.5/0.5, 9.0/1.0, 8.5/1.5 and 8.0/2.0 g/g. The addition of PU tended to decrease the peak, breakdown and final viscosity of the composite system. The increasing tanδ of the dynamic viscoelastic measurement suggested that PU enhanced the liquid-like properties of TS gels. The decreased setback values and the slower increase of the storage modulus at 4 °C indicated that the short-term retrogradation of TS was restrained. Meanwhile, the peaks of X-ray diffraction became lower and wider, revealing that PU could inhibit the long-term retrogradation of TS. The FTIR spectra showed that the absorption peak of the O−H stretching gradually redshifted with increasing PU content, suggesting that a strong intermolecular hydrogen binding occurred between TS and PU. From low-field nuclear magnetic resonance, the spin-spin relaxation time decreased from 1687 ms (TS/PU = 10.0/0) to 1427 ms (TS/PU = 8.0/2.0), illustrating that the addition of PU promotes the water retention ability of the TS paste. Therefore, PU had an effect on TS gelatinization and retrogradation.

Electrospun fibers from blends of pea (Pisum sativum) protein and pullulan

Pea protein isolate (PPI) was evaluated to produce food grade fibers through the electrospinning technique. The presence of pullulan (PUL) and Tween 80 (T) on the spinnability, morphology, physicochemical and mechanical properties were addressed. The presence of PUL in the polymeric aqueous solutions of PPI resulted in decreasing of electrical conductivity and increasing of the viscosity and surface tension. Electrospun mats formed by fiber structures were obtained with PPI:PUL 20:80 and 50:50 containing 20 wt % of Tween 80 (50:50T20) with diameters of 170 and 149 nm, respectively. FTIR data indicated that interactions were present between the hydroxyl groups of pullulan and the amino groups of pea protein and that the presence of Tween 80 favored these interactions. The mechanical properties of the electrospun mats showed that the increase of pullulan in blend increased Young's modulus, maximum stress and strain at rupture.