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BACKGROUND Curcumin has become increasingly popular in functional foods and beverages field as a result of its high biological activity. Nevertheless, the application of curcumin is usually limited by its poor water solubility, low absorption, rapid metabolism and instability. Accordingly, the development of an appropriate wall material is crucial for its effective use. In the present study, curcumin-octenyl succinic anhydride modified pullulan (Cur-OSAP) micelles were successfully prepared by an anti-solvent co-precipitation method. RESULTS Octenyl succinic anhydride modified pullulan (OSAP) micelles exhibited the highest encapsulation efficiency (57.31%) and loading capacity (5.73%) of curcumin when the mass ratio of OSAP to curcumin was 10:1 and the degree of substitution of OSAP was 0.0469, at which point Cur-OSAP micelles formed via hydrogen binding and hydrophobic interactions, as confirmed by Fourier transform infrared and fluorescence techniques. The transmission electron microscopy results showed that the Cur-OSAP micelles were roughly spherical in shape with diameters in the approximate range 30-60 nm. CONCLUSION The encapsulation of OSAP greatly improved photostability and sustained release properties of curcumin in Cur-OSAP micelles. These findings suggest that OSAP can be used as a carrier to encapsulate and protect hydrophobic food ingredients. © 2021 Society of Chemical Industry.

In this study, clove extract loaded nanofibers consisting of pullulan and whey protein were produced by electrospinning method in order to improve antibacterial and antioxidant activity. Diameters of the produced nanofibers varied between 215.7 and 536.8 nm. Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD) and differential scanning calorimeter (DSC) results showed that there was a close interaction between the obtained nanofibers and clove extract. Although the antioxidant activity of the nanofibers varied between 1535 and 4174 mu mol TE/g dm, the antioxidant activity of the clove extract loaded nanofibers tended to increase when compared to their polymer solutions. The total phenolic content of the nanofiber samples varied between 4.93 and 45.05 mg GAE/g dm. It has been determined that clove extract loaded nanofibers showed antibacterial activity against Staphylococcus aureus (ATCC29113) and Micrococcus luteus (NCIMB). As a result, it was seen that the addition of clove extract improved the antioxidant and antibacterial properties of the fibers and these fibers could be a potential coating and packaging material that could have a potential use in food preservation.

To evaluate the dental pulp response to a novel mineral trioxide aggregate containing phosphorylated pullulan (MTAPPL) in rats after direct pulp capping.Ninety-six cavities were prepared in the maxillary first molars of 56 male Wistar rats. The dental pulps were intentionally exposed and randomly divided into four groups according to the application of pulp capping materials: MTAPPL; phosphorylated pullulan (PPL); a conventional MTA (Nex-Cem MTA, NCMTA; positive control); and Super-Bond (SB; negative control). All cavities were restored with SB and observed for pulpal responses at 1-, 3-, 7- and 28-day intervals using a histological scoring system. Statistical analysis was performed using Kruskal-Wallis and Mann-Whitney U-test with Bonferroni's correction, and the level of significance was set at 0.05. DMP1 and CD34 antigen were used to evaluate odontoblast differentiation and pulpal vascularization, respectively.On day 1, mild inflammatory cells were present in MTAPPL and NCMTA groups; fewer inflammatory cells were present in the PPL, whereas SB was associated with a mild-to-moderate inflammatory response. A significant difference was observed between PPL and SB (p .05). No mineralized tissue deposition was observed. On day 3, moderate-to-severe inflammatory cells were present in PPL and SB, whereas MTAPPL and NCMTA had a mild inflammatory response. Initial mineralized tissue deposition was observed in the NCMTA, MTAPPL and SB. A significant difference was observed between MTAPPL and PPL (p .05). On day 7, a thin layer of mineralized tissue was observed in all tested groups with no or mild inflammatory response. On day 28, no inflammatory response was observed in MTAPPL, whereas NCMTA, PPL and SB had mild inflammatory responses. A significant difference was observed between MTAPPL and SB (p .05). Complete mineralized tissue barrier formation was observed in MTAPPL, NCMTA and PPL with no significant difference (p .05). SB exhibited incomplete mineralized tissue barriers, significantly different from NCMTA, MTAPPL and PPL (p .05). The staining with CD34 was positive in all the groups on all observation days.The favourable pulpal responses and induction of mineralized tissue formation associated with MTAPPL indicate its potential application as a direct pulp capping material.

Microbeads consisting of pullulan and dextran supplemented with hydroxyapatite have recently been developed for bone tissue engineering applications. Here, we evaluate the bone formation in two different preclinical models after injection of microbeads reconstituted with either saline buffer or autologous blood. Addition of saline solution or autologous blood to dried microbeads packaged into syringes allowed an easy injection. In the first rat bone defect model performed in the femoral condyle, microcomputed tomography performed after 30 and 60 days revealed an important mineralization process occurring around and within the core of the microbeads in both conditions. Bone volume/total volume measurements revealed no significant differences between the saline solution and the autologous blood groups. Histologically, osteoid tissue was evidenced around and in contact of the microbeads in both conditions. Using the sinus lift model performed in sheep, cone beam computed tomography revealed an important mineralization inside the sinus cavity for both groups after 3 months of implantation. Representative Masson trichrome staining images showed that bone formation occurs at the periphery and inside the microbeads in both conditions. Quantitative evaluation of the new bone formation displayed no significant differences between groups. In conclusion, reconstitution of microbeads with autologous blood did not enhance the regenerative capacity of these microbeads compared to the saline buffer group. This study is of particular interest for clinical applications in oral and maxillofacial surgery.

BACKGROUND To overcome the problems associated with niosomes, proniosomes - a dry powder - was prepared to nanoencapsulate catechins using Span 60 as surfactant, cholesterol as stabilizer and maltodextrin, lactose monohydrate and pullulan as wall materials. The proniosomes were made by the thin-film hydration technique, and were characterized for fortification in milk beverages. Scanning and atomic force microscopic images showed the varying morphology and ultrastructure of the proniosomes. The mean hydrodynamic diameter of 193.57-262.52 nm, polydispersity index of 0.24-0.25 and zeta potential of -15.8 to -24.73 were suggestive of the size, homogeneity and stability of the catechin-loaded proniosomes. X-ray powder diffractograms and Fourier transform infrared (FTIR) spectra provided insight about the interaction between catechins and wall materials. Entrapment efficiency and in vitro release were calculated to determine the extent of nanoencapsulation of catechins and their bioavailability, respectively. The nanoencapsulates were fortified in milk and yogurt to find their organoleptic acceptability. RESULTS Moisture content was found to be 20-30 g kg-1 , indicating longer stability of the proniosomes. Scanning electron microscopic and atomic force microscopic images revealed the ultrastructure and spherical-shaped morphology of proniosomes. Entrapment efficiency of catechins using pullulan as wall material was as high as 83.43%. In vitro release studies revealed the sustained release of catechins from the proniosomes. FTIR and X-ray diffraction spectra revealed the absence of chemical interactions between catechins and encapsulants. CONCLUSION Food-grade proniosomes are a good vehicle for fortification of milk and yogurt without noticeable adverse changes in their organoleptic and physicochemical properties, thus increasing the potential for bioavailability of catechins in the gastrointestinal tract. © 2020 Society of Chemical Industry.

Background Whey protein concentrate (WPC)/pullulan (PUL) hydrogel is applied as a microencapsulation wall material to protect probiotics. However, the interactions between WPC and PUL during gelation have not been clarified. In the present study, the effects of PUL concentration and pH on the interactions between WPC and PUL during gelation were evaluated with respect to appearance, zeta-potential, sulfhydryl group amount, surface hydrophobicity and infrared spectroscopy measurements. The rheological properties of WPC/PUL gels were also determined. Results The results obtained showed that a proper concentration (0.40 g mL-1 ) of PUL could improve the gel by enhancing the strength of hydrogen bonding, electrostatic interactions and exposure of hydrophobic groups, whereas too much PUL inhibited the formation of disulfide bonds. Furthermore, hydrophobic interactions, disulfide bonds and hydrogen bonds were destroyed in varying degrees under an alkaline environment. The rheological results also demonstrated a similar effect of PUL concentration and pH on the storage modulus (G') of WPC/PUL gels. Conclusion When the WPC/PUL gel was formed at PUL concentration of 0.40 g mL-1 and pH 7.0, the interaction between WPC and PUL could be enhanced, which is beneficial for the future application of WPC/PUL gels in the food industry. © 2020 Society of Chemical Industry.

Pullulan is a polysaccharide polymer commonly used to produce edible films. However, pure pullulan film is usually brittle and very hydrophilic, which limit its use in both food and pharmaceutical fields. The objective of this research is to improve the structural and mechanical properties of pullulan film by incorporating globular protein lactoferrin (LF).The incorporation of LF increased the surface hydrophobicity and decreased the water vapor permeability (WVP) of pullulan film. The presence of low concentrations of LF (0.03%) has no significant influence on tensile strength (TS) and elongation at break (EAB) of pullulan film. However, further increase of LF concentration to levels0.03% resulted in a film-weakening effect. LF molecules aggregated with each other during the film-producing process, which presented as spherical particles from the observation of microscopy. LF aggregates dispersed homogeneously throughout the pullulan matrix and their size increased with increasing concentration. Analysis from Fourier transform infrared spectroscopy indicated that the secondary structure of LF molecules was modified during the drying process.It is possible to increase the hydrophobicity of pullulan film while maintaining its mechanical properties. The produced composite film can be potentially used for food and medical packaging. © 2019 Society of Chemical Industry.

The self-assembly of a novel double hydrophilic block copolymer in water without the application of external triggers is described, namely pullulan-b-poly(2-ethyl-2-oxazoline) (Pull-b-PEtOx). The biomacromolecules, Pull (8–38 kg mol−1), is modified and conjugated to biocompatible PEtOx (22 kg mol−1) via modular conjugation. Moreover, the molecular weight of the Pull blocks are varied to investigate the effect of molecular weight on the self-assembly behavior. Spherical particles with sizes between 300 and 500 nm are formed in diluted aqueous solution (0.1–1.0 wt %) as observed via dynamic light scattering and static light scattering. Additionally, cryo scanning electron microscopy and laser scanning confocal microscopy are performed to support the finding from light scattering. The block ratio study shows an optimum ratio of Pull and PEtOx of 0.4/0.6 for self-assembly in water in the concentration range of 0.1–1.0 wt %. At higher concentrations of 20 wt %, vesicular structures with sizes above 1 µm can be observed via optical microscopy. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017

Aim Osseointegration of titanium implants is predictable, but can be improved via surface functionalization. Materials and Methods One hundred and twenty implants were installed in parietal bone of 12 domestic pigs and left to heal for 1 or 3 months. Five groups were defined according surface treatments: immersion in water (H2O), 10% polyphosphoric acid (PPA10), 1% phosphorylated pullulan (PPL1), 10% phosphorylated pullulan (PPL10) or 10% phosphorylated pullulan + 1 μg bone morphogenetic protein-2 (PPL10 BMP). As primary outcome, implant osseointegration was evaluated by quantitative histology, namely peri-implant bone formation (B/T in %) and bone-to-implant contact (BIC in %) for each healing period. The Wilcoxon signed-rank test and Mann–Whitney U-test with α = 0.05 were performed. Results PPL10 and PPA10 groups showed significantly higher B/T and BIC results than the control (H2O) group at 1-month (p .05). After 3 months, no experimental group showed a significant difference compared to the control group (H2O) for both investigated parameters (B/T and BIC; p > .05). Conclusion Functionalizing titanium implants with inorganic or organic phosphate-containing polymers at 10 wt% concentration may stimulate peri-implant bone formation and implant osseointegration at early healing times.

The effects of pullulan (PUL) addition (0, 1.0 and 2.0% w/ v) on the physicochemical properties of yoghurts stored at 4 °C for 28 days were investigated. Fortification of the yoghurt with 1% PUL had a weakening effect on the gel network structure, leading to a decrease in initial viscosity and an extreme increase in syneresis, when compared to control. The addition of 2% PUL largely improved product stability by increasing gel firmness and adhesiveness, showing higher viscosity and consistency index values as well as displaying the lowest syneresis. Storage time increased yoghurt gel firmness and susceptibility to serum separation.

RNA is a functionally versatile polymer but suffers from susceptibility to spontaneous and RNase-catalyzed degradation. This vulnerability makes it difficult to preserve RNA for extended periods of time, thus limiting its use in various contexts, including practical applications as functional nucleic acids. Here we present a simple method to preserve RNA by pullulan (a complex sugar produced by Aureobasidium pullulans fungus) film formation. This strategy can markedly suppress both spontaneous and RNase degradation. Importantly, the pullulan film readily dissolves in aqueous solution, thus allowing retrieval of fully functional RNA species. In order to illustrate the advantage of this protective method in a practical application, we engineered a simple paper sensor containing a bacteria-detecting RNA-cleaving DNAzyme. This detection capability of the device was unchanged after storage at room temperature for six months.

With the high rate of mortality associated with breast cancer among women, breast cancer treatment has attracted great deal of attention globally. To reduce the exposure of body organs to high cytotoxicity of the common chemotherapeutic drugs, local co-delivery of selected chemotherapeutics has emerged as a solution. In this work, an electrospun composite including a co-drug-loaded graphene oxide-based nanocarrier is fabricated for local anticancer applications. To increase dispersion and cellular uptake of graphene oxide (GO), first, the hydroxyl groups at the edges of GO are grafted by poly (epichlorohydrin) (PCH) to form GO-PCH. Then, the hydroxyl end groups of PCH are grafted (g) with hyperbranched polyglycerol (HPG) leading to formation of oxygen-rich nanocarrier (GO-PCH-g-HPG). Paclitaxel (PTX) and curcumin (Cur) are loaded into the GO-PCH-g-HPG nanocarrier, and are encapsulated into pullulan nanofibers using electrospinning. SEM, FTIR, and UV–Vis characterizations confirmed the presence and distribution of the nanocarrier inside the nanofibers. The drugs showed a sustained release (93 h) from the nanocarrier-loaded nanofibers in the neutral pH (7.4). According to MTT assay and optical microscopy, a synergistic cytotoxicity effect of PTX and Cur is observed. The nanocarrier-encapsulated nanofiber system represents a novel tunable drug delivery system for local chemotherapeutic applications.

BACKGROUND Poly(β-malic acid) (PMA) and pullulan are two valuable biopolymers that can be produced simultaneously by certain strains of pullulans. In previous studies, PMA production was successfully improved with reducing pullulan production. In the present study, a DO-shift control strategy was developed to obtain simultaneous high yields of PMA and pullulan. RESULTS PMA and pullulan co-production at various constant DO levels ranging from 10% to 90% saturation were carried out in a 5 L stirred tank fermentor. Based on the analysis of batch fermentation kinetics at various constant DO levels, a DO-shift control strategy that shifted the DO level from 30% saturation to 70% saturation at 72 h was developed to effectively obtain high productions of both PMA and pullulan. CONCLUSION By applying the DO-shift control strategy, the maximum concentrations of PMA and pullulan reached 118.6 g L−1 and 27.2 g L−1, respectively. PMA production was successfully improved without sacrificing pullulan production. © 2016 Society of Chemical Industry

Pullulan based films possess several advantages, including high transparency, low toxicity, good biodegradability, good mechanical properties, and low oxygen permeability, are preferable for food packaging. The application of pullulan films on food packaging, however, has inherent disadvantage of high water solubility. In this study, glutaraldehyde and glycerol were used as the cross-linking reagent and the plasticizer respectively to improve water resistance and physical properties of the pullulan films. Effects of cross-linking degree on physical properties, including water absorptions, swelling behaviors, water vapor permeability and tensile strengths of films were evaluated. FTIR results demonstrated that the pullulan films were successfully cross-linked by glutaraldehyde. The tensile strength of pullulan films could be enhanced significantly (P < 0.05) when glutaraldehyde was between 1% and 5% (w/w); nevertheless, the amount of glutaraldehyde above 20% (w/w) led to films brittleness. With the addition of glycerol as a plasticizer enhanced the extensibility of films as well as the hydrophilicity, resulting in higher water vapor permeability.

Freeze-casted nanofiber based sponges or aerogels exhibit a hierarchical porous structure. Pore formation is only partially understood. Therefore, we studied the underlying solid templating mechanism. We were able to tailor the secondary pore size between 9.5 and 123 µm while retaining the smaller primary pores known from electrospun nanofiber membranes. To understand the effect of microstructure on the sponges’ bulk properties, mass flow through the pores and interaction with the sponges’ internal surface were investigated. By solely altering the sponges’ microstructure we indeed found tunability in permeability by a factor 7 and in filtration efficiency by a factor of 220. Hence, pore architecture of nanofiber based sponges is a key element for their performance. The selected pullulan/PVA polymer blends and aqueous electrospinning conditions are benign and allow the facile adaptation of these ultralight highly porous sponges for a large number of applications.

The food enzyme pullulanase (pullulan 6‐α‐glucanohydrolase; EC 3.2.1.41) is produced with a genetically modified Bacillus licheniformis (strain DP‐Dzp39) by Danisco US Inc. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its recombinant DNA. This pullulanase is intended to be used in brewing processes, starch processing for glucose syrups production and distilled alcohol production. Residual amounts of total organic solids (TOS) are removed by distillation and by the purification steps applied during the production of glucose syrups, consequently, dietary exposure was not calculated for these food processes. For brewery products, based on the maximum use level recommended for the brewing processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme–TOS was estimated to be up to 0.053 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests with the food enzyme did not raise concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no‐observed‐adverse‐effect level at the highest dose of 500 mg TOS/kg bw per day that, compared to the estimated dietary exposure, results in sufficiently high margin of exposure (at least 9,400). The amino acid sequence of the food enzyme did not match those of known allergens. The Panel considered that, under the intended condition of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is considered low. Based on the data provided, the Panel concluded that this food enzyme does not raise safety concerns under the intended conditions of use.

Introduction Wound-dressing materials that promote wound healing while protecting wounds from infections are advantageous for clinical applications. Hence, we developed a cholesterol-bearing pullulan (CHP) nanogel that stimulated wound healing; however, it was mechanically weak and difficult to handle. Thus, the purpose of this study was to examine precisely the effects of a mechanically reinforced nanogel-crosslinked (NanoClik) membrane on wound healing. Materials and Methods NanoClik was prepared by mixing a thiol-terminated polyethylene glycol solution and an acryloyl group-modified CHP nanogel solution. A thin silicone sheet membrane, which was combined with NanoClik, was prepared. The NanoClick membranes and both positive and negative control membranes (collagen combined with silicone membrane and silicone membrane alone, respectively) were tested in vivo using a dorsal skin defect rat model. The rate and extent of wound healing was compared between groups after 7 and 14 days of implantation. Results In the NanoClik membrane group, the wound area was significantly reduced and neoepithelialization was promoted, compared with that observed in the other groups. In addition, extension and accumulation of collagen fibers were evident in the NanoClik membrane group. Conclusion The NanoClik membrane is a strong candidate for use as an effective and safe wound-dressing material. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2015.

International audience; Various pullulan-b-Jeffamine block copolymers (Jeffamine is a PEO-b-PPO copolymer (PEO = poly(ethylene oxide), PPO = poly(propylene oxide)) are used as surfactants for vinyl acetate miniemulsion polymerization. Their influence on the kinetics, average molecular weights and colloidal stability, is compared with that of Pluronic F68, when using either organosoluble or hydrosoluble initiators. In the first case, conversions and molecular weights are not affected by the polymer surfactant, in contrast to what is observed when using the hydrosoluble system, that may be due to a reaction between the incoming radicals and the sugar units of the pullulan chain. Long-term colloidal stability between 4 degrees C and 40 degrees C over more than 4 months is observed and is not affected by the addition of salts when using pullulan-based surfactant.

Highly porous composites of poly(glycidyl methacrylate-co-1,3-butanediol dimethacrylate) and pullulan were prepared as semi-interpenetrating polymer networks by cross-linking of high internal phase emulsions (HIPEs). HIPEs were prepared by using an aqueous phase made of pullulan dissolved in deionised water. Instead of conventional cross-linkers such as divinyl benzene, 1,3-butanediol dimethacrylate was used as a flexible co-monomer. Morphological and mechanical properties of the porous composites with an initial pullulan loading ranging from 1 to 10 wt % have been investigated. Resulting composites were tested in terms of uniaxial compression stress and it was found out that the use of pullulan and flexible co-monomer in the HIPE preparation increases the compression modulus from 13.53 to 30.8 MPa. Textural analyses of the resulting composites show that in all cases open cellular foams composed of primary cavities (approximate to 112-74m) connected with secondary interconnected pores were produced with a specific surface area in the range of 1.7-3.0m(2) g(-1). In addition, the influence of using porogen in the oil phase was investigated. It was found that using a porogen reduces the cavity size diameter from approximate to 112 to 20 mu m and increases the compression modulus from 13.53 to 47.06 MPa. POLYM. ENG. SCI., 55:2636-2642, 2015. (c) 2015 Society of Plastics Engineers

Edible active coatings (EACs) based on pectin, pullulan, and chitosan incorporated with sodium benzoate and potassium sorbate were employed to improve the quality and shelf life of strawberries. Fruits were washed, disinfected, coated by dipping, packed, and stored at 4 °C for 15 d. Application of EACs reduced (P 0.05) throughout storage, and ascorbic acid content was maintained in pectin-EAC coated strawberries. Microbiological analyses showed that application of EACs reduced (P < 0.05) microbial growth (total aerobic counts, molds, and yeasts) on strawberries. Chitosan-EAC coated strawberries presented the best results in microbial growth assays. Sensory quality (color, flavor, texture, and acceptance) improved and decay rate decreased (P < 0.05) in pectin-EAC, pullulan-EAC, and chitosan-EAC coated strawberries. In conclusion, EACs based on polysaccharides improved the physicochemical, microbiological, and sensory characteristics, increasing the shelf life of strawberries from 6 (control) to 15 d (coated fruits).

In this work, the effectiveness of atmospheric pressure non-equilibrium plasma, generated by a dielectric barrier discharge and operated in air, in successfully crosslinking water soluble pullulan electrospun fibers is demonstrated. After being exposed to plasma, the fibers show a remarkable capability to preserve structural stability and fiber morphology upon water exposure. The gel fraction and the water absorption are quantified to evaluate the crosslinking efficiency of different plasma operating conditions, and ATR-FTIR characterization provided some insights into plasma-induced modification of pullulan chemical structure.

There has been a growing interest in the use of natural materials as a delivery mechanism for antimicrobials and coatings in foods. The aim of the present study was to evaluate the effectiveness of pullulan coatings to improve internal quality and shelf-life of fresh eggs during 10 wk of storage at 25 and 4 °C. Three treatments of eggs were evaluated as follows; non-coated (control; C), coated with pullulan (P), and coated with pullulan containing nisin (N). The effects of the pullulan coatings on microbiological qualities, physical properties, and freshness parameters were investigated and compared with non-coated eggs. For non-coated eggs, as storage time increased, yolk index, albumen index, and Haugh unit value decreased and weight loss increased. However, pullulan coatings (P or N) minimized weight loss (

International audience; The food enzyme considered in this opinion is a pullulanase (pullulan 6-alpha-glucanohydrolase; EC 3.2.1.41) produced with a non-genetically modified Pullulanibacillus naganoensis (strain AE-PL) by Amano Enzyme Inc. (Japan). The pullulanase food enzyme is intended to be used in starch processing for the production of glucose syrups. Since residual amounts of total organic solid (TOS) in glucose syrups are removed by filtration and purification during starch processing, dietary exposure assessment was not performed. Genotoxicity tests made with the food enzyme indicated no genotoxic potential. A repeated dose 90-day oral toxicity study in rodents, carried out with the food enzyme, showed minor effects that were considered to be of no biological relevance. The allergenicity was evaluated by comparing the amino acid sequence to those of known allergens and no match was found. The Panel considered that there are no indications for food allergic reactions to dietary intake of this food enzyme. Based on the removal of residual amounts of TOS from glucose syrups, consumer exposure is not expected. In addition, the safety of the manufacturing process, the compositional and biochemical data lead the Panel to conclude that the food enzyme pullulanase from P. naganoensis (strain AE-PL) does not give rise to safety concerns under the intended conditions of use.

BACKGROUND Pullulan with high molecular weight (Mw) is attractive for industrial application. Previous studies indicated that high yield and high Mw of pullulan cannot be simultaneously obtained under constant agitation speeds or constant dissolved oxygen levels during batch pullulan fermentation. In this study, a two-stage agitation speed control strategy was developed to obtain pullulan with both high yield and high Mw. RESULTS Batch pullulan production at various constant agitation speeds ranging from 200 to 600 rpm was carried out in a 5 L stirred tank fermentor. Based on kinetic analysis of batch processes, activity assays of the enzymes associated with pullulan biosynthesis and biodegradation, and intracellular energy supply under different constant agitation speeds, a two-stage control strategy that shifted the agitation speed from 500 to 400 rpm at 18 h proved to effectively achieve simultaneous high yield of high Mw pullulan. CONCLUSION A maximum pullulan concentration of 27.4 g L−1 and final Mw of 3.09 × 106 Da were obtained using the two-stage agitation speed control strategy, which were both greatly improved compared with those obtained at constant agitation speeds. © 2014 Society of Chemical Industry

In this study, the barrier properties, crystalline structure and morphology of pullulan–sodium alginate-based films were investigated. Among the tested samples, pure pullulan films exhibited the lowest water vapor permeability and oxygen permeability values (7.053 × 10−6 g.m/Pa.h.m2 and 0.48x10−6 mL.mm/m2day.pa, respectively). Incorporating pullulan into alginate, the absorbance bands of COO− groups shifted significantly to higher wavenumbers (1,640 and 1,413 cm−1, respectively). This could be attributed to the disruption of intermolecular hydrogen bonds present originally between the carboxylic groups caused by added pullulan. The diffractogram of pullulan films indicated that pullulan was a fully amorphous polymer, whereas that of sodium alginate exhibited semi-crystalline features. Furthermore, the morphology of pullulan–alginate-based film was studied by atomic force microscopy. Roughness parameter (Rq and Ra) values increased significantly with the incorporation of alginate content into pullulan. Practical Applications Because pure pullulan, alginate and their blend film are highly water soluble, transparent, tasteless, odorless and heat sealable and poses low permeability to oxygen, they could potentially be a secondary packaging material for beverage products such as instant coffees and cocoa.

Pullulanase from Klebsiella pneumoniae was entrapped into calcium alginate beads. Its activity was estimated by the determination of number–average molar masses using two different methods: a colorimetric assay of reducing ends (REs) and a size-exclusion chromatography/multiangle light scattering/differential refractive index. The second method also provided weight–average molar masses of hydrolyzed pullulan and the quantity of maltotriose (DP3) and its multiples (DP6 and DP9) produced by the enzymatic treatment. The alginate beads showed a good retention of the loaded pullulanase (30%), and the system showed a downturn of hydrolysis kinetics in comparison with free pullulanase due to the limiting access of substrate–enzyme. On the contrary with the results obtained from free enzyme hydrolysis, for which a large distribution of pullulan fragments is observed during the treatment, the immobilized enzyme system has evidenced, during the enzymatic treatment, the coexistence of native or only slightly degraded pullulan chains together with maltotriose units. Complete hydrolysis of pullulan chains was achieved once diffused into the gel. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 938–944, 2014.

Cells perceive their microenvironment through physical and mechanical cues, such as extracellular matrix topography or stiffness. In this study, we developed a polysaccharide scaffold that can provide combined substrate topography and matrix compliance signals to direct cell fate. Pullulan/dextran (P/D) nanofibers were fabricated with variable stiffness by in situ crosslinking during electrospinning. By varying the chemical crosslinking content between 10, 12, 14, and 16%, (denoted as STMP10, STMP12, STMP14, and STMP16 respectively), scaffold mechanical stiffness was altered. We characterized substrate stiffness by various methods. Under hydrated conditions, atomic force microscopy and tensile tests of bulk scaffolds were conducted. Under dry conditions, tensile tests of scaffolds and single nanofibers were examined. In addition, we evaluated the efficacy of the scaffolds in directing stem cell differentiation. Using human first trimester mesenchymal stem cells (fMSCs) cultured on STMP14 P/D scaffolds (Young's modulus: 7.84 kPa) in serum-free neuronal differentiation medium exhibited greatest extent of differentiation. Cells showed morphological changes and significantly higher expression of motor neuron markers. Further analyses by western blotting also revealed the enhanced expression of choline acetyltransferase on STMP14 (7.84 kPa) and STMP16 (11.08 kPa) samples as compared to STMP12 (7.19 kPa). Taken together, this study demonstrates that the stiffness of P/D nanofibers can be altered by differential in situ crosslinking during electrospinning and suggests the feasibility of using such polysaccharide nanofibers in supporting fMSC neuronal commitment. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 959–968, 2015.

A simple and inexpensive method is reported for the long-term stabilization of enzymes and other unstable reagents in premeasured quantities in water-soluble tablets (cast, not compressed) made with pullulan, a nonionic polysaccharide that forms an oxygen impermeable solid upon drying. The pullulan tablets dissolve in aqueous solutions in seconds, thereby facilitating the easy execution of bioassays at remote sites with no need for special reagent handling and liquid pipetting. This approach is modular in nature, thus allowing the creation of individual tablets for enzymes and their substrates. Proof-of-principle demonstrations include a Taq polymerase tablet for DNA amplification through PCR and a pesticide assay kit consisting of separate tablets for acetylcholinesterase and its chromogenic substrate, indoxyl acetate, both of which are highly unstable. The encapsulated reagents remain stable at room temperature for months, thus enabling the room-temperature shipping and storage of bioassay components.

Pullulanase is a debranching enzyme that specifically hydrolyzes the α-1,6 glycosidic linkage of α-glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18-11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and Km for pullulan of PulA are 750 U mg(-1) and 16.4 μmol L(-1), respectively. PulA has a half-life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75-2.22 A resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β-strand and the third α-helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate-binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain.

Pullulan-based edible coatings were used to extend the shelf life of strawberries (Fragaria ananassa cv. Camarosa) under cold storage conditions in perforated package. Some physicochemical properties of the strawberry fruits such as weight loss, firmness, colour, ascorbic acid, total carotenoids and fungal decay were determined during the storage. The coatings delayed mould formation and decreased weight loss, softening and degradation of ascorbic acid and carotenoids in the fruits. The most marked difference was recorded in the firmness value of the fruits coated with a 10% pullulan-based emulsion. Coating with a 10% pullulan-based emulsion successfully extended the shelf life of the strawberries for 5 days longer under cold storage conditions. Copyright © 2014 John Wiley & Sons, Ltd.

BACKGROUND Consumption of different dietary fibers may influence the digestibility of carbohydrates and other nutrients. Therefore the objectives of this experiment were to determine the effect of novel fiber ingredients on the apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD) of gross energy (GE), dry matter (DM), crude protein (CP) and total dietary fiber (TDF) in pigs and to calculate the standardized digestibility of analyzed TDF in four novel fiber ingredients. RESULTS The AID of DM and GE in diets containing novel fiber ingredients was less (P

This research evaluated the antimicrobial efficacy of pullulan films containing caraway essential oil (CEO). The films were prepared from a 10% of pullulan, containing from 0.12% to 10.0% of CEO. The composition of the CEO was analyzed with the use of gas chromatography. The antimicrobial activity of the CEO was evaluated with the method of serial microdilutions, and the films containing CEO—with the agar diffusion method against selected Gram-negative, Gram-positive bacteria, and fungi. The structure of the film surface and its cross-section were analyzed using a scanning electron microscope (SEM). Analyses were also carried out to determine the efficacy of a pullulan coating with 10% CEO on baby carrots experimentally inoculated with Salmonella enteritidis, Staphylococcus aureus, Saccharomyces cerevisiae, or Aspergillus niger and stored at a room temperature for 7 d. At a concentration of 0.12%, CEO inhibited the growth of all the tested microorganisms. Pullulan films containing 8% to 10% of CEO were active against all tested microorganisms. Populations of S. aureus on carrot samples were reduced by approximately 3 log CFU/g, while those of A. niger and S. cerevisiae by, respectively, 5 and 4 log CFU/g, after 7 d of storage. S. enteritidis was the most resistant among the tested species, since it was not significantly reduced after 7 d of storage. At the end of storage, samples treated with pullulan–caraway oil coating maintained better visual acceptability than control samples. Results of this study suggest the feasibility of applying a pullulan film with incorporated CEO to extend the microbiological stability of minimally processed foods. Practical Application This study demonstrates the efficacy of a pullulan film containing caraway essential oil in the inhibition of the growth of microorganisms in foods. The results of this study may form the basis for the application of the packing system with such antimicrobial effects to extend the shelf-life of minimally processed food products.

Screen printing method was employed to produce microbial time temperature indicators (TTI). Bio-pastes containing lactic acid bacteria loaded Ca-alginate microparticles (LCAMs) and suitable for printing on polymer films have been produced. Through a spray-solidification method, polysaccharide gel microparticles allowed for the efficient encapsulation of the lactic acid bacteria, which chromatically induced a colour change in the pH indicator. As the alginate concentration of LCAMs increased, the size of the microparticles decreased. The average diameter of LCAMs ranged from (1.67 ± 0.15) × 103 to (2.93 ± 0.31) × 103 nm. For the evaluation of bio-pastes, the contact angles and lactic acid production properties were determined. Lower contact angles were obtained with decreasing pullulan concentration, indicating the increase in wettability for printing. The curve of lactic acid production by alginate immobilized cells was determined to take place as a zero-order reaction favourable to TTI colour change. Visibility of TTIs was greatly improved at microencapsulation sites. As the size of the LCAMs was decreased, the visibility was found to be improved. The Arrhenius activation energy (Ea) of CIFP009-based TTI was 117 kJ/mol. The results show that the developed manufacturing method would be used for an industrialized, simple and low-cost manufacturing method for microbial TTIs. Copyright © 2013 John Wiley & Sons, Ltd.

pH-sensitive pullulan-doxorubicin (DOX) conjugates were synthesized by attaching DOX onto pullulan derivate through hydrazone bond that was stable under neutral environment but readily cleaved under mildly acidic condition. By changing the feed ratio of DOX to the pullulan derivate, conjugates with drug-loading content up to 30 wt % were obtained. In aqueous solution, the conjugates spontaneously formed uniform core-shell structured nanoparticles with DOX as core and pullulan as shell. The diameters of the nanoparticles ranged from 50 to 110 nm according to the drug-loading content. In vitro releasing experiments showed that more than 75% DOX released within 2 h at pH 5.0, while less than 15% DOX released after 12 h at pH 7.4. This pH-responsive manner of DOX release might assist the quick diffusion of DOX from the acidic endosome/lysosome and the intracellular transfer into the nucleus. Pullulan on the nanoparticles surface provided the nanoparticles with active targeting property to hepatic cells through specific interaction with asialoglycoprotein receptors on the membrane of hepatic cells, without the necessity of introducing any extra ligand. These pullulan-DOX conjugate nanoparticles were expected to be promising drug delivery system for liver targeting antitumor chemotherapy.

The gelatin was added into pullulan films to improve their performances and lower their cost. The gelatin addition raised the tensile strength of the gelatin–pullulan composite films, and reduced the oxygen permeability. The cost of composite films was reduced comparing to that of the pullulan films. Moreover, the molecular interaction of the composite film was evaluated. The interactions of gelatin and pullulan in the composite films were detected, such as (1) formation of 2 glycosylated proteins; (2) improvement of β-sheet content; (3) formation of the interchain hydrogen bond and a semicrystalline region. Therefore, the molecular interaction was the main reason for the performance improvement of the composite films. Practical Application The pullulan films could not meet the commercial use as inner food packages due to their poor performances and high cost. The gelatin addition raised the tensile strength of the gelatin–pullulan composite films, and reduced their oxygen permeability and cost. The pullulan–gelatin composite film might be an inner packages for foods.

Summary: In this study, the rheological behaviors of media fermented by two Aureobasidium pullulans strains (IOC 3467 and IOC 3011) were evaluated in different nutritional conditions. The media consisted of crystal sugar (sucrose), as the main carbon source, and different nitrogen sources (ammonium sulfate, sodium nitrate, ammonium nitrate, urea or residual brewery yeast - RBY). Viscosity measurements were performed on cell-free supernatants, from 48-hour fermentation assays, at 25 °C. Shear rates ranged between 0.1000 and 500 s−1. All samples showed pseudoplastic behavior. Nevertheless, the viscosimetric profile of each one varied according to the nitrogen source, its concentration and the strain used. The maximum viscosity of 0.06 Pa.s. was achieved at 15.6 s−1 for IOC 3011 strain grown on RBY as nitrogen source.

New hybrid poly(ethylene glycol) (PEG) hydrogels crosslinked with both nanogels and nanogel-coated liposome complexes are obtained by Michael addition of the acryloyl group of a cholesterol-bearing pullulan (CHP) nanogel to the thiol group of pentaerythritol tetra(mercaptoethyl) polyoxyethylene. The nanogel-coated liposome complex is stably retained after gelation and the complexes are well dispersed in the hybrid gel. Microrheological measurements show that the strength and gelation time of the hybrid hydrogel can be controlled by changing the liposome:nanogel ratio. The hydrogel is gradually degraded by hydrolysis under physiological conditions. In this process, the nanogel is released first, followed by the nanogel-coated liposomes. Hybrid hydrogels that can incorporate various molecules into the nanogel and liposomes, and release them in a two-step controllable manner, represent a new functional scaffold capable of delivering multiple drugs, proteins or DNA.