Design and Preparation of a Biobased Colorimetric pH Indicator from Cellulose and Pigments of Bacterial Origin, for Potential Application as Smart Food Packaging.

Nowadays, worldwide challenges such as global warming, pollution, unsustainable consumption patterns, and scarcity of natural resources are key drivers toward future-oriented bioeconomy strategies, which rely on renewable biobased resources, such as bacterial pigments and bacterial cellulose (BC), for materials production. Therefore, the purpose of this study was to functionalize bacterial cellulose with violacein, flexirubin-type pigment, and prodigiosin and test their suitability as pH indicators, due to the pigments' sensitivity to pH alterations. The screening of the most suitable conditions to obtain the BC-pigment indicators was achieved using a full factorial design, for a more sustainable functionalization process. Then, the pH response of functionalized BC to buffer solutions was assessed, with color changes at acidic pH (BC-violacein indicator) and at alkaline pH (BC-violacein, BC-prodigiosin, and BC-flexirubin-type pigment indicators). Moreover, the indicators also revealed sensitivity to acid and base vapors. Furthermore, leaching evaluation of the produced indicators showed higher suitability for aqueous foods. Additionally, color stability of the functionalized BC indicators was carried out, after light exposure and storage at 4 °C, to evaluate the indicators' capacity to maintain color/sensitivity. Thus, BC membranes functionalized with bacterial pigments have the potential to be further developed and used as pH indicators.

Characterization of Bioactive Colored Materials Produced from Bacterial Cellulose and Bacterial Pigments.

A Bacterial Cellulose (BC) film was developed and characterized as a potential functional bioactive material. BC films, obtained from a microbial consortium of bacteria and yeast species, were functionalized with the bacterial pigment prodigiosin, produced by Serratia plymuthica, and flexirubin-type pigment, from Chryseobacterium shigense, which exhibit a wide range of biological properties. BC was successfully functionalized at 15% over the weight of the fiber at 40 °C during 60 min, and a color strength of 1.00 ± 0.01 was obtained for BC_prodigiosin and 0.38 ± 0.02 for BC_flexirubin-type pigment. Moreover, the BC films showed moderate hydrophilic character following alkaline treatment, which was maintained after both pigments were incorporated. The porosity and mechanical performance of the functionalized BC samples also remained unaffected. Furthermore, the BC samples functionalized with prodigiosin presented antibacterial activity and were able to inhibit the growth of pathogenic bacteria Staphylococcus aureus and Pseudomonas aeruginosa, with inhibition rates of 97.89 ± 0.60% and 85.12 ± 0.17%, respectively, while BC samples functionalized with flexirubin-type pigment exhibited the highest antioxidant activity, at 38.96 ± 0.49%. This research provides an eco-friendly approach to grant BC film-based material with color and advantageous bioactive properties, which can find application in several fields, especially for medical purposes.

Antimicrobial Food Packaging Based on Prodigiosin-Incorporated Double-Layered Bacterial Cellulose and Chitosan Composites.

Nowadays, food packaging systems have shifted from a passive to an active role in which the incorporation of antimicrobial compounds into biopolymers can promote a sustainable way to reduce food spoilage and its environmental impact. Accordingly, composite materials based on oxidized-bacterial cellulose (BC) and poly(vinyl alcohol)-chitosan (PVA-CH) nanofibers were produced by needleless electrospinning and functionalized with the bacterial pigment prodigiosin (PG). Two strategies were explored, in the first approach PG was incorporated in the electrospun PVA-CH layer, and TEMPO-oxidized BC was the substrate for nanofibers deposition (BC/PVA-CH_PG composite). In the second approach, TEMPO-oxidized BC was functionalized with PG, and afterward, the PVA-CH layer was electrospun (BC_PG/PVA-CH composite). The double-layer composites obtained were characterized and the nanofibrous layers displayed smooth fibers with average diameters of 139.63 ± 65.52 nm and 140.17 ± 57.04 nm, with and without pigment incorporation, respectively. FTIR-ATR analysis confirmed BC oxidation and revealed increased intensity at specific wavelengths, after pigment incorporation. Moreover, the moderate hydrophilic behavior, as well as the high porosity exhibited by each layer, remained mostly unaffected after PG incorporation. The composites' mechanical performance and the water vapor transmission rate (WVTR) evaluation indicated the suitability of the materials for certain food packaging solutions, especially for fresh products. Additionally, the red color provided by the bacterial pigment PG on the external surface of a food packaging material is also a desirable effect, to attract the consumers' attention, creating a multifunctional material. Furthermore, the antimicrobial activity was evaluated and, PVA-CH_PG, and BC_PG layers exhibited the highest antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. Thus, the fabricated composites can be considered for application in active food packaging, owing to PG antimicrobial potential, to prevent foodborne pathogens (with PG incorporated into the inner layer of the food packaging material, BC/PVA-CH_PG composite), but also to prevent external contamination, by tackling the exterior of food packaging materials (with PG added to the outer layer, BC_PG/PVA-CH composite).

Chromatographic HPV-16 E6/E7 plasmid vaccine purification employing L-histidine and 1-benzyl-L-histidine affinity ligands.

Affinity chromatography based on amino acids as interacting ligands was already indicated as an alternative compared to ion exchange or hydrophobic interaction for plasmid DNA purification. Understanding the recognition mechanisms occurring between histidine-based ligands and nucleic acids enables more efficient purification of a DNA vaccine, as the binding and elution conditions can be adjusted in order to enhance the purification performance. Decreasing pH to slightly acidic conditions increases the positive charge of histidine ligand, what influences the type of interaction between chromatographic support and analytes. This was proven in this work, where hydrophobic effects established in the presence of ammonium sulfate were affected at pH 5.0 in comparison to pH 8.0, while electrostatic and cation-π interactions were intensified. Histidine ligand at pH 5.0 interacts with phosphate groups or aromatic rings of plasmid DNA. Due to different responses of RNA and pDNA on mobile phase changes, the elution order between RNA and pDNA was changed with mobile phase pH decrease from 8.0 to 5.0. The phenomenon was more evident with L-histidine ligand due to more hydrophilic character, leading to an improved selectivity of L-histidine-modified chromatographic monolith, allowing the product recovery with 99% of purity (RNA removal). With the 1-benzyl- L-histidine ligand, stronger and less selective interactions with the nucleic acids were observed due to the additional hydrophobicity associated with the phenyl aromatic ring. Optimization of sample displacement chromatography parameters (especially (NH4 )2 SO4 concentration) at slightly acidic pH enabled excellent isolation of pDNA, by the removal of RNA in a negative mode, with binding capacities above 1.5 mg pDNA per mL of chromatographic support.

Screening of L-histidine-based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform.

The growing demand of pharmaceutical-grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l-histidine and its derivatives, 1-benzyl-L-histidine and 1-methyl-L-histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l-histidine and its derivatives was high (KD > 10(-8) M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K(D) = 1.1 × 10(-10) M and KD = 3.34 × 10(-10) M for open circular and linear plasmid isoforms, respectively). L-Histidine and 1-benzyl-L-histidine were immobilized on monolithic matrices. Chromatographic studies of L-histidine and 1-benzyl-L-histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1-benzyl-L-histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l-histidine monolith was 29-fold higher than that obtained with conventional L-histidine agarose. Overall, the combination of either L-histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications.