4.1. γ-PGA Production, Purification, and Characterization

Bacterial γ-PGA was produced by B. licheniformis ATCC 9455a in a 5 L fermenter (Electrolab, Tewkesbury, UK) as reported earlier [49]. Infrared spectra were recorded on a Nicolet 380 FT-IR (Thermo Fisher Scientific Inc., Wilmington, DE, USA) with 32 scans and 4 cm⁻¹ resolution. Samples were ground with KBr (Sigma Aldrich, Milano, Italy) (1 mg sample/100 mg KBr), and pelletized under pressure.

TGA measurements were carried out using a TA-TGA2950 (TA Instruments, New Castle, DE, USA). Analyses were performed at 10 °C/min from room temperature to 600 °C both under air and under nitrogen flow. DSC measurements were performed using a TA-DSC-Q100 (TA Instruments, New Castle, DE, USA) apparatus equipped with a liquid nitrogen cooling system (LNCS) accessory. Heating scans were run at 20 °C/min, from −100 °C to 200 °C in a helium atmosphere. Between heating scans, quench cooling was applied. Melting temperature (T_m) was taken at the peak maximum of endotherm, while the glass-transition temperature (T_g) was taken as the midpoint of the stepwise increase of the specific heat associated with the transition.

Wide-angle X-ray diffraction measurements (WAXS) were carried out at room temperature with a PANalytical X’Pert PRO diffractometer equipped with an X’Celerator detector for ultrafast data collection (PANalytical B.V., Almelo, The Netherlands). A Cu anode was used as X-ray source (KR radiation: λ = 0.154 18 nm, 40 kV, 40 mA), and ¼ divergence slit was used to collect the data in 2θ range from 2° to 60°. The degree of crystallinity (χ_c) was evaluated as the ratio of the crystalline peak areas to the total area under the scattering curve.

Dynamic mechanical measurements (DMTA) were carried out in dual cantilever mode, at 3 °C/min and 3 Hz from −120 °C to 280 °C using a DMTA MkII (Polymer Laboratories Ltd., Church Stretton, UK). A strip (8 mm wide) cut from a glass fiber nonwoven mat (Whatman GF/C, Sigma Aldrich, Milano, Italy) was used to support the polymer. Polymer deposition was carried out by means of multiple immersions of the glass-fiber support in a 1% aqueous solution of Sample 2. A solvent-evaporation step (in oven under vacuum at 80 °C) was performed between consecutive immersions. The final glass-fiber strip coated with the polymer was subjected to DMTA measurement.

Rheological measurements were carried out at 25 °C using an Anton Paar MCR102 Rheometer (Anton Paar GmbH, Graz, Austria) with cone-plate CP50-1-SN19392 geometry (d = 0.102 mm, acquisition time 100 s, shear rate from 0.01 s⁻¹ to 200 s⁻¹).

Static water-contact angle measurements were performed on samples coated on glass slides (from a 0.8% aqueous solution) by means of a Laurell (WS-650-23NPP) spin coater (Laurell Technologies Corporation, North Wales, PA, USA) under ambient conditions. The contact-angle experiments were run with an optical contact angle and surface-tension meter KSV’s CAM 100 (KSV, Espoo, Finland) using distilled water. The water-drop profile images were collected every second in a time range of 0–30 s. Four drops per sample were analyzed.

The enantiomeric composition of the produced γ-PGA was investigated using HPLC (Thermo Scientific, Schwerte, Germany). The hydrolysis of γ-PGA was performed according to many studies [50,51]. Initially, the lyophilized polymer was resuspended in 6 M HCl (Sigma, Irvine, UK) at the concentration of 1 mg/mL, and hydrolyzed at 110 °C for 24 h. The hydrolyzed samples were then lyophilized again and dissolved in water and methanol (30:70) to the final concentration of 300 μg/mL. After that, 10 μL of the samples was injected into a Chirobiotic T column, 25 cm × 4.6 mm I.D., 5 μm particles (Astec, Sigma-Aldrich, Irvine, UK) at 25 °C using a mobile phase: water; methanol (Thermo Fisher Scientific, Altrincham, UK); and formic acid (Thermo Fisher Scientific, Altrincham, UK) (30:70:0.02, respectively) at a flow rate of 1 mL/min according to the manufacturer’s instructions. Chromatography was performed using a Dionex Ultimate 3000 series HPLC (Thermo Scientific, Schwerte, Germany) connected to an ultraviolet (UV) spectrometer (Thermo Scientific, Schwerte, Germany). The analytical software used was Chromeleon 7 (Thermo Scientific, Schwerte, Germany). In order to determine retention time, standards of d- and l-form of glutamic acid were first tested. After that, the mass of glutamic acid was used to determine the amounts of d- and l-glutamic acid by peak integration. Number average molar mass M_n was determined by conventional aqueous-based GPC at Smithers Rapra in Shrewsbury, United Kingdom. An MZ Hema guard plus 2× Hema Linear column (Cognis Performance Chemicals Ltd., Southampton, UK) was used for analysis. The GPC experiments were carried out in 0.2 M NaNO₃, 0.01 M NaH₂PO₄, at pH = 7, with a flow rate of 1 mL/min at 30 °C. The GPC system used was calibrated with polyacrylate standards. The data were collected and analyzed using Polymer Laboratories’ “Cirrus 3.0” software Supplied by Polymer Laboratories (Salop, UK).