Heto powerdry ll1500

V. floribundum Kunth fruit samples were collected between March and April 2019 in Sanchez‐Carriòn province (La Libertad, Peru) and gathered at the National University of San Marcos (Lima, Peru), where their taxonomy was certified by the Herbario San Marcos (National University of San Marcos, Lima, Peru). Berries were mashed, freeze‐dried by a Heto PowerDry LL1500 (Thermo Fisher), finely ground in a mortar, and stored at −20°C until use. Optima® MS grade water, methanol (MeOH), and acetonitrile (ACN) were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Acetone, acetic acid, formic acid, sodium hypochlorite, sodium acetate, 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) diammonium salt (ABTS), and Folin–Ciocalteu's phenol reagent were purchased from Merck (Kenilworth, New Jersey, USA). L. plantarum starters were supplied by the American Type Culture Collection (ATCC, Manassas, Virginia, USA). De Man, Rogosa, and Sharpe (MRS) agar powder and yeast from Saccharomyces cerevisiae were purchased from Sigma‐Aldrich (St. Louis, Missouri, USA). L. plantarum at 5% was activated in 10 mL of MRS agar broth for 16 h at 30°C in anaerobic conditions, diluted to 1% with an additional 40 mL MRS agar broth, and activated for a further 16 h under the same conditions.

For sample preparation, the hydrogel (control and strained) was firstly frozen at −80 °C for 24 h and then lyophilized by using a freeze-dryer (Heto PowerDry LL1500, Thermo Scientific) at room temperature for 12 h. The freeze-dried specimens were submerged into liquid nitrogen for about 5 minutes, and then fractured with a scalpel blade. The pore structure of hydrogels were sputter coated with platinum and then examined using a S-3000N, HITACHI scanning electron microscope (SEM). To test the porosity of hydrogels, hydrogel scaffolds were first thawed and hydrated for 24 hours. Hydrated scaffolds were weighed on a scale, and a Kimwipe was lightly applied to the scaffold surface for 40 s to wick away loosely held water, and the mass was again recorded. The interconnected volume was calculated as the mass of water wicked away divided by the total hydrated mass.

Each material was decocted in water, following the traditional instructions in the Chinese Materia Medica [40 ]. The decoction was filtered on cellulose 3 times, and the filtrate was lyophilized (Heto PowerDry LL1500, Thermo Fisher Scientific Inc., Waltham, MA, USA) and stored at −20 °C until further use. The extracts were dissolved and diluted with a complete culture medium to the required concentrations.

Solvent extraction method was used for the extraction of PHAs from the cells of P. aeruginosa as this method does not degrade the polymer (Jacquel et al. 2008 (link)). The cells were collected by centrifugation at 10,000 rpm and 4 °C for 10 min. Acetone washing was used to remove any organics from the cell surface (Brown 1996 ). The acetone washed cells were subjected to re-washing with an anionic surfactant Triton X-100 to make cells permeable and extract the organelles (Koley and Bard 2010 (link)). Then, the cells were freeze dried for overnight using Heto Power Dry LL1500 freeze dryer (Thermo Electron Corporation, USA). These cells were then re-suspended to 50 ml chloroform, shifted in a 250 ml flask and placed on the orbital shaker at 180 rpm at 30 °C for 24 h for the extraction of bio-polymer. Then, the cell debris was removed through Whatman^® filter paper and the filtrate was concentrated using the rotary evaporator. Then, chilled methanol was added (1:3) drop wise to precipitate PHA polymer (Tan et al. 2014 (link)). A milky precipitate containing solution (chloroform and methanol) was rotary evaporated and the concentrate containing the PHA polymers was weighed to a constant mass at 60 °C (Jiang et al. 2008 (link)).

TBF hydrochloride (10 g; 0.03 mmol) was dissolved in a 70 mL biphasic mixture of HPLC water and dichloromethane (DCM) (47:53), and to this, solid sodium carbonate (1.87 g) was added. The mixture was then stirred for 1 h at 20–25 °C. After this period, the organic layer was separated, and the aqueous layer washed three times with DCM (3 × 5 mL). The organic layers were then combined and dried with anhydrous magnesium sulphate. Subsequently, the solvent was evaporated with a rotary evaporator (Heidolph, Schwabach, Germany), leaving TBF as the free base (8.2 g; 0.028 mmol) with the appearance of a thick oil at a yield of 93.8%. The oil was treated with methanol and freeze-dried (Heto Power Dry LL 1500, Thermo Electron Corporation, Warwickshire, UK) to yield TBF base as a white solid. The methodology was adapted from Korean patent publication no. KR100979903B1 with slight modifications [21 ]. NMR analysis was subsequently carried out with a Bruker Avance 500 MHz NMR (Bruker Corporation, Billerica, MA, USA) equipped with broadband and selective (¹H) inverse probes. The analysis was conducted at a temperature of 300 K and chloroform-D (CDCl₃) was used as the solvent.