Modulyo freeze dryer

All spectra analysis were done on a Jasco UV-Vis spectrophotometer model-(V-530) (Japan), a Jasco Fourier Transform Infrared (FT/IR) Spectrometer model-4100 (Japan) and a JASCO spectrofluorometer model-FP-6300 (Japan). Edwards Modulyo Freeze Dryer for lyophilization of prepared samples before FTIR measurments. Gold nanospheres core diameter was measured by High Resolution Transmission Electron Microscopy (HRTEM) model-(JEOL JEM-2100), while nanospheres hydrodynamic diameter and surface charge (zeta potential) analyzed by Malvern Zetasizer (Nano-ZS90). PH measurements measured by WTW inoLab $\circledR$ Multi 9620 IDS Multiparameter Benchtop Meter. Automatic Water Still. Electronic Balance. Micro Quartz Cuvette (1 ml). 250 ml single-neck flask with flat base. Distillation and reflux condenser. Thermo Fisher Scientific^TM hotplate and stirrer.

Fresh leaves (approx. 6) from each developmental stage were collected and freeze-dried. The leaves were trimmed into segments (4 mm × 4 mm) and quenched in subcooled liquid nitrogen. Leaf segments were freeze-dried using an Edwards–Modulyo freeze dryer for 72 h (−60 °C in a vacuum of 10–2 Torr). The segments were mounted and secured onto brass stubs using carbon conductive tape. The segments were coated in gold using a Polaron SC500 Sputter Coater and viewed on a LEO 1450 SEM at a working distance of 14–19 mm. Smart SEM version 5.03.06. was used for image capture.

Fluorescent chitosan NS were obtained by labeling freeze-dried pre-formed chitosan NS with fluorescein isothiocyanate (FITC), which was previously reported. For this purpose, an FITC dimethylsulfoxide solution (10 mg/mL) was added to the chitosan nanosphere suspension under stirring for 24 h at room temperature in the dark. At the end of the labeling procedure, an aliquote of NS aqueous nano-suspension was freeze-dried using a Modulyo Freeze-dryer (Edwards, Crawley, UK) to obtain a solid powder.

A top-down method was used to generate the GSH/pH-NS from a coarse powder. First, a weighted amount of the GSH/pH-NS was suspended in saline solution (0.9% w/v NaCl) at a concentration of 10 mg/mL while stirring at room temperature. The suspension was then dispersed using a high shear homogenizer (Ultraturrax^®, IKA, Konigswinter, Germany) for 5 minutes at 24,000 rpm. The samples were subjected to HPH for 90 minutes at a back pressure of 500 bar using an EmulsiFlex C5 instrument (Avestin, Mannheim, Germany) to further reduce the sizes of the NS and obtain a homogenous distribution. The GSH/pH-NS were then purified by dialysis (Spectrapore cellulose membrane, MWCO: 12,000 Da) and stored at 4°C. A fraction of the GSH/pH-NS was freeze-dried using a Modulyo Freeze-Dryer (Edwards, Crawley, UK).

B21P2 and IS1 were streaked onto GYM agar plates and grown for 7 days. Production of the bioactivity was assessed by using the plug assay. Agar from the plates was harvested, disrupted by passaging through a 50 ml syringe and frozen overnight at −20°C. A water-based extract was produced by squeezing the thawed slurry through muslin cheesecloth and filtering the resultant liquid through Whatman filter paper. A concentrated extract was obtained by freeze drying the filtered extract in an Edwards ‘Modulyo’ freeze dryer and resuspending the lyophilised material in an appropriate volume of water. The agar–cell mixture produced was resuspended in a volume of acetone equal to the initial volume of water-based extract and incubated overnight at 4°C. Acetone-based extract was produced by squeezing the mixture through muslin cheesecloth, filtering it and removal of acetone using a Büchi vacuum rotary evaporator to generate a concentrated extract comprising the residual water present in the spent agar–cell slurry.

The whole root and shoot samples were ground into a fine powder using a Freezer Mill (SPEX SamplePrep) following the manufacturer’s instructions. Samples were then lyophilized in a Modulyo freeze dryer (Edwards, West Sussex, UK) for at least 48 h or until thoroughly dried, and then submitted for chemical analysis. The total N content of dried root or shoot samples was measured by the LECO CN628 combustion analyser following the manufacturer’s protocol. For elemental analysis, root and shoot samples were first digested using 85:15 (v/v) nitric acid:perchloric acid in an open tube digestion block. After volatilization of the acids, the residues were dissolved in 5% (v/v) nitric acid. Sample analysis was carried out with an Agilent 5900 SVDV inductively coupled plasma-optical emission spectrometer (ICP-OES).

Burned inner and outer chestnut shells (CS) were kindly provided by Ingino s.r.l. food factory (Montoro Inferiore, Avellino, Italy). CS were dried in oven at 55 °C until reaching constant weight and powdered using a food homogenizer (type 8557-54, Tefal, France). Three different extractions, using water as a solvent, were carried out for the recovery of the bioactive molecules: 1) CLE in boiling water under continuous and vigorous stirring; 2) UAE at 60 °C in ultrasonic bath frequency 28–34 kHz, power 80–180 W, 230 V) (Ultrasonic Falc, Treviglio, Bergamo, Italy); 3) MAE (frequency 2450MHz, power 1000 W, input 1080 W, output 700 W). All the extractions were conducted for 60 min using 5% (w/v) CS. After the extraction process, each suspension was cooled on ice, centrifuged at 3220× g for 1 h at 4 °C (Eppendorf 5810R), and the supernatant was recovered. The remaining solid residue was rinsed with the same volume of water lost during the extraction, the resulting suspension was centrifuged as above, and the supernatant was added to the previous one in order to restore the original volume. The clear solutions were freeze-dried in an Edwards Modulyo freeze-dryer (Edwards, Cinisello Balsamo, Milano, Italy), and the powder chestnut shell dry extract (CSDE) was stored at room temperature (RT) until use.