Heto powerdry ll3000

The EEB was prepared with distilled water (33 g/L). The solution was heated in the Uniclave 99 (A.J. Costa (Irmãos), Agualva-Cacém, Portugal) autoclave at 121 °C for 15 min and then put in a cold chamber at 4 °C until it cooled. It was then filtered using Whatman filter paper number 1 and gauze, in order to recover as much extract as possible. After that, the solution was placed in the freezer at −20 °C and dried in the Heto^® PowerDry LL3000 (Thermo Fisher Scientific, Milford, OH, USA) freeze dryer.

Milled cassava sample was incubated at 40 °C for 4 hours in a 0.1 M NaAc buffer containing 5 mM calcium and pH equal to 5.0 ± 0.05, both in the absence and presence of the NSPase product at 250 ppm. Afterwards, samples were centrifuged, and the supernatant was filtered (1.2 µm) and freeze-dried (Heto PowerDry LL3000, Thermo Fisher Scientific, USA). Subsequently, 10 mg of lyophilizate was resuspended in 1 ml of Milli-Q water. Aliquots of 0.7 µl of sample mixed 1:1 with THAP matrix (20 mg/ml in a 50% methanol solution) was applied to an anchorchip target plate. The analysis was run on MALDI MS (UltrafleXtreme TOF/TOF, Bruker, Germany), equipped with a 355 nm Smartbeam II Nd:YAG (neodymium-doped yttrium aluminium garnet) laser operated at 2 kHz in reflectron positive-ion mode (reflector 5.7x). Mass range was set between 100–4500 m/z. Maltodextrin (1 mg/ml) was used for mass calibration.
Data acquisition and analysis of oligosaccharides generated by treatment of cassava with the NSPase were performed using the Flex software suite (FlexControl 3.4 and FlexAnalysis 3.4, Bruker Daltonik, Germany).

Fibroin was extracted and purified from the raw Bombyx mori silkworm cocoons, following our previous studies (Pham et al., 2018 , 2019 (link), 2020 ). For this, the cocoons were degummed (i.e. sericin removal) using 0.5% (w/v) Na₂CO₃, washed with de-ionized water, and air dried. Then, the degummed silk (10 g) was cut into small pieces, dissolved in a hot (85 °C) mixture of CaCl₂:H₂O:Ca(NO₃)₂:EtOH (30:45:5:20 w/w/w/w), dialyzed against de-ionized water to remove residual salts, and centrifuged at 10,000 rpm for 30 min. Finally, the fibroin solution was freeze-dried (Heto PowerDry LL3000, Thermo Fisher, USA) at 10⁻⁴Torr and −55 °C. The fibroin powder was stored at −20 °C for further experiments.

The aqueous extract was prepared by boiling under optimum conditions, and the filtrate was freeze-dried with a Heto PowerDry LL3000 freeze dryer (Thermo, USA) and stored at −80°C. The components and content of Eucommia leaf extracts were determined by HPLC. The standard chemicals (aucubin, geniposidic acid, carnosine, catechin, isoquercitrin, chlorogenic acid, and astragaloside) were purchased from Shanghai Yuanye Biotechnology Co., Ltd (Yuanye, China). HPLC analysis of ELAE was performed using a 2695 liquid chromatographic system equipped with an inverted C18 column (Waters, USA). The mobile phase was a gradient elution system composed of water containing acetonitrile (A)-0.1% phosphoric acid solution (B), with a flow rate of 1 ml/min, and a column temperature of 35°C. Acetonitrile (A)-0.1% phosphoric acid solution (B) was eluted in a gradient manner (0–7 min, 5% A; 7–9 min, 5% A ⟶ 8% A; 9–28 min, 8% A; 28–30 min, 8% A ⟶ 20% A; 30–42 min, 20% A; 42–43 min, 20% A ⟶ 50% A; 43–63 min, 50% A; 63–64 min, 50% A ⟶ 5% A). A photodiode array (PDA) detector was set at 254 nm, and the online UV spectrum was recorded within the range of 200–300 nm.

BioTe with its synthesizer A. pittii D120 was observed by using an SEM Regulus 8230 (Hitachi, Tokyo, Japan). Purified BioTe was analyzed by using an HR-TEM coupled with energy dispersive X-ray spectrometry JEM-F200 (JEOL, Tokyo, Japan). The particle size of BioTe was measured by using a software image J according to images of HR-TEM. To analyze the crystal structure, BioTe solution was concentrated and dried to powder by using a vacuum freeze dryer Heto PowerDry LL3000 (Thermo Fisher Scientific, Waltham, MA, USA). BioTe powder was analyzed by using an XRD Empyrean S3 (Rigaku, Tokyo, Japan). The surface charge of purified BioTe was quantified by using Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK). For the zeta potential analysis, 500 μL BioTe solution was analyzed by using a Zetasizer Nano ZS. A He-Ne laser (633 nm) was used as the light source, the scattering angle was 90 degrees, and the temperature was 28 °C. The experiment was repeated three times.