Pectinex

Pectinex (9500 polygalacturonase units per millilitre; PGU ml⁻¹) was obtained from Novozymes (Pty) Ltd (Johannesburg, SA), amoxicillin-clavulanate from Sandoz (SA) and ciprofloxacin from Adcock Ingram (SA).

Scenedesmus obtusiusculus Hydrolysate (SH) was prepared by hydrolyzing autoclaved algae biomass. Briefly, dry biomass samples weighing 50 g were transferred to 2 L glass bottles containing 1 L of 50.0 mM sodium acetate buffer, pH 5.0. Different hydrolytic enzyme mixtures were examined, including Cellic-Ctec2 (Novozymes, Denmark), Celli-Htec2 (Novozymes, Denmark), Pectinex (Novozymes, Denmark) and Fungamyl (Novozymes, Denmark). Hydrolytic Reactions were initiated by adding the enzyme solution and incubating the mixture at 50 °C for 72 h. Buffer and enzymes were sterile-filtered prior to hydrolysis. Samples were then spun down for 30 min. Cross-filtration using a 10 kDa membrane made from regenerated cellulose was completed under the following parameters: Inlet-Pressure (P1) of 2 bar, Repentant-Pressure (P2) of 0.3–0.5 bar and permeate was open to atmospheric pressure. Flow-Rates of retentate and permeate were adjusted to 2 L/min and 0.1 L/min respectively. A 0.2 µm filter capsules were installed at the outlet to sterilize the resulted hydrolysate. Biological triplicates of the SH were prepared.

The fucoidan from sea mustard (Undaria pinnatifida) was obtained from Bion F&B (Korea), which was estimated as 31-34% sulfate contents according to a certificate of chemical analysis provided by the company. Three different commercial carbohydrate-hydrolyzing enzymes, AMG (rich in amylase), Pectinex (rich in pectinase), and Viscozyme (rich in cellulase, β-glucosidase, xylanase), were purchased from Novozymes (Bagsvaerd, Denmark) for enzymatic biotransformation of fucoidan. Macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-Β ligand (RANKL) for the differentiation of osteoclast were purchased from PeproTech (UK). α-Minimal essential medium (MEM) containing 100 μg/ml streptomycin, 100 U/ml penicillin, and fetal bovine serum (FBS) were purchased from Gibco (USA). The ascorbic acid and β-glycerophosphate used for inducing the osteoblast differentiation and tartrate-resistant acid phosphatase (TRAP) staining kit for detecting the osteoclast differentiation were purchased from Sigma Chemicals (USA). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was purchased from Duchefa (Netherland). To isolate and purify total RNA, TRIzol reagent was used (Life Technologies, USA).