Microfiltration membrane

Dried aerial parts were grounded (10 g), mixed with the 50% (v/v) ethanol in water as solvent, and shaken for 5 minutes for the extraction process. Next, the extraction was performed by ultrasonic-assisted extraction (UAE) using a sonication water bath (Elma Transsonic T 460, Germany) at 35 kHz for 90 min. After these steps, extracts were filtered through the filter paper under vacuum and microfiltered through 0.45 μm pore size microfiltration membrane (Millipore). Nanofiltration was applied to concentrate biologically valuable components and obtain polyphenolic-rich extracts. The nanofiltration was performed with a tangential filtration KOCH Membrane laboratory unit equipped with an NF90 (Dow Filmtec, Sterlitech Company, USA), with a molecular weight cut-off of 200–300 Da, made of polyamide material.

Several kinds of proportion of water soluble royal jelly protein and intestinal enzyme mixture had been tried as following: enzyme 15 mg and WSRJ 20 mg(lane B), enzyme 15 mg and WSRJ 40 mg(lane C), enzyme 15 mg and WSRJ 60 mg (lane D), enzyme 15 mg and WSRJ 80 mg(lane F).The WSRJ was about 42.7 mg/ml while intestinal enzyme mixture was about 31.9 mg/ml. The total volume was 10 ml, except for those two components, we added PBS to 10 ml. The optimized digestion condition for the intestinal enzyme solution and water-soluble royal jelly protein (1:3 mg/mg) mixture was 37 °C, pH 8.5 for 24 hours in a 2000-mL container. The reaction was stopped by placing the mixture in an ice bath, and the crude royal jelly peptides were obtained. This solution was centrifuged at 10000 g at 4 °C for 10 min to remove the insoluble components. Additionally, a 10 μm (Millipore, USA) microfiltration membrane was used to eliminate impure contents. Afterwards, crude RJPs were preliminarily purified through a 5-kDa, 3-kDa and 1-kDa ultra-filtration column and RJPs with MW < 1 kDa, 1-3 kDa, and 3-5 kDa were obtained (MSM-2008, Shanghai Mosu Science Equipment, China). Then, the digests were lyophilized and reconstituted in distilled water. In addition, the digests were filtered using a 0.22 μm (Millipore, USA) membrane, and the protein concentration was measured using a BCA assay.