An efficient measure for the isolation of chenodeoxycholic acid from chicken biles using enzyme-assisted extraction and macroporous resins refining

Abstract

Chicken bile is a by-product of chicken processing, rich in chenodeoxycholic acid (CDCA), an active pharmaceutical raw material. In this study, a green measure for the extraction and purification of CDCA from chicken biles by enzymatic hydrolysis and macroporous resins refining was established. For the assisted extraction of CDCA, the active bile salt hydrolase (BSH) from Bifidobacterium was heterologously expressed and applied, its activities on GCDCA and TCDCA were 4.96 ± 0.32 U/mg and 3.07 ± 0.031 U/mg and optimal catalytic conditions for the extraction of CDCA were determined as 0.04 g/g of the enzyme dosage, pH 5.0 and 38 °C. Through validation of the conditions, the yield of CDCA was up to 5.32 %, which was equivalent to that by saponification method. In order to further refine CDCA from the extract obtained by enzyme-assisted extraction, a more preferable resin, AB-8 was selected for the purification of CDCA, which had a good adsorption capacity of 61.06 ± 0.57 mg/g for CDCA. Besides, the obtained CDCA extract was purified through AB-8 resin, the purity of CDCA was improved from 51.7 % to 91.4 % and the recovery yield of CDCA was 87.8 %. The advantages of energy conservation, time saving, economy and environmental friendliness make the measure using enzyme-assisted extraction and macroporous resins refining a promising candidate for isolation of CDCA from chicken bile.

Keywords: Adsorption; Bile salt hydrolase; Chenodeoxycholic acid; Macroporous resin; Purification.

Fig. 1

Schematic diagram of an efficient measure for the isolation of chenodeoxycholic acid from chicken biles using enzyme-assisted extraction and macroporous resins refining.

Fig. 2

SDS-PAGE of recombinant protein. M: Standard Marker; 1: un-induced E.coli BL21 (DE3) (pET-22b (+)) products; 2: the supernatant obtained by breaking cell walls of induced E.coli BL21 (DE3) (pET-22b (+)); 3: induced E.coli BL21 (DE3) (pET-22b (+)); 4: the precipitation obtained by breaking cell walls of induced E.coli BL21 (DE3) (pET-22b (+)).

Fig. 3

Response surfaces and contour plots show the effects of dosage of enzyme (A), pH (B) and enzymolysis temperature (°C) on the yield of CDCA. (a) Dosage of enzyme and pH; (b) Dosage of enzyme and enzymolysis temperature; (c) Enzymolysis temperature and pH.

Fig. 4

Static adsorption capacities and desorption ratios of ten macroporous resins.

Fig. 5

Isothermal adsorption curves of AB-8 for CDCA at different temperatures (a) and dynamic absorption breakthrough curves of CDCA adsorption at different loading concentrations (b) and flow rates (c).

Fig. 6

Dynamic desorption ratios of AB-8 macroporous resins. (a) Investigation of gradient ethanol dolution concentration. (b) Gradient elution curve of CDCA. 0–24 BV was obtained by eluting with 40.0% ethanol, and 25–45 BV was obtained by eluting with 50.0% ethanol. (c) The amount of CDCA in the elution fractions detected by TLC. S was a CDCA extract sample before purification, 1–24 (40%) and 0.5–22 (50%) were the BV numbers of collecting tube was eluted by 40% and 50.0% ethanol, 3 mL (0.5 BV) for each tube.