Chemicals and reagents
HPLC-grade acetonitrile was purchased from Merck KGaA Co. Ltd (Darmstadt, Germany), and purified water was provided by Hangzhou Wahaha Co. Ltd (Zhejiang, China). Analytical-grade methanol, n-butanol, petroleum ether, ethanol, KH2PO4 and K2HPO4 were purchased from Beijing Chemical Reagents Co. Ltd (Beijing, China). Enterodiol Standard was purchased from Sigma Chemical Co. (St. Louis, MO., USA). Amberlite XAD-2 macroporous resin (20-60 mesh size, 330 m2 g-1 average surface area) was purchased from Supelco, Sigma-Aldrich Co. Ltd (Bellefonte, USA). Optical rotations were measured in MeOH solutions with a DIP-360 automatic polarimeter (Jasco Co., Tokyo) at 25°C, and CD spectra were determined with a JASCO J 805 spectropolarimeter (Jasco Co.).
Flaxseed samples were collected from Bei-An County of Heilongjiang Province, China, and were identified as the dried seeds of Linum usitatissimum L. by author. Voucher specimens (sample no. 071024) were deposited was deposited in the herbarium of pharmacognosy research group, School of Pharmaceutical Sciences, Peking University Health Science Center. They were ground into powder (pass 40 mesh sieve) and then defatted by petroleum ether prior to use.
Culture media and bacterial culture
Cooked meat medium base and Luria-Bertani (LB) nutrient agar were purchased from Beijing Land Bridge technology Co. Ltd (Beijing, China). Medium A contained tryptone 30 g, yeast extract 5 g, beef powder 5 g, glucose 3 g, NaH2PO4 5 g and amidulin 2 g, and the volume was made up to 1 liter with distilled water. Medium B was designed to lack any carbon source in the medium except defatted flaxseeds (see below), containing the following reagents (in one liter): NaCl 3 g, KH2PO4 2.6 g, K2HPO4 1.85 g, 1% (v/v) reducing solution (30 g/l L-aminothiopropionic acid and 30 g/l sodium hyposulfite, dissolved in PBS), and 1 g NH4Cl. Medium C was the same as medium B except the absence of any nitrogen source.
Culture was conducted as follows: 0.3 g of defatted flaxseeds was added into each of tubes containing either medium A, B or C (3 ml), which were then sealed with liquid paraffin and autoclaved at 121°C for 15 min. Into the medium, 0.3 g of fresh human feces was added and incubated at 37°C for 72 h. Supernatant of the cultures was then inspected for the appearance of END.
Collection and processing of fecal samples
Initially, fresh fecal specimens (ca. 4.0 g each), obtained from 28 healthy young subjects (fourteen females and fourteen males, 22-33 years old), were suspended in 20 ml sterile phosphate buffer saline (PBS, 2.6 g l-1 KH2PO4, 1.85 g l-1 K2HPO4, PH 7.4) and 2 ml such fecal suspension was transferred to 20 ml medium, followed by incubation at 37°C for 36 h. During the fecal collection and culture preparation, no strictly anaerobic techniques or instruments were used. The fecal specimen that we used for END production was from a 33 years old female.
High-performance liquid chromatography (HPLC)
The HPLC system consisted of Agilent 1200 series HPLC apparatus (Agilent Technologies, USA), including high-pressure binary-gradient solvent-delivery pump, DAD detector, autosampler, thermostat column compartment and chemstation (9.01 edition). Zorbax SB-C18 column (4.6 mm × 250 mm, 5 μm) was used to analyze all of the samples. Mobile phase consisted of water (A) and acetonitrile (B) in a linear gradient change from 100% A to 50% A and 50% B in 30 min. Detection wavelength was 280 nm, and the temperature of the column oven was 25°C with a flow rate of 1.0 ml min-1.
Calibration of the END and SECO curves
The stock solutions of END standard (1.98 mg ml-1) and SECO standard (175.5 μg ml-1) were prepared by accurately weighing and transferring each of them into a volumetric flask (1 ml) and dissolving it in methanol. Solutions for END calibration (0.0198 ~ 1.98 mg ml-1) and SECO calibration (175.5 ~ 2.74 μg ml-1) were prepared by dilution of the stock solutions with methanol, with six dilution series being analyzed (1.98, 0.99, 0.396, 0.198, 0.099, 0.0198 mg ml-1) for END calibration and seven dilution series being analyzed (175.5, 87.75, 43.86, 21.94, 10.97, 5.48, 2.74 μg ml-1) for SECO calibration. For each calibration curve, independent dilutions were analyzed. The calibration equation of END was obtained by plotting HPLC peak areas (Y) versus the concentration of calibrators (X, mg ml-1), which was as follows: Y = 4433.46 X + 63.86 (R2 = 0.9999), with a good linearity over the range from 0.0198 mg ml-1 to 1.98 mg ml-1, and the calibration equation of SECO was obtained by plotting HPLC peak areas (Y) versus the concentration of calibrators (X, μg ml-1), which was as follows: Y = 12.59 X - 1.40 (R2 = 0.9998), with a good linearity over the range from 2.74 μg ml-1 to 175.5 μg ml-1.
Limits of detection and quantification
Stock solutions of END and SECO standards were separately diluted to make a series of solutions with methanol and analyzed by HPLC. On the basis of signal-to-noise ratio (S/N), the limits of detection (LOD) and quantification (LOQ) of END standard were determined to be 0.699 μg ml-1 (S/N = 3) and 1.398 μg ml-1 (S/N = 10), respectively. The LOD and LOQ of SECO standard were determined to be 0.690 μg ml-1 (S/N = 3) and 1.370 μg ml-1 (S/N = 10), respectively.
Sampling of the cultures
A volume of 200 μl of culture was sampled every 24 h and extracted with 400 μl n-butanol saturated with water. A portion of n-butanol extracts (320 μl) was transferred to a centrifuge tube and evaporated to dryness by N2. The residue was dissolved in 200 μl methanol and centrifuged for 3 min (12500 r min-1), and then 20 μl of the supernatant was filtered and analyzed by HPLC.
Successive passages of cultures for sustained production of END
A culture was started with a fecal specimen at 37°C and sampled every 24 hours for analysis by HPLC. As END could be detected in the culture as early as within the first 24 hours at concentrations of 31.45 ± 1.51 mg l-1 and the yields remained relatively stable for 6 days (starting to decline on day 9; data not shown), we used an interval of 6 days for successive passages of the culture by 1:10 dilutions in medium B without paraffin, as strict anaerobic culture conditions were not necessary (see above). A portion of the first fecal culture was stocked on day 6 from the initiation of the culture in 25% (v/v) glycerol at -80°C as "passage 1" (designated as END-1); a portion of each of all successive subcultures was stocked on the 6th day of the culture in the same way and was designated as END-2, END-3, and so on. To identify the bacteria that were involved in the biotransformation of flaxseed lignans into END, we first needed to select them out of the initial bacterial mixture in the fecal specimen. Our general strategy was to dilute the cultures in which END was produced and use the highest dilution of the bacterial culture that still produced END for successive passages in medium B, which would support only the bacteria that use defatted flaxseeds as a carbon source.
Pulsed field gel electrophoresis (PFGE)
The endonucleases I-CeuI, AvrII, XbaI and SpeI were purchased from New England Biolabs. PFGE was performed in a CHEF - DRII system (Bio-Rad). Preparation and digestion of high molecular weight genomic DNA, digestion of DNA in agarose blocks and separation of DNA by PFGE, were as reported [30, 31].