2.2. Methods

Unadulterated cultures of B. breve ATCC 15700and B. longum BB536 were used. Gram staining was used to check the purity of bacterial cultures. The standard bacterial culture was proliferated and stored in 40% glycerol at −80 °C for further use. Bifidobacteria grow anaerobically. Anaerobic environment was obtained with Anaero Gen sachets (Oxoid Ltd., West Heidelberg/Vic., Australia).

Soymilk was produced following the procedure described by Hou et al. (2000) with few changes. Soybean grains were firstly cleaned up and soaked overnight in distilled water. The soaked soybeans were added to ten times the weight of (100 g dry soya bean to 1000 ml water) distilled water and boiled for 30 min at 95 °C in a water bath. Further it was blended for 5 min. The obtained slurry was then purified through double-layered cheesecloth to yield soymilk (New England Cheese making supply company, South Deerfield, MA, USA). Soymilk was autoclaved at 121 °C for 15 min and stored in a refrigerator (4 °C).

Viable cell counts of B. breve and B. longum were established in duplicate using the pour plate method on BHI agar medium. Each fermented soymilk was added to 90 ml sterile 0.85% saline (w/v) and vortexed for 30 s. Resultant suspension was serially diluted with sterile 9ml saline and 1 ml of the proper dilution was used for selective enumeration by the pour plate technique. The cell growth of each organism was assessed by enumerating a bacterial population on BHI agar at 0, 12, 24, 36 and 48 h of fermentation. To be effective, plates containing 30–300 colonies were counted and recorded as CFU per ml of fermented soymilk.

Bacterial species (B. breve ATCC 15700, B. longum BB536) were activated in BHI medium by relocating three times in 10 ml of BHI broth and incubation at 37 °C 20 h followed by collecting bacterial cells by centrifuging (3000 × g for 15 min). To get bacterial co-culture cell suspensions, the two cell suspensions were mixed at a volume ratio of 1:1. Inoculums of the bacterial single and co-culture were set by using 100 ml of sterile soymilk and incubation for 20 h at 37 °C.

B. longum BB536 and B. breve ATCC15700 were activated by incubating in 10 ml of BHI broth. Incubation was carried out at 37 °C for 20 h. Bacterial cells were collected by centrifugation at 3000 × g for 15 min. The inoculum of single culture for every bacteriological strain was made with 50 ml of sterile soymilk and incubation for 20 h at 37 °C. Ten milliliters of the vigorous culture were injected into 250 ml of each soymilk (5% v/v) batches of and incubated for 48 h at 37 °C. Fifty milliliters were withdrawn aseptically from every inoculum at 12, 24, 36 and 48 h of incubation to measure the enzyme activity. β-Glucosidase activity of the bacterial strains was evaluated by identifying the degree of hydrolysis of the substrate ρ-NPG. It was prepared in 100 mM sodium phosphate buffer (pH 7.0) (Millipore Sigma, Chemical Co., St. Louis, Mo– U.S.A). One milliliter of ρ NPG (5 mM) was added to 10 ml of each aliquot and incubated at 37 °C for 30 min (Otieno et al., 2006; Scalabrini et al., 1998). The reaction was ended by adding of 500 μl from 1 M cold sodium carbonate. The aliquot was transferred to centrifuge tube followed by centrifugation (14,000 g for 30 min) using Eppendorf refrigerated centrifuge (Model 5810 R). The quantity of ρ-nitro-phenol relieved was determined by Perkin Elmer spectrophotometer (Model: Lambda 25 UV/VIS Spectrophotometer) at 420 nm. One unit of the enzyme was defined as the amount of enzyme that released1 μ mol of ρ-nitro-phenol from the substrate ρ NPG, per ml per min under assay conditions.

The fermentation process was executed in 1 L volume bioreactor BIOSTAT QDCU3 (Sartorius BBI System GmbH, Melsungen, Germany) and controlling of temperature was achieved using water bath (Jeio Tech Desk Top, Seoul, South Korea) and an electronic stirrer (Gas-Col Ltd, Northvale, NJ 07647, USA). The temperature was set at 37 °C. Anaerobic condition for fermentation was conserved by flushing oxygen-free nitrogen gas through the medium. No control stood for pH. The stirring speed for all batch fermentation was set at 200 rpm/min. One hundred ml inoculums of single culture for each bacterial strain (B. longum BB536 and B. breve ATCC 15700) in sterile soymilk were transferred to the fermenter to inoculate the soymilk in a 2-L vessel (with 1 L working volume). Samples of fermented soymilk were taken at 0, 24 and 48 h into sterile universal bottles to examine changes on isoflavones concentrations.

Fermented soymilk (2 ml) was added to 80% methanol (8 ml) and stirred for 2 h at 25 °C.

Then, the blend was centrifuged at 9000 rpm for 20 min. The supernatant was clarified using a 0.22 μm syringe membrane into HPLC vials and kept at -20 °C for HPLC investigation.

HPLC protocol was in accordance with the method mentioned by Elghali et al. (2012) with some alterations. Twenty microliters of sample were injected into high-performance liquid chromatography (HPLC) (Model CO-2065 JASCO Corporation Hachioji, Tokyo, Japan) equipped with C18 reversed-phase column (25 cm × 4.5 cm × 5 μ) (Ascentis–Supelco, Sigma-Aldrich Co. LLC. L, USA), diode array ultraviolet (UV) visible detector, vacuum degasser, and thermostatically controlled column compartment. Column temperature was set at 27 °C. HPLC gradient elution was composed of 10% acetonitrile solution in water (solution A) and 90% acetonitrile solution in water (solution B). The elution program was as follows: solution B was run at 30% for 15 min, linearly increased to 50% for 10 min, and then linearly increased to 70% for 5 min. The flow rate was at 1 ml/min. A diode array UV-visible detector was set at 270 nm. UV spectra and retention times of the metabolites produced from daidzin and daidzein by bacteria were compared with those of the standard compounds daidzin, daidzein and equol in HPLC chromatograms.

Commercial sugars and prebiotics were screened for ability to enhance equol production from fermented soymilk. They were: glucose (≥99.5%) and sucrose (≥99.5%) purity [Sigma, Louis, USA], inulin and fructo-oligosaccharides (OraftiPty. Ltd, Tienen, Belgium). The inulin used was Raftiline ST with a purity of 92% and an average degree of polymerization (DP) of 10. The fructo-oligosaccharide (FOS) which utilized was Raftilose P95 that formed from 5% of glucose, fructose and sucrose. It also composed of oligo-fructose with DP ranging from 2-7 with an average of 4. One hundred ml of sterile soymilk supplemented with Inulin, FOS, Glucose and Sucrose (1%w/v) individually was inoculated with activated culture of (B. breve ATCC15700 and B. longum BB536) and incubated anaerobically at 37 °C for 48 h. The soymilk medium was set to contain a final concentration 1% (w/v). Trials of inoculated soymilk were taken at 12, 24, 36 and 48 h to measure the quantity of isoflavones by the usage of HPLC (see section 2.2.8).