Dm il led

¹H NMR and ¹³C NMR were measured using a nuclear magnetic resonance spectrometer (AVANCE III 500 MHz, Bruker, Germany). ESI-HRMS was recorded using high-resolution mass spectrometry (Thermo Scientific Q Exactive, Bruker, Germany). IR absorption spectra were recorded in the range of 4000–400 cm−1 on a WQF-510A FTIR spectrophotometer. UV/Vis spectra were recorded on a UV-3600i plus spectrophotometer (UV/Vis/NIR spectrophotometer, Shimadzu, Japan). CD spectra were recorded by circular dichromatic spectrometer (Chirascan/V100, Applied Photophysics, UK). In the MTT assay, the absorbance of each compound was measured at 570 nm using Multiskan GO (Thermofisher, Thermo Fisher Scientific, Waltham, MA, USA). AO/EB staining was performed under an inverted fluorescence microscope (DMIL LED, Leica, Germany). Cell apoptosis and ROS intensity were detected using the flow cytometry system (Beckman coulter CytoFLEX, Beckman Coulter, Pasadena, CA, USA). In addition, ROS intensity was also detected using an inverted fluorescence microscope (DMIL LED, Leica, Germany).

After 2 days of culture, the cells were washed twice with PBS and fixed with 4% Paraformaldehyde (PFA, Sigma–Aldrich) for 10 min at room temperature followed by three PBS washes. Plates were then sealed with parafilm and stored in the fridge until immunofluorescence. Cells were permeabilized with PBS containing 0.1% Triton (Sigma–Aldrich; TPBS) for 10 min and washed in PBS three times before being incubated with a blocking solution containing 10% donkey serum (Merck) in TPBS for 30 min. The blocking solution was then aspirated and the samples were washed three times in TPBS washes. The cells were then incubated with osteocalcin (1:200, Abcam, Cambridge, UK) in 10% donkey serum and 0.1% TPBS overnight at 4 °C. After 24 h, the primary antibody solution was decanted and the cells were washed three times with PBS and incubated with the secondary antibody Alexa Fluor 488 (1:500, Abcam) in 5% donkey serum and 0.2% TPBS for 1.5 h at room temperature. Following this, cells were incubated for 5 min with Hoechst (1:5000, Life Technologies, Scoresby, Australia) and three PBS washes were performed prior to mounting the coverslips on a glass slide. Cells were then visualized under a fluorescence microscope (Inverted Laboratory Microscope, Leica DM IL LED, Wetzlar, Germany).

Light micrographs of the yogurt-based beverages were obtained using a Leica DM IL LED inverted laboratory microscope equipped with a Leica DFC295 digital color camera (Leica microsystems Ltd., Milton Keynes, UK). The samples were observed through a 40×dry objective lens. Pictures were taken using the in-built 3 MP digital camera and picture analysis was performed using Leica application suite software (V.3.6.0).