Foods 2020 , 9 , 1147
4of 19
The study product was manufactured at Teagasc, Moorepark, under HACCP Food Quality Standards. No chemical additives were used, and the product was free of sulfites, sulfates and aflatoxins. The source material was produced in an ISO 22000 and HAACP compliant facility. 2.3. Sample Preparation and Mass Spectrometry Analysis Samples (100 µ L) were desalted and concentrated using 30-kDa Spin-X UF centrifugal concentrators (Corning Inc., Lowell, MA, USA). Each flow-through was then acidified in formic acid and cleaned of contaminants by solid phase extraction (SPE) on an Empore 96-well disk plate with C18-SD sorbent (3M, St. Paul, MN, USA). Eluates were lyophilized and resuspended in 0.1% TFA for analysis. Samples were analyzed by nano LC-MS / MS with a Waters NanoACQUITY HPLC system (Waters Corporation, Milford, MA, USA) interfaced to a ThermoFisher Q Exactive (ThermoFisher Scientific Inc., Canoga Park, CA, USA). Peptides were loaded on a trapping column and eluted over a 75 µ m analytical column with a 1 h gradient at a flow rate of 350 nL min − 1 . Both columns were packed with Luna C18 resin (Phenomenex, Torrance, CA, USA). The mass spectrometer was operated in data-dependent mode, with MS and MS / MS performed in the Orbitrap at 70,000 FWHM and 17,500 FWHM resolution, respectively. From the MS scan, the fifteen most intense ions were selected for MS / MS. To confirm the presence of predicted peptides in rice NPN, raw data from the Q Exactive was processed by MaxQuant version 1.5.3.17 (Martinsried, Germany) [37] with the Andromeda search engine [38]. MS / MS spectra were searched against the Uniprot Oryza sativa L . subsp. japonica database. All searches were carried out in unspecific digestion mode with a minimum peptide length of 5 amino acids and a fragment mass tolerance of 20 ppm. A false discovery rate (FDR) of 0.01 was selected for both peptides and proteins. Database searches were performed with no fixed modifications and with oxidation (M) as a variable modification. 2.4. Cell Culture, Di ff erentiation of Monocytes and Inflammation ELISA Assays Human monocytic leukemia (THP-1) cells (ECACC collection; Sigma-Aldrich, St Louis, MO, USA) were maintained in culture in Roswell Park Memorial Institute medium (RPMI 1640, Lonza, Basel, Switzerland) supplemented with 1% L-glutamine, 10% heat-inactivated FBS and 1% penicillin–streptomycin. Cells were kept viable at 37 ◦ Cand5%CO 2 . To di ff erentiate into macrophages, THP-1 cells (1x10 6 cells / mL) were seeded in 24 well plates and treated with 100 nM phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich, St Louis, MO, USA) for 72 h at 37 ◦ C, 5% CO2. After incubation, non-attached cells were aspirated, and adherent cells were treated with peptides (0.5 µ g / mL), peptide network (0.5 and 5 µ g / mL) or remained untreated. Docosahexaenoic acid (DHA) at 32.8 µ g / mL was used as a positive control [39]. Following incubation for 24 h, LPS from Escherichia coli O127:B8 (Sigma-Aldrich, St Louis, MO, USA) was added at 100 ng / mL for 24 h at 37 ◦ C, 5%CO 2 . Cell supernatants were collected and TNF- α concentrations were determined by ELISA according to the manufacturer’s instructions (BioLegend, San Diego, CA, USA). Absorbance was measured at 450 nm with a microplate spectrophotometer (SpectraMax M3, Molecular Devices, Sunnyvale, CA, USA). 2.5. Human Randomised, Double Blinded, Parallel Group, Placebo Controlled Clinical Trial 2.5.1. Participants and Study Design Forty volunteers participated in a randomized, double-blind, placebo-controlled, parallel study carried out in accordance with the Declaration of Helsinki. Written, informed consent was obtained from all participants and ethical approval was granted by the clinical research ethics committee of the Cork Teaching Hospitals. The trial was carried out under the supervision of Prof. Fergus Shanahan and was carried out at Atlantia Food Clinical Trials sites at Blackrock and Mallow, County Cork, Ireland (trial protocol NCT04450979). Eligible participants were healthy males and females aged 65–75 years with a BMI < 30 kg / m 2 . Participants were excluded if they had a chronic or infectious disease, were taking anti-inflammatory medications or hormone replacement therapy, or had an allergy
Powered by FlippingBook