2.1. ChemicalsAldehyde and 4-heptanone standards, bovine albumin serum,biuret reagent, HPLC grade methanol, water, formic acid, acetonitrile,ferrous sulfate heptahydrate and sodium phosphate dibasicanhydrous were purchased from Fisher Scientific (Tustin, CA, USA).Sodium phosphate monobasic was from Spectrum Chemical (Gardena,CA, USA). Ellman's reagent 5, 5'-dithiobis(2-nitrobenzoicacid), EDTA, ferrozine, trichloroacetic acid (TCA), 2-Thiobarbituricacid (TBA), and 1, 1, 3, 3,-tetramethoxypropane (TMP) were purchasedfrom SigmaeAldrich (St. Louis, MO, USA).2.2. Preparation of coffee and pork treatmentsGreen Colombia Primeval coffee beans obtained from Rose ParkRoasters (Long Beach, CA, USA) were roasted to produce a light(10 min 32 s to 210 C) and dark (12 min 8 s to 235 C) roast. Wholecoffee beans were ground (Cuisinart “Grind Central” Coffee Grinder,East Windsor, NJ, USA) and passed through a 1.0 mm sieve (18-mesh size) resulting in WGC. Lyophilized brewed coffee was preparedfollowing the methods indicated by Budryn and Nebesny(2013) with modifications. Coffee brew was prepared by heatingwater to 90 C, then adding ground coffee to water at a 1 to 6 ratio.The solution was held at 90 C for five min with constant stirring,filtered with a paper coffee filter to yield liquid brew, which wasthen lyophilized (Dura-Dry mP manifold lyophilizer, FTS Systems,model #FD2085C0000, Stone Ridge, NY, USA) to yield LBC. Theremaining solid grounds from coffee brew extraction was lyophilizedand used as SCG. Coffee was stored at <0 C before use. Beforeincorporation into minced pork, all coffee forms were passedthrough a 1.0 mm sieve (18-mesh size).Meat was prepared in accordance to AOAC Official method983.18 (AOAC., 2010) with modifications. Minced sirloin pork chopsfrom Butcher hogs, averaging 6 months in age, 95.34 kg live weightat the time of slaughter,was supplied and prepared at Farmer John®facilities (Vernon, CA, USA). Pork meat was minced in an industrialsizedchopper, and refrigerated (<4 C) overnight prior to additionof coffee or rosemary oleoresin the following day. All treatments,negative control (NC), rosemary oleoresin [RO; Herbalox® HT-25from Kalsec Inc. (Kalamazoo, MI, USA); (2 g/kg)], SCG, WGC, andLBC of light (1 g/kg) and dark (1 g/kg) roasts were mixed using aHobart Legacy HL200 20 Qt mixer (Troy, OH, USA) for two minresulting in eight different treatment samples with uniform processing.This process was repeated per treatment in order to achievetrue duplicates. The pork was transferred to polyethyleneplastic bags, placed into cardboard boxes, and transported 56 kmfrom Vernon to Orange, CA, USA.2.3. Cooked pork preparationPork meat was stored at 4 C until ready to be formed into porkpatties (~3 h). Pork was prepared and cooked following theResearch Guidelines for Cookery, Sensory Evaluation, and InstrumentalTenderness Measurements of Fresh Meat (AMSA., 1995)with modifications. Pork patties (100 ± 1 g) were molded (11.5 cmdiameter, 1.25 cm thickness) then cooked on two electric griddles(ToastMaster®, Model #TG21W & # TM161GR, St. Louis, MO, USA)set to 205 C for 3.5 min on each side or until internal temperaturereached a minimum of 72 C in the center of the patty. Patties werecooled at 22e25 C before being individually placed into oxygenpermeable zipper bags (polyethylene, 16.5 cm 14.9 cm). Pattieswere stored at 18 C until ready to be analyzed. Patties from eachtreatment were transferred from 18 C to 4 C to thaw for 12 h,and were hand mixed for 30 s prior to analysis.2.4. Chlorogenic, Maillard reaction products, and iron chelatingability quantificationSpent, ground, and lyophilized brew of light, medium, and darkroasted coffee were added to deionized water at 1 g/100 mL to testMRPs and 0.1 g/100 mL to test iron chelating ability and CGA, thenincubated for 2 h at 22 C. Quantification of CGA in the variousforms of coffee were measured via HPLC following protocols by Linet al. (2015). A C18 column (Kinetex, 2.6u C18 100A, 100 4.60 mm,Phenomenx, Torrance, CA, USA) was used at 30 C using a flow rateof 1.5 mL/min with mobile phase (A) 1 mg/mL formic acid in HPLCwater and (B) HPLC grade acetonitrile. Sample was injected (5 mL)with starting conditions of A/B, 95/5 held for 10 min. Solvent Awaslinearly decreased to 85% within 1 min and held for 0.5 min before
returning back to starting conditions within 2.5 min. A standard
curve of chlorogenic acid (0e0.6 mM) was used to quantify
chlorogenic acid detected at 330 nm. Quantification of MRPs and
ferrous iron chelating ability were measured following protocols by
Teets and Were (2008).
2.5. Thiobarbituric acid reactive substances (TBARS) measurement
The TBARS assay was prepared as described by Lin et al. (2015)
with modifications. The supernatant (5 mL) was reacted with 5 mL
of 0.02 M TBA solution in glass test tubes. Recovery values were
determined by spiking additional meat samples (ra
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