Nopal and yellow prickly pear fruits from (Opuntia ficus-indica plants, cv. Milpa Alta) were obtained from commercial plantations in Milpa Alta, DF, Mexico. The prickly pear cactus stems (about 15-20 cm long; respiration rate of 11.2 μL kg-1 s-1) and fruits (about 140-190 g; respiration rate of 19.7 μL kg-1 s-1) were manually harvest (a day before being processed) by cutting the articulation with the 'mother cladode' during afternoon at end July, 2010. The cut zones were immersed in a solution of ascorbic acid (100 ppm) for 15 min. Both stems and tunas were placed in a cover carton containers and transported to the laboratory by land. They were washed in a NaOCl solution (200 ppm) at 4°C for 15 min and dried very fast using a fan. Further they were selected and classified according to size, uniformity and freedom of defects. The respiration rate of the stems and fruits were determined using a closed system according to the methodology established by  and adapted to cladodes by . Reagents 6-hydroxy-2,5,7,8-tetrametilcromano-2-carboxylic acid (Trolox), 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfunic acid) diammonium salt (ABTS), 1,4-bis(sulfanyl)butane-2,3-diol (dithiothreitol) and standards of (2R)-2-[(1S)-1,2-dihydroxyethyl]-4,5-dihydroxyfuran-3-one (ascorbic acid), (5R)-5-[(1S)-1,2-dihydroxyethyl]oxolane-2,3,4-trione (dehydroascorbic acid), (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraen-1-ol (all-trans-retinol), 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4-dihydrochromen-6-ol (α-tocopherol) and 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one (quercetin), were obtained from Sigma-Aldrich (St. Louis, MO). Methanol, propan-2-one (acetone), hexane and ethanol (HPLC grade) were purchased from JT Baker (Baker Mallinckrodt, Mexico).
Nopal previously blanched by immersion in water (90°C, 15 s) was immediately despinned using knives and grinded using a food processor (Osteriser® model 9110, Jarden Corporation, USA) at high speed for 45 s until a dispersion was obtained.
Prickly pear fruit jam (PPJ)
It consisted of a jam made with 62% prickly pear fruit pulp, 30% sucrose, 7% water, 0.05% pectin and 0.04% citric acid. The jam development process was carried out according to standard procedures reported previously .
Three type of bars were produced. Basic formulation included: 33% wheat flour (11% of protein content, Molinos Mexico), 19.8% oat bran (Quaker), 0.3% sucrose (Danisco), 1.1% vegetable oil (Unilever), 0.3% salt (Sanudo), 0.1% calcium carbonate (Calymar), 10.6% liquid egg (Inova Alimentos), 1.9% butter (Unilever), 0.1% pectin (Texture innovation company), 0.3% glucose syrup and 0.4% maltodextrins (Corn Products S.A de C.V.), all companies from Mexico. Nopal-based bars included in addition 32% grounded nopal. The wet ingredients were mixed and then poured into pre-mixed dry ingredients. The resultant mixture was mixed thoroughly by hand, and thirty five grams were weighed out and pressed into rectangle-shaped casts (10 cm × 10 cm × 2 cm) and rolled on a hollow cylinder, which was removed once bars were baked (180°C, 20 min) using a TEDESCO oven (FTT 300, Coaxias do Sul/RS Brasil). A third type of bar was obtained by adding 15 g of PPJ inside the bars. The bars were packed at room temperature in polyethylene bags of high density and final weight was 40 g. The elaboration process was repeated every seven days.
Nopal based tortillas were made initially mixing 51.3% corn flour nixtamalized (Maseca®), 0.7% texturizer and softener (MEXELIN®, Mexico) and 48% of ground nopal using a mixer (LENIN, Mexico) with a capacity of 50 kg for 10 min. The resulting dough was allowed to steep for 5 min prior to processing it into tortilla disk and baked on a triple-pass, gas-fired baking oven (model MLR 30, LENIN, Mexico) at an average temperature of 280°C for 60 s. The tortillas were vacuum-packed in Freshness Plus™ bags (Sealed Air, Duncan, SC, U.S.A.) containing 500 g of tortillas using a vacuum packaging (EVD-4, TOREY, Mexico) at room temperature before being physicochemical and nutritionally evaluated. A batch of 40 kg of tortillas was made every week throughout intake period.
Physicochemical analysis in bars and tortillas
The content of total phenolic compounds (free and conjugated) expressed as mg quercetin equivalents (QE) was determined according to a previously described methodology . The total dietary fiber (method 985.29) , ash (method 940.26) , protein (method 2001.1) , lipids (method 2003.05) , moisture (method 925.10)  and aw (method 978.18)  were also assessed.
The color in the samples was determined using a Minolta colorimeter (CR-300, Konica Minolta Sensing Inc., Osaka Japan) and was expressed as the total color difference (ΔE*) according to the formula:
Where L*, a* and b* represent the parameters of luminosity, red-green and yellow-blue, respectively, between nopal enhanced and control products (without nopal).
This parameter was determined as the maximum force (N) required to penetrate 5 mm into bars and tortillas (folded transversely into four parts), using a texture analyzer (Texture Technologies Corp, NY) with a circular probe of 14 mm diameter at a speed of 1 mm/s .
Subjects and study design
Twenty-eight volunteers (16 women and 12 men) were recruited with prior consent. The health of the participants was determined by anthropometric measures and a questionnaire under the following criteria: 1) no history of gastrointestinal, kidney, liver, or cardiovascular disease, 2) not using antibiotics or vitamin or mineral supplements six weeks before the start of the study and 3) no smoking. The protocol was approved by the Ethics Committee for Human Studies of the San Luis Potosí Autonomous University (UASLP).
By a double treatment, and completely random cross-linked, 28 healthy volunteers supplemented their diet with 100 g of nopal-based tortilla (5 pieces) or 40 g of nopal-based bar (1 piece) filled with prickly pear fruit jam (PPJ), twice daily for three weeks with no supplementation period of six weeks between each treatment. Subjects were allowed to continue their usual eating habits, with the restriction of omitting any horticultural products in their diet.
Blood samples from the volunteers were collected after an overnight fast ≥ 8 h by venipuncture in tubes containing EDTA (1 mg/mL) before (baseline) and at the end of each supplementation period. The samples were stored at 4°C and subjected within the next 2 h to centrifugation at 3000 × g for 10 min to separate the plasma from erythrocytes. Samples were stored at -40°C for subsequent biochemical analysis (TEAC, total polyphenols, vitamins, malondialdehyde, glucose, cholesterol, HDL, LDL, triglycerides). The red blood cells were washed with phosphate buffered saline (5 mmol/L, pH 7.4) and were analyzed immediately.
A HPLC system (Varian Inc., Walnut Creek, CA) composed by a ternary pump (Solvent Delivery System Model 9012), UV/Vis detector (Model 9050), Refractive Index detector (Model Star 9040) and a Fluorometric detector (Model 1200) was used.
Oxidative status indicators
The trolox-equivalent antioxidant capacity (TEAC) was determined according to a previously reported method .
Total polyphenols in plasma
Total polyphenols were measured using the Folin-Ciocalteau method . The absorption at 376 nm was measured spectrophotometrically. The total polyphenol content was expressed as quercetin equivalent per liter.
The (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraen-1-ol (all-trans-retinol) and 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4-dihydrochromen-6-ol (α-tocopherol) were determined using HPLC as reported before . (2R)-2-[(1S)-1,2-dihydroxyethyl]-4,5-dihydroxyfuran-3-one (ascorbic acid) was determined in 500 μL of plasma using 1.0 mM of 1,4-bis(sulfanyl)butane-2,3-diol (dithiothreitol) as reducing agent. The extraction, HPLC separation and spectrophotometric detection at 266 nm were carried out as previously reported .
Propanedial (Malondialdehyde, MDA)
It was evaluated in plasma by colorimetric reaction with 2-sulfanylidene-1,3-diazinane-4,6-dione (thiobarbituric acid) as previously reported .
(2S)-2-amino-5-[[(2R)-1-(carboxymethylamino)-1-oxo-3-sulfanylpropan-2-yl]amino]-5-oxopenta-noic acid (Glutathione)
GSH and GSSG in red blood cells were assessed by HPLC ion exchange reverse phase using an aminopropyl silica column (250 × 30 mm inside diameter, Supelco) according to the method described before .
Glucose, cholesterol and triglycerides
Glucose levels were determined in serum using the GAGO20 kit (Sigma Aldrich), while triglycerides levels, total cholesterol and HDL and LDL fractions were determined using the kits ETGA-200, E2CH-100 and E2HL-100 (Bioassays Systems, California, USA), respectively. In all cases procedures specified by the supplier were followed.
The experiments were carried out using a completely randomized design. Values are expressed as mean ± SD of three physicochemical analyses and twenty-eight independent analysis of biofunctional activity. Significant difference p < 0.05 was determined using paired t test.