Black rice (Oryza sativa L.) was cultivated from a local farm in Tianzhong Township, Changhua County, Taiwan. The black rice was milled and passed through a 100-µm sieve and stored in an air-tight aluminum container before further analysis. The analysis of moisture (Association of Official Agricultural [AOAC] Official Method 925.10), crude protein (AOAC Official Method AOAC 920.87), and ash content (AOAC Official Method AOAC 923.03) of the floor followed the methods of the AOAC (1995). The moisture, crude protein, and ash content of black rice flour were 12.48%, 9.82%, and 1.84%, respectively.
Vedan Enterprise (Taichung, Taiwan) provided the rice protein powder (67% of protein, dry basis) with a moisture content of 4.75%, fat content of 8.63%, and carbohydrate content of 7.0%, respectively. The other basic baking ingredients, such as wheat flour, margarine, sugar powder, cornflour, lecithin, and salt, were purchased from a local market in Yuanlian City, Changhua Country, Taiwan. All chemicals used in this study were American Chemical Society (ACS) certified of analytical grade.
Flaky rolls preparation
Flaky rolls were prepared by designing three different wheat/black rice flour combinations with the following ratios: 100:0 (control), 95:5 (5BR), 90:10 (10BR), and 80:20 (20BR), shown in Table 1. First, margarine and sugar powder were mixed in a mixer until the mixture was fluffy. Next, the wheat flour, black rice flour, rice protein, cornflour, lecithin, salt, and deionized water were added to the mixture. All ingredients were mixed completely to form a smooth flowing batter. The dough underwent further analysis to determine the pasting properties and rheological characteristics. For the flaky roll preparation, the circular metal pan was preheated to 150°C. Next, a spoonful of the batter was spread on the pan. The lid was closed and followed by baking for 30 sec. The flaky roll was folded one-quarter on each side, then rolled around a flat rod to produce a flaky roll (baking temperature was 180oC for 10 min).
The Rapid Visco Analyzer (Model RVA-3D, Newport Scientific, Sydney, Australia) was used to examine the pasting properties of flour samples with 3 replicates (Lu et al. 2019). A 3-g amount of flour sample and distilled water were prepared in the test canister and continued to enter a heating-cooling cycle. The starch slurry was equilibrated (50°C, 1 min) at the beginning, then heated to 95°C at 5°C/min, held for 5 min, and continued by cooling at 50°C (5°C/min), then the temperature was controlled at 50°C for 2 min. The viscoamylograph clarified the pasting profiles of the sample as well as the peak viscosity, breakdown, setback, and final viscosity, from the diagram of Rapid Visco Analyzer curves. Three replication tests of each sample were carried out.
The Brabender farinograph (Brabender, Duisburg, Germany) was used to investigate the mixing properties of dough corresponding to the standard method of the American Association for Clinical Chemistry (AACC) 54-21 (2000). Wheat flour with different proportions of black rice flour at 0%, 5%, 10%, and 20% was formulated before analyzing and with the following modifications: 1) weighting the formulated flour at 100 g with 0.2 g salt added and pre-mixing time 5 min; 2) adjusting the water addition to the 500 Brabender units (BU) line; and 3) agitating until the graphs of farinograph significantly changed and prolonged to 12 min. Water absorption, arrival time, departure time, stability, peak time, tolerance index, and valorimeter unit were recorded. Water absorption was defined as the percentage of water addition absorbed by the dough to the 500 BU line. Arrival time was defined as the time in minutes measured from the addition of water to the point of the curve band at the 500 BU line. Departure time was defined as the time in minutes measured from the peak of the curve band departing to the 500 BU line. Stability was defined as the difference between arrival time and departure time. Peak time was defined as the time in minutes to the peak of the curved band. The tolerance index was defined as the BU unit difference from the peak of the curve band to another curve band after 12 min. The valorimeter value measured by the farinograph scale was defined as the quality of the dough gluten.
The extensible properties of dough were investigated by using the Brabender extensograph (Brabender, Duisburg, Germany) by referring to the standard method of the AACC 54-10 (2000). For the farinograph measurements, the dough was cut into two parts, every part is 150 g. Next, the dough was passed through the balling and mold unit of the extensograph. Stretching the dough every time after 45 min of resting in the fermentation cabinet. Balling and molding operations were repeated after fermentation. The test was carried out in triplicate. The results are expressed as the maximum resistance (BU) to the extension. The extensibility (E) is described as the distance traveled by the recorder paper from the moment the hook touches the test piece until the rupture of the test piece.
Nutritional Composition Analysis
The approximate nutritional composition of flaky rolls supplemented with black rice flour moisture, total sugar, crude protein, crude lipid, total dietary fiber (TDF), soluble dietary fiber (SDF), insoluble dietary fiber (IDF), ash, free sugar, and cholesterol content were studied in 3 replicates and corresponded to the official methods of AOAC 935.39. The moisture content was evaluated by the hot-air oven method; ash content was analyzed by the method of incinerating samples in a muffle furnace at 550-600°C; crude protein content was studied by the Kjeldahl method or Kjeldahl digestion method; fat content was measured by the acid hydrolysis method; cholesterol content was tested by the digitonin method, and fiber content was studied by the enzymatic–gravimetric method.
Characteristics and texture analysis
A vernier caliper was used to measure the length (cm), width (cm), and thickness (cm) of the flaky roll supplemented with black rice flour. The other textural properties, such as hardness (N), fracturability (mm), springiness (mm), cohesiveness, and adhesiveness (N.S), were analyzed by a 3-point bend rig (HDP/3 PB) which was equipped with a 5-kg load cell, and heavy-duty platform of the TAXTPlus texture analyzer (Stable Micro Systems, UK). The test speed was set at 3.0 mm/s and the trigger force was set automatically at 50 g. The hardness value represents the maximum force applied, while the fracturability value exhibited the distance at the point of break (Chan et al. 2020).
The color of the flaky roll samples was analyzed by the Color Meter ZE-2000 (Nippon Denshku Industries, Tokyo). L* (lightness measurement), a* (greenness-redness value), and b* (blueness-yellowness value) value was studied to determine the quality changes. Calibration of the instrument involved using a standard black-and-white ceramic tile before measurement. Color measurements were carried out at room temperature in triplicate.
In total, 31 panelists (16 females, age range 25 to 35 years) from the Department of Food and Nutrition, Providence University, were randomly selected to participate in the sensory evaluation session. The evaluation was carried out in the sensory laboratory at room temperature and strictly followed the GB/T 13662-2008 and ISO 4121 criteria to evaluate the sensory characteristics of flaky roll supplemented with black rice flour in terms of appearance, aroma, taste, texture, and total acceptability. The panelists were required to attend the tutorial and training classes to be able to identify and comprehend the rating scales for each attribute before testing. The flaky roll samples for each formulation were cut into a size of 3.0 cm2. Samples were selected randomly and placed on white disposable polyform plates marked with a random 3-digit number. All samples were covered with food wrap until testing. The flaky rolls were evaluated by quantitative descriptive analysis involving a 6-point interval scale with scores from 0 to 5 for each attribute, 0 indicating no value and 5 indicating extremely strong value (Chan et al. 2020).
Determination of total phenolic content (TPC)
The total phenolic content of the sample was studied by the Folin-Ciocalteu method (Li et al. 2020). First, the sample was extracted with 80% methanol. Next, 1 mL extract was added to 9 mL distilled water in a 25-mL volumetric flask. Double distilled water (ddH2O) was used as a blank reagent. Then 0.5 mL Folin-Ciocalteu phenol reagent and 5 mL of 5% sodium carbonate (Na2CO3) solution were added to the mixture solution and mixed thoroughly. The mixtures were diluted with distilled water to 25 mL and allowed to stand for 60 min. Wavelength 750 nm was used to measure the absorbance versus the blank. Total polyphenol content was calculated by comparing with a standard curve of gallic acid, and the results are expressed as milligram gallic acid equivalent (GAE) per g dry weight (mg GAE g−1 DW).
Determination of total anthocyanins
Total anthocyanin content was determined as described (Ziegler et al. 2017) with some modifications. A 500-mg amount of flaky roll samples was placed into a 15-mL Falcon™ tube, then 10 mL acidified methanol with a ratio of 85% methanol: 15% 1 N HCl, was added for extraction. The mixture was homogenized for 30 sec by vortex mixing, followed by 30 min of continuous stirring at 200 × g on an orbital shaker. Next, the sample mixture was centrifuged at 7600 × g for 25 min to collect the supernatant. The absorbance was measured at wavelength 535 nm. Total anthocyanin was expressed as cyanidin-3-O-glucoside (CGE) equivalent, and calculated by the formula:
C = (A/ε) × (vol/1000) × MW × (1/sample wt) × 106
Where C = concentration of total anthocyanin (mg/100g); A = absorbance reading at 535 nm; Ɛ = molar absorptivity (cyanidin-3-O-glucoside =25,965 cm−1 M−1); vol = total volume of anthocyanin extract; MW = molecular weight of cyanidin-3-O-glucoside (449.2).
Determination of total antioxidant capacity by 2,2-diphenyl-1-picrylhydrazyl (DPPH)
The free radical scavenging activity of the sample and the standard was evaluated by the free radical scavenging effect of the stable DPPH free radical activity (Li et al. 2020). Gallic acid was used as the standard solution, and 0.1 mM DPPH was diluted in methanol. A 2-mL amount of 0.1 mM DPPH was mixed with a 1 mL sample solution or standard solution. These mixtures were kept in the dark for 30 min and the absorbance was measured at a wavelength of 518 nm. The measurements were tested in triplicate.
Oxygen radical absorbance capacity (ORAC)
The ORAC analysis method used, with fluorescein (FL) as the “fluorescent probe,” was as described (Ou et al. 2001; Folch-Cano et al. 2010; Zulueta et al. 2009) with modifications. The 96-well microplates were used, and the absorbance was read at wavelengths 485 nm (excitation) and 530 nm (emission). The reaction was carried out at 37°C as the reaction was started by thermal decomposition of 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) in 75 mM phosphate buffer (pH 7.0). In brief, in each of the 96 -wells was placed 50 μL of 78 nM FL and 50 μL of sample; the blank was the PBS, and 20 μM Trolox was the standard; then 25 μL of 221 mM AAPH was added. To avoid variations in measurement among wells due to the low conductivity of the 96-wells plates, plates were heated up to a temperature of 37°C for 15 min before adding the AAPH. The fluorescence was measured as the relative fluorescence intensity (FI%), measurements were carried out every 5 min until the value was less than 5% of the value of the initial reading. Each analysis and measurement were taken in triplicate.
In vitro starch digestibility
The in vitro starch digestibility of the flaky rolls sample was examined as described (Tang et al. 2019) with slight modifications. A 200-mg amount of the sample was mixed with 15 mL of 0.2 M sodium acetate buffer (pH 5.2). Next, following by adding 10 mL of the formerly prepared enzyme solution (porcine pancreatic α-amylase [290 U/mL] and glucoamylase [15 U/mL]). The mixture was shaken in a water bath with a temperature of 37°C at 160 revolutions/min. A 0.50-mL amount of the sample solution was collected at different times (0, 20, 40, 60, 80, 100, 120, and 180 min), and the enzymes were inactivated by added in 4.5 mL of ethanol. Glucose content (mg/g) was analyzed by the 3.5-dinitrosalicylic acid (DNS) method.
Determination of free asparagine content
Free asparagine content was measured as described (Žilić et al. 2020) with modification. A 1-g amount of the test flaky roll sample was extracted with 20 mL of 10 mM formic acid solution and vortexed mixing for 3 min. Carrez I and II solutions were added to the mixture solution to clarify it. The mixture was centrifuged at 15,000 g for 10 min, and the supernatants were stored at -80°C. All extractions were accomplished in triplicate for each sample. The supernatant collected was diluted with an equal volume of acetonitrile and centrifuged at 15,000 g for 10 min. Then, the supernatant was passed through a 0.45-µm nylon filter and collected in a vial before further analysis. The Waters Acquity UPLC system attached to a triple quadrupole detector was applied to investigate the free asparagine content of the sample. Thermo Scientific Syncronis HILIC column (100 × 2.1 mm × 1.7 µm) with a gradient mixture of Solvent A (5 mM ammonium formate in water with formic acid), and Solvent B (5 mM ammonium formate in water: acetonitrile (v/v 1:9) with formic acid) as the mobile phase at a flow rate of 0.7 mL/min at 40°C. The mobile phase gradient was programmed to start with 0% Solvent A, then gradually rose to 80% in 8 min and held for 5 min, then declined regularly to the initial conditions (0% Solvent A) in 2 min and held for 10 min. The electrospray source parameter was as follows: capillary voltage 3.5 kV, cone voltage 20 V, extractor voltage 3 V, source temperature 120°C, desolvation temperature 370°C, and desolvation gas (nitrogen) flow 900 L/h. Calibration curves were built, and quantifications were from 0.05 to 3.0 mg/L. Theanine as a standard for both working standards and extracts. The results are expressed as mg per kg.
Determination of acrylamide content
A 1-g amount of ground flaky roll sample was extracted with 20 mL of 10 mM formic acid in 20 mL water and vortexed mixing for 3 min. The extraction and preparation method were mentioned in Section 2.15. The supernatant collected from the extraction was passed through a preconditioned Oasis MCX solid-phase extraction cartridge, and the pure extract was analyzed by using LC-MS/MS. The extracts for acrylamide were studied by the Waters Acquity H Class UPLC system (Millford, MA) attached to a TQ detector with electrospray ionization operated in a positive mode. The chromatographic separations were performed at a flow rate of 0.5mL/min on a Thermo Scientific Hypercarb column (100 × 2.1 mm × 3 µm) with the formic acid solution as the mobile phase. The column was equilibrated at 50°C, while the electrospray source was settings with 2.00 kV of capillary voltage, 23 V of cone voltage, 4 V of extractor voltage, and 120°C of sources temperature, 400°C of desolvation temperature, and desolvation gas (nitrogen) flow 900 L/h. Calibration curves were built, and quantifications were from 1 to 40 ng/mL (1, 2, 5, 10, 20, 40 ng/mL). The results are expressed as μg per kg.
Determination of HMF and furfural
HMF and furfural content was determined as described (Arribas-Lorenzo et al. 2009; Rufián Henares et al. 2006) with modifications. A 500-mg amount of ground sample was suspended in 5 mL deionized water clarified with 0.25 mL potassium ferrocyanide (15% w/v) and 0.25 mL zinc acetate (30% w/v) solutions in a centrifuged tube. The mixture was centrifuged at 4,500 rpm for 15 min at 5°C. The supernatant was collected and filtered by using a 0.45-µm syringe filter before analysis by HPLC (Shimadzu, Kyoto, Japan). The Synergy 4 µm Hydro-RP 80A, 250 x 4.6 mm (Phenomenex) was used as the column. The mobile phase was a mixture of acetonitrile in water (5% v/v) at a flow rate of 1 mL/min under isocratic conditions. The UV detector was set at 280 nm, and HMF was quantified by using the external standard method within the concentration range of 0.025 to 75 mg/L. All the analyses were performed in triplicate and the results are expressed as mg/kg samples.
2.18. Determination of dicarbonyl compounds, glyoxal (GO), and methylglyoxal (MGO)
In this study, GO and MGO contents were analyzed correspondingly to quinoxalines (Q) and 2-methylquinoxaline (2-MQ) content, respectively, by using a reverse-phase high-performance liquid chromatography (RP-HPLC) procedure attached to UV detection at wavelength 315 nm (Arribas-Lorenzo and Morales 2010). An LC system (Kyoto, Japan) equipped with an ACEC18 column (5 μm, 250 × 4.6 mm, Advanced Chromatography Technologies, Aberdeen, UK), low-pressure gradient former, pump, and a DAD detector was used. Elution was accomplished at a mixture of 0.5% (v/v) acetic acid in water and methanol (40:60, v/v) with a detection wavelength of 317 nm. Standard stock solutions of Q and 2-MQ were prepared in deionized water to a concentration of 5 mg/mL.
The data are reported as mean ± standard deviation (SD) of triplicate independent experiments. All data were studied by single-factor ANOVA to determine significant differences at P<0.05.