2.1. Materials and chemicals
Whole grain HB was purchased from Xining Gaogu Trading Co. Ltd. (Qinghai, China). Porcine pancreatic alpha-amylase (EC 3.2.1.1), pepsin (EC 3.2.1.3) and trypsin (EC 3.4.23.1) were purchased from Shanghai Yuanye Biotechnology Co. Ltd. (Shanghai, China). Alpha-glucosidase (EC 3.2.1.20), total starch assay kit and β-glucan assay kit were purchased from Megazyme Inter-national Ireland Ltd. (Bray, Ierland). Analytical chemicals were at analytical grade. Phosphate-buffered solution (PBS, pH 8.0), bovine serum albumin (BSA), glutamic acid, bile salt and ninhydrin were purchased from Shanghai Yuanye Biotechnology Co. Ltd (Shanghai, China). Sodium acetate buffer (pH 5.2) and coomassie brilliant blue R250 were purchased from Shanghai McLean Biochemical Technology Co. Ltd. (Shanghai, China). DNS solution and absolute ethanol were purchased from Beijing Coolaber Technology Co. Ltd. (Beijing, China). Hydrochloric acid and potassium hydroxide were purchased from Shanghai GaoXin Chemical Glass Instrument Co. Ltd. (Shanghai, China). BCA protein concentration assay kit, protein loading buffer and Tris-Gly prefabricated glue were purchased Shanghai Beyotime Biotechnology Co. Ltd. (Shanghai, China)
2.2. Thermal processing
Atmospheric boiling (AB), high-pressure boiling (HPB), and baking (B) were used to thermally-treat HB, respectively. Mixture of HB and water (1:2, m/m) was steamed at 100 ℃ under atmospheric pressure for 30 mins in an electric cooker (ET30BS01, Zhejiang Supor Co. Ltd, Zhejiang, China); or at 120 ℃ at high-pressure for 30 mins in high-pressure cooker (YL203, Zhejiang Supor Co. Ltd, Zhejiang, China); or baking oven (T1-109F, Guangdong Midea Network Technology Co. Ltd, Guangdong, China) at 180 ℃ for 30 mins. After thermal processing, thermally-treated HB were labeled as: AB-HB (atmospheric boiling, highland barley), HPB-HB (high-pressure boiling, highland barley), and B-HB (baking, highland barley). After thermal treatments, whole grain HB were dried through air oven at 40 ℃ till to constant weight, milled and sieved through 80 mesh before the following analysis.
2.3. Proximate Analysis of HB
The proximate analysis of HB including moisture content (MC), crude fat, protein, and ash was evaluated following the previous methods including AOAC 934.01, AOAC 2003.05, AOAC 2001.11, and AOAC 923.03, respectively. All measurements were tested for three replicates. Content of β-glucan and starch was determined by β-glucan assay kit and starch assay kit, respectively. All measurements were tested for three replicates.
2.4. Content of amylose
The amylose content of HB was determined according to the method of Liu et al. (2019)
with modification [10]. 100 mg HB was solubilized in 3 mL of 1 M KOH for 10 hrs at 4°C with occasional mixing. 3 mL of 1 M HCl and 2 mL of H2O was added to neutralized the starch solution. 1 mL solution containing diluted starch in the range of 0.5-1 mg and 100 µL of iodine solution (0.0025 mol/L I2 and 0.0065 mol/L KI)) was prepared and the optical density was measured at 620 nm. The amylose content was determined according to the standard curve of pure amylose (Y = 5.7531x + 0.0147). Each experiment was tested for three replicates.
2.5. Physicochemical Properties of HB
2.5.1. Scanning electron microscope (SEM)
The microstructure of HB was studied by using field emission scanning electron microscopy (FESEM) (SU5000, Hitachi, Japan). Gold was sprayed on HB sample prior to the test. SEM was operated at 10 kV accelerated voltage. Magnification was 500×.
2.5.2. Color Measurement
HB was measured with a Hunter-Lab spectrophotometer (Model YS6010, Beijing, China). The colorimeter was calibrated against standard white and standard black tile. Values of lightness (L*), redness/greenness (a*), and yellowness/blueness (b*) were obtained. Each treatment was detected for three replicates. Whiteness index (WI) and △E were calculated. Each treatment was tested for three replicates.
△E= [(L*-L0)2+(a*-a0)2+(b*-b0)2]1/2
WI = 100-[(100-L*)2+a*2+b*2]1/2
Wherein, the L0, a0, and b0 represented the color values of HB without heat treatment.
2.5.3. Rapid Viscosity Analysis (RVA)
Pasting properties of HB were determined by a rapid viscosity analyzer (RVA, Model Super-4, Perten Instruments Ltd., Beijing, China) and followed by our previous method [11]. Each treatment was tested for four replicates.
2.5.4. Differential Scanning Calorimetry (DSC)
Thermal property of HB was studied using a Differential Scanning Calorimetry (DSC Q2000 TA instruments, New Castle, DE, USA) equipped with a thermal analysis data station. The method was followed by Guo et al. [12]. Each treatment was tested for three replicates.
2.5.5. X-ray Diffraction (XRD) Analysis
The crystallinity of HB was scanned using X-ray diffractometer (PANalytical X’pert pro X-ray Diffractor, Beijing, China). The analysis was operated at current 40 mA, Cu target, tube pressure at 40 kV, step length at 0.02°, and scanning speed at 2 °/min. 2θ angle was from 4 to 40°. The relative crystallinity (%) was calculated with Highscore software. The relative crystallinity of samples was calculated by the following Formula:
Xc (%) = 100Ac/(Ac + Aa)
Wherein, Xc is the degree of crystallization, Ac is the area of the crystalline peak and Aa is the area of the amorphous regions.
2.5.6. Fourier Transform Infrared Spectroscopy (FTIR)
The FTIR analysis of HB was tested through FTIR spectrometer (FTIR650, Tianjin Gangdong Sci. &Tech. Development Co., Ltd, Tianjin, China). HB was mixed with KBr and pressed into tablets. The pure KBr pellet was used as reference. The spectra were collected within a range of 4000 − 400 cm− 1, with a resolution of 4 cm− 1 and a scanning speed of 2 cm− 1/s.
2.6. Starch digestibility in vitro
The in vitro digestibility of native and thermally-treated HB was determined according to the method Van Hung et al. with some modifications [13]. 0.5 g HB was weighted and mixed with 15 mL of sodium acetate buffer (pH 5.2) using vortex mixer (Vortex-2, Shanghai Huxi Industrial Co. Ltd, Shanghai, China). Then HB was equilibrated at 37°C for 15 mins. 5 mL of porcine pancreatic alpha-amylase (1400 U/ml) and alpha-glucosidase (800 U/ml) were added, followed by incubation in a thermostatic oscillator (HZQ-X300C, Shanghai-Heng Scientific Instrument Co. Ltd, Shanghai, China) at 37°C with continuous shaking at 220 rpm. After 0 (G0), 20 (G20) and 120 (G120) mins of incubation, 0.5 mL hydrolysate was removed and 4.5 mL absolute ethanol was added to stop the enzymatic reaction. The reaction solution treated with the inactivated enzyme, was centrifuged at 4000 r/min for 20 mins using centrifuge (TD25-WS, Suqian Huazhiheng E-commerce Co. Ltd, Suqian, China). 1 mL of the supernatant was mixed with 2 mL of DNS solution, placed in the boiling water bath for 5 mins in electric-heated thermostatic water bath (DK-S22, Shanghai Jinghong Experimental Equipment Co. Ltd, Shanghai, China) and finally fixed the volume to 15 mL. The absorbance was measured at 540 nm using multi-scan spectrum (Synergy HTX, American Berten Instrument Co. Lid, Vermont, USA), and the glucose content was calculated according to the standard curve (Y = 1.6x + 0.079, R2 = 0.9963). The test was repeated for three replicates. Content of rapidly digestible starch (RDS, %), content of slowly digestible starch (SDS, %), and content of resistant starch (RS, %) of each treatment were calculated from the values of G20 (glucose content at 20 min), G120 (glucose content at 120 min), G0 (glucose content at 0 min) and TS (total starch content) as follows:
RDS% = (G20-G0)×0.9×100%/TS
SDS% = (G120-G20)×0.9×100%/TS
RS% = 100%-RDS%-RS%
The in vitro starch hydrolysis was followed by the method described by Huang et al. [7]. 0.2 g HB was mixed with 6 mL of deionized water and 2 mL pepsin solution (10.0 mg/ml, 20.0 mg pepsin dissolved in 2 mL pH 2.0 hydrochloric acid-potassium chloride buffer) at 37 ℃ at continuous shaking of 220 rpm (HZQ-X300C, Shanghai-Heng Scientific Instrument Co. Ltd, Shanghai, China) for 30 mins. The suspension was neutralized with 2 M sodium hydroxide solution. 3 mL porcine pancreatic alpha-amylase (290U/mL) and alpha-glucosidase (60U/mL) mixture (0.079g alpha-amylase and 0.06 mL alpha-glucosidase dissolved in pH 5.2 sodium acetate buffer) was added and shaken (HZQ-X300C, Shanghai-Heng Scientific Instrument Co. Ltd, Shanghai, China) at 37 ℃ for 180 mins. The 0.5 mL of hydrolysates was collected at digestion time of 30, 60, 90, 120, 150 and 180 min and 4.5 mL absolute ethanol was added to stop the enzymatic reaction. The hydrolysate was treated with the inactivated enzyme and centrifuged at 4000 r/min for 20 mins (TD25-WS, Suqian Huazhiheng E-commerce Co. Ltd, Suqian, China). After centrifugation, the glucose content in the supernatant was determined by the 2, 4-dinitrosalicyclic acid (DNS) method. The test was repeated for three replicates. The hydrolysis rate, hydrolysis index (HI) and estimate glycemic index (eGI) was calculated from the values of Gt (glucose content at t min), TS (total starch content), AUC− 1 (Area under the hydrolysis curve of sample) and AUC− 0 (Area under the hydrolysis curve of fresh white bread) as follows:
Hydrolysis rate =\(\frac{\text{G}\text{t}\times 0.9}{TS}\)
HI = AUC− 1×100/AUC− 0
eGI = 39.7་0.549×HI
2.7. Protein digestibility in vitro
In vitro protein digestion test was followed by Huang et al. with some modifications [14]. Simulated gastric fluid (SGF): 0.007 g pepsin (2000U/ mL) was dissolved in 10 mL deionized water. Simulated intestinal fluid (SIF): 1.0 g trypsin (200U/mL) and 0.12 g bile salt (10 mmol/L) were dissolved in 20 mL deionized water. The above three enzyme solutions were prepared and preheated to 37 ℃ before the test. 0.5 g HB and 10 mL gastric SGF was added and the pH of was adjusted to 3.0 with 2 M hydrochloric acid to start digestion. After shaking at 37 ℃ for 120 mins, the pH of reaction was adjusted to 7.5 with 2 M potassium hydroxide solution to end the gastric digestion.
20 mL SIF was added to start intestinal digestion and then shaking at 37 ℃ for 120 mins. After gastrointestinal digestion, the digestive enzymes were inactivated in a boiling water bath and then stored at -20°C for amino acid composition analysis and SDS-PAGE. The 0.5 mL of hydrolysate was collected at digestion time (at 30, 60, 90, and 120 min of gastric and intestinal digestion reaction) and boiled for 5 mins to stop the enzymatic reactions. The hydrolysates were diluted with 4.5 mL deionized water and centrifuged at 4000 r/min for 20 mins. The supernatant was used to determine the content of water-soluble protein and free amino acid. The analysis was repeated for three replicates.
Determination of water-soluble protein content
The water-soluble protein content in the supernatant was determined by BCA protein concentration assay kit [15]. Bovine serum albumin (BSA) was used for the standard calibration curve (Y = 0.6857x + 0.0714, R2 = 0.9953). The detection limit was 5 mg/mL. In vitro protein digestibility (%) was calculated as the following:
Protein digestibility (%) = \(\frac{\text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t} \text{i}\text{n} \text{t}\text{h}\text{e} \text{s}\text{u}\text{p}\text{e}\text{r}\text{n}\text{a}\text{t}\text{a}\text{n}\text{t}}{\text{t}\text{o}\text{t}\text{a}\text{l} \text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}}\)×100%
Each treatment was tested for three replicates.
Determination of free amino acid content
The free amino acid content was determined by ninhydrin method published by Huang et al. [14]. 1 mL of diluted supernatant was added with 0.5 mL PBS and 0.5 mL 2% ninhydrin solution, heated in a boiling water bath for 15 mins, cooled and fixed volume to 25 mL. The absorbance was measured at 570 nm after 10 min at room temperature. Glutamic acid was used for the standard calibration curve (Y = 3.4657x + 0.0247, R2 = 0.9951). The free amino acid content was calculated according to the standard curve. Each test was repeated for three replicates.
Determination of amino acid profile
Amino acid compositions of digestates were determined using a high-speed amino acid autoanalyzer (Hitachi L8800, Tokyo, Japan). Each treatment was tested for three replicates. Separation was performed using an ion exchange resin. The temperature of analytical column was set up as 57 ℃, and the injection volume was 20 µL. The flow rate of pump 1 and pump 2 channels was 0.4 mL/min and 0.35mL/min, respectively.
2.8. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE)
SDS–PAGE under reducing conditions was performed according to the method described by Sun et al. [16], using an AE-6450 electrophoresis system (Atto Corporation, Tokyo, Japan). The digestates were directly dissolved (1:1, v/v) in the sample buffer, containing 5 mL 0.5 mol/L Tris–HCl buffer (pH 6.8), 2.5 mL bmercaptoethanol, 12.5 mL 60 mmol/L Na2EDTA, 2.5 g SDS, 250 mL bromophenol blue and 30 g glycerol in 50 mL distilled water (pH 6.8). After boiling at 100 ℃ for 2 min, the digested samples were centrifuged at 10,000 g for 20 min. The 15 µL digestibility samples and 15 µL marker solutions were placed into wells. Gel electrophoresis was run on 15% acrylamide SDS-PAGE gels using a discontinuous buffer system at 150 V for 1.5 h until the tracking dye, bromophenol blue, reached the bottom of the resolving gel. The gels were stained using Coomassie Brilliant Blue R-250 method and distained with 40% methanol and 10% acetic. The appearances of the gels were recorded by an image forming system (Fluor Chem. FC2, Alpha Innotech Corporation, USA).
2.9. Statistical analysis
Data was analyzed using SAS.9.4 analysis software. Statistical significance of difference was evaluated using the Tukey’s range test (P ≤ 0.05).