The Effect of Thermal Modifications on the Physicochemical, Structural, Functional Properties and In Vitro Digestibility of Black Wheat Kernel and Whole-Grain Flour

Abstract

Whole grains, due to their intact structure, retain more nutrients and offer significant health benefits. Thermal modification is commonly applied to modify cereal grains. This study aimed to investigate the effects of thermal treatments (microwaving (abbreviation MW-BW), roasting (RST-BW), and an emerging technology, heat fluidization (HFL-BW)) on whole-grain black wheat flour. The results showed minimal loss in proximate composition and increased anthocyanin content (from 38.78 mg/kg (BW) to 39.57 (HFL-BW) and 46.06 mg/kg (MW-BW)) relative to the control. Analysis of physical properties and microstructure revealed that all thermal treatments caused kernel swelling, darkened the flour color, decreased the kernel hardness, and disrupted the starch microstructure. All thermal treatments disrupted starch short-range order and reduced crystallinity (from 26.75% (BW) to 2.56 (HFL-BW) and 15.74% (RST-BW)), resulting in a transformation to a V-type structure. The protein secondary structure (mainly for α-helix) was disrupted, and gluten was denatured and aggregated in all thermal-treatment groups. Thermal treatments decreased gelatinization enthalpy (from 4.76 J/g (BW) to 0.59 (HFL-BW) and 4.44 J/g (RST-BW)) and altered pasting viscosity. The viscoelasticity of pastes made from thermal treatments was improved. In vitro digestibility results showed that thermal treatments decreased starch digestibility, decreased the protein bioavailability, and increased resistant starch content (from 20.1% (BW) to 30.9 (MW-BW) and 39.6% (RST-BW)). Altogether, heat fluidization had the most pronounced effect among the treatments. Thermal modifications—particularly heat fluidization—are promising technologies for enhancing the quality of whole-grain black wheat flour and developing functional foods.

1. Introduction

Whole grains have high nutritional value owing to their intact structure, which comprises the bran, endosperm, and germ in proportions consistent with the original grain seed [1]. Numerous studies have shown that increased intake of whole grains is associated with a reduced risk of diseases, including cardiovascular disease, hypertension, type 2 diabetes, mental disorders, and lung cancer [2,3,4,5,6]. Wheat is a major global grain used to produce staple foods such as bread, pasta, and Chinese steamed bread. Colored wheats (blue, purple, and black), which contain more phytochemicals than conventional white wheat, have attracted increasing research attention. The most obvious difference between colored wheat and traditional wheat is the pigments in the bran. Microscopic analysis has revealed that the blue and black wheat donors develop color in the aleurone layer, whereas the purple and black wheat donors exhibit pigmentation in the pericarp [7]. Although they produce low grain yield and thousand-grain weight, colored wheats possess a superior nutritional content—including carbohydrates, protein, dietary fiber, vitamins, phenolic acids, and anthocyanins—while maintaining processing parameters similar to common wheat, facilitating their commercialization for functional food applications. Breads made from colored wheats possess greater antioxidant capacity than those made from conventional wheat [8,9,10,11]. Studies have demonstrated that foods produced from colored wheat exhibit significantly higher nutritional value and unique sensory characteristics compared to those made from conventional wheat [12]. Utilizing black-, purple-, and blue-colored wheats as novel ingredients offers significant potential for the food industry to create value-added products with enhanced appeal and functionality. Black wheat contains a higher protein content than that of common bread wheats. It has high-molecular-weight glutenin subunits, which make it a source for making bread [9]. Like common wheat, black wheat contains bran, endosperm, and germ. The pigments in the pericarp give the visually black color. Higher nutrients (such as protein and dietary fiber) were reported for this grain compared to common wheat. Similar to other colored wheats, with the high nutritional value and bioactivity, there is huge potential for using black wheat to develop functional foods [7].

Thermal processing is a widely used technique in food manufacturing. Thermal treatment plays an important role in prolonging the shelf life of foods, guaranteeing food safety, and improving product quality [13,14,15]. Common thermal treatment methods include microwave heating, roasting, and infrared heating. Microwave processing offers higher energy efficiency and more uniform heating in a shorter time than conventional methods such as heat–moisture treatment or hot-air ovens [16]. Microwave heating reduces enzyme activity, inhibits color deterioration, and preserves the antioxidant capacity of whole-wheat flour [13]. Roasting can increase kernel volume and reduce density. This is achieved by generating inner porosity, cavities, and cracks, which compromise the structural integrity of the whole-wheat kernels. In addition, roasting can increase water absorption capacity and decrease gluten extensibility of whole-grain wheat flour, and these changes improve bread quality and extend the shelf life [17,18]. Infrared heating was reported to have the advantages of causing less thermal damage, providing more uniform heating, and increasing antioxidant activity of cereal grain [19,20]. In addition to the heating methods mentioned above, a new heating technology, heat fluidization, has emerged in recent years. Heat fluidization technology uniformly heats materials using a combination of infrared and hot-air heating systems [21]. This technology has gradually gained attention due to its excellent heating effect. Recent studies have shown that heat fluidization effectively modifies the processing properties of whole-grain flours, such as those from highland barley and black Tartary buckwheat. This technology significantly increases bioactive compounds, reduces kernel hardness, elevates resistant starch content, and improves the texture of resulting products [21,22,23].

Previous research on colored wheat has primarily focused on its biological activities and applications in processed foods, with a notable lack of studies about how heat processing affects wheat flour properties. To address this research gap, this study investigates the effects of thermal treatments on the properties of whole-grain black wheat flour. Three thermal treatments (heat fluidization, microwaving, and roasting) were selected to heat the whole-grain black wheat. The physical characteristics of grains and the properties of whole-grain black wheat flour (including proximate composition, color values, microstructural characterization, starch short-range-ordered structure, crystallinity, protein secondary structure, gluten protein molecular weight distribution, thermal properties, pasting properties, and in vitro digestibility) were analyzed. This research will advance the understanding of the processing properties of thermally modified whole-grain black wheat flour and provide theoretical support for producing subsequent modified whole-grain foods.

2. Materials and Methods

2.1. Samples and Chemical Reagents

The whole-grain black wheat was purchased from Henan Xudu Dongli Agricultural Development Co., Ltd. (Xuchang, China). Amyloglucosidase was purchased from Solarbio Technology Co., Ltd. (Beijing, China). α-Amylase from porcine pancreas was obtained from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). GOPOD reagent (D-Glucose Assay Kit (KGLUC) was obtained from Megazyme (Bray, Wicklow, Ireland). Unless otherwise stated, all reagents used in this research were of analytical grade.

2.2. Preparation of Thermally Treated Whole-Grain Black Wheat Flour

The whole-grain black wheat samples were divided into four groups: one untreated group and three groups subjected to different thermal treatments. Three independent batch samples were prepared (n = 3). The specific processes of various treatments were as follows: (i) Heat fluidization treatment: the sample was fed into industrial heat fluidization equipment (GW-100, Number Times Technology (Huai’an) Co., Ltd., Huai’an, China). The sample was subjected to a superficial air velocity of 3.0 m/s and gradient heating from 165°C to 115°C, with the following temperature setpoints: 165°C, 155°C, 150°C, 140°C, 135°C, and 115°C. The entire heating process was divided into 6 stages, with the temperature decreasing gradually, for a total duration of 21 min [22]. (ii) Microwave treatment: an appropriate amount of sample was evenly spread in a glass dish and heated in a microwave oven (Midea microwave oven, EG720FA4-NR, Guangdong Midea Kitchen Appliance Manufacturing Co., Ltd., Foshan, China) at 700 watts for 3 min [24]. (iii) Roasting treatment: an appropriate amount of sample was evenly spread on a tray and roasted in an electric oven (DELMCA electric oven, XN-60S, Foshan Beier Electric Appliance Co., Ltd., Foshan, China) for 5 min. Both the upper and lower heating tubes (elements) were 220 °C [25]. (iv) The whole-grain black wheat without any thermal treatment was set as the control group, which was abbreviated to BW. The groups of heat fluidization treatment, microwave treatment, and roasting treatment were abbreviated to HFL-BW, MW-BW, and RST-BW, respectively. To further explore the effect of thermal treatment on flour, the thermal-treated whole-grain black wheat was ground into 100-mesh flour using a mill (DLFl8, Wenzhou Dingli Medical Instruments Co., Ltd., Wenzhou, China). The flour sample was then stored at 4 °C for subsequent research.

2.3. Proximate Composition Analysis

The content of crude proteins, fat, total dietary fiber, and ash was determined according to the methods from National Standards of the People’s Republic of China GB 5009.5–2016 [26], GB 5009.6–2016 [27], GB 5009.88–2014 [28], and GB 5009.4–2016 [29], respectively. The flour sample was dried at 105 °C for 90 min to measure the moisture content using an electric moisture meter (DHS-16A, LICHEN, Shanghai, China). The method for protein content determination was based on the Kjeldahl method (AutoKjeldahl Unit K-370, Buchi, Switzerland) with a conversion coefficient of 6.25. The fat content was determined using the Soxhlet extraction method. The total dietary fiber was determined based on the enzymatic–gravimetric method. To determine the ash content, the sample was incinerated in a muffle furnace (SX-4-10, Shanghai Techeng Machinery Equipment Co., Ltd., Shanghai, China) at 550°C for 4 h. The total starch content was analyzed using the total starch assay kit (Megazyme, Ireland) according to AOAC Method 996.11 [30].

The total anthocyanin content of whole-grain black wheat was also measured using a previously established method [8]. The black wheat was milled to make 50-mesh flour. A sample of 1 g was mixed with 10 mL acidified methanol (CH₃OH:HCl, 85:15, v/v). After shaking in the dark at 28°C for 12 h, the mixture was centrifuged at 7000 rpm and 4°C for 30 min. The supernatant was filtered through a 0.45 μm filter membrane, and the absorbance was measured at 520 nm. The anthocyanin content was expressed as cyanidin-3-glucoside equivalents, calculated according to the formula described by Young et al. [31], which is as follows:

$$\mathrm{C}= {\displaystyle \frac{\mathrm{A}}{\mathsf{\epsilon }}}\times {\displaystyle \frac{\mathrm{V}}{1000}}\times \mathrm{M}\mathrm{W}\times {\displaystyle \frac{1}{\mathrm{w}\mathrm{t}}}\times {10}^6,$$ (1)

where C is the total anthocyanin content (mg/kg), A is the absorbance of the sample, ε is the molar absorption coefficient of cyanidin 3-glucoside (25965 cm⁻¹ M⁻¹), V is the total volume of the extraction solution, MW is the molecular weight of cyanidin 3-glucoside (449 g/mol), and wt is the sample weight.

2.4. Physical Properties Analysis

The physical properties (thousand-seed weight, bulk density, puffing index, and hardness) of whole-grain black wheat were analyzed. Thousand-seed weight was measured using the method specified in GB/T 5519–2018 [32]. The bulk density was determined as a ratio of the mass of whole-grain black wheat to its volume (g/L). The sample was poured into a graduated cylinder, tapped gently 10 times, and filled with grains to the 100 mL mark. The grains were weighed, and the test weight (weight per unit volume) was calculated [33]. The puffing index was expressed as the ratio of the bulk density of the control group sample to that of the thermally treated sample. Hardness was measured using a Texture Analyzer (TMS-Pro, Food Technology Corporation, Sterling, VA, USA) equipped with a 50 N intelligent load cell and a 36 mm cylindrical probe. The probe was set to compress the grains at 30 mm/min, with a target deformation of 15%. The trigger force was set to be 0.3 N [21].

2.5. Color Characteristics

The color characteristics of whole-grain black wheat flour were analyzed using a High-Quality Colorimeter (NH300, 3NH Technology Co., Ltd., Shenzhen, China) according to the instrument’s manual. The color values were expressed as L* value (0–100, darkness to whiteness), a* value (+red, -green), and b* value (+yellow, -blue). Each analysis was performed in six replicates. The total color difference value (ΔE) was expressed as

$$\Delta \mathrm{E}=\sqrt{{\left(\mathrm{L}\mathrm{*}-{\mathrm{L}}_0\right)}^2+{\left(\mathrm{a}\mathrm{*}-{\mathrm{a}}_0\right)}^2+{\left(\mathrm{b}\mathrm{*}-{\mathrm{b}}_0\right)}^2}$$ (2)

where the subscript “0” represents the color value of the untreated sample.

2.6. Scanning Electron Microscope (SEM) Analysis

The morphological characteristics and microstructure of whole-grain black wheat flour were observed using a Scanning Electron Microscope (SEM, Regulus SU8100, Hitachi, Hitachinaka, Japan). The freeze-dried samples were coated with platinum to ensure conductivity. The micrographs were obtained at an accelerating voltage of 10 kV at magnifications of 500× and 1500×.

2.7. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

The FTIR spectrometer (Nicolet Summit X, Thermo Fisher Scientific Inc., USA) in conjunction with an accessory of attenuated total reflectance (ATR) was used to obtain the infrared spectra of whole-grain black wheat flour. Spectra were collected over a wavenumber range of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹ with 32 accumulated scans. The spectral data were processed using OMNIC 9.2.86 software (Thermo Fisher Scientific Inc., Waltham, MA, USA) to analyze the short-range order and protein secondary structure of flour. Fourier deconvolution was used to extract information on short-range order (wavenumbers: 1200–900 cm⁻¹) from spectral data. The peak width and enhancement factor were set to 40 and 1.9, respectively. To analyze the protein secondary structure, Gaussian deconvolution and second-derivative analysis of the Amide I band (1700–1600 cm⁻¹) were performed using PeakFit 4.12 software (SYSTAT Software inc., San Jose, CA, USA).

2.8. X-Ray Diffraction (XRD) Analysis

The XRD patterns of whole-grain black wheat flour were measured using an X-ray diffractometer (D8-Advance, Bruker AXS, Karlsruhe, Germany). Diffractograms were acquired at an operating voltage of 40 kV and a current of 40 mA. The scan range was 5° to 50° at a rate of 5°/min. The diffractogram data were analyzed by Origin 2022 software (OriginLab Corporation, Northampton, MA, USA). The relative crystallinity (RC, %) of the samples was calculated as the ratio of the area corresponding to the crystalline peaks to the total area of the diffractogram, following previous research [34,35].

2.9. Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The molecular weight distribution of protein subunits in whole-grain black wheat flour was analyzed by SDS-PAGE, following a previously described method with modifications [36]. The 25 mg sample was mixed with 0.5 mL loading buffer (0.06 mol/L Tris-HCl, pH 6.8; 25 % (v/v) glycerol; 2% (w/v) SDS; and 0.1 % (w/v) bromophenol blue). For reduced SDS-PAGE, 5% (v/v) β-mercaptoethanol was added to the loading buffer. The sample buffer was shaken at room temperature for 4 h and then centrifuged at 12,000 rpm for 10 min. After heating in a 100 °C water bath for 4 min, the supernatant was used for the electrophoresis analysis using electrophoresis apparatus (DYY-7C, Liuyi Biotech Co., Ltd., Beijing, China) at 110 V for 80 min.

2.10. Thermal Properties Analysis

A 4 mg sample of whole-grain black wheat flour was mixed with 12 μL of deionized water in a hermetically sealed aluminum crucible. The mixture was equilibrated at room temperature for 12 h. An empty aluminum crucible was the reference material. The thermal properties of the samples were measured using Differential Scanning Calorimetry (DSC, TA Q20, TA Instruments, New Castle, DE, USA) at a heating rate of 10°C/min from 30°C to 110°C under a nitrogen flow rate of 50 mL/min. Finally, the DSC curves were analyzed to obtain the onset (T_o), peak (T_p), and conclusion (T_c) temperatures, as well as the enthalpy change (ΔH) [37].

2.11. Pasting Properties and Viscoelastic Properties Analysis

The pasting properties of whole-grain black wheat flour were determined using a Rapid Visco Analyzer (RVA4500, Perten Instruments, Stockholm, Sweden). A 3.5 g sample (14% moisture) was thoroughly mixed with 25 mL of deionized water in an aluminum container to form a slurry. The slurry was stirred at 960 rpm for 10 s, followed by a maintained stirring rate of 160 rpm for the remainder of the process. The temperature protocol was as follows: the slurry was held at 50°C for 60 s, then heated to 95°C at 12°C/min, held at 95°C for 2.5 min, and finally cooled to 50°C at 12°C/min. The process concluded by keeping the slurry at 50°C for 2 min. According to the pasting curve, the pasting temperature (PT), peak viscosity (PV), trough viscosity (TV), breakdown viscosity (BV), final viscosity (FV), and setback viscosity (SV) of the samples were recorded [38].

In addition, the viscoelastic properties of whole-grain black wheat flour were determined using a Modular Compact Rheometer (MCR 302e, Anton Paar companies, Ashland, VA, USA) according to previous methods [39]. Mix 3.5 g of flour (14% moisture) with 25 mL of deionized water, heat in a water bath at 95°C for 30 min while stirring thoroughly, then rapidly cool to 25°C. The flour paste samples were conducted with strain sweeps using a rheometer to identify the linear viscoelastic region. The storage modulus (G’) and loss modulus (G’’) were determined by frequency sweeps at 25°C (angular frequency: 0.1–100 rad/s; gap size: 1 mm).

2.12. In Vitro Digestibility Analysis

The in vitro digestibility of whole-grain black wheat flour was determined using previous methods [40,41]. A sample of 200 mg was thoroughly mixed with 15 mL of sodium acetate buffer (0.2 mol/L; pH 5.2). The mixed solution was gelatinized in a water bath at 100°C for 30 min and then cooled to room temperature. Subsequently, enzyme solutions of amyloglucosidase (50 U/mL, 5 mL) and porcine pancreas α-amylase (290 U/mL, 5 mL) were prepared and incubated in a 37°C water bath for 6 min. Concurrently, the cooled sample solution was incubated under the same temperature and time conditions. After that, the enzyme and sample solutions were mixed and shaken at 180 rpm and 37°C. Aliquots (0.5 mL) of the mixture were taken at 0, 20, 60, 90, 120, and 180 min during hydrolysis and immediately added to 4.5 mL of anhydrous ethanol to quench the reaction. After centrifugation at 4000 rpm for 5 min, the supernatant was reacted with the GOPOD reagent. Absorbance at 510 nm was measured to determine the extent of starch hydrolysis. The ratios of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) were calculated as previously described [41].

In addition, the gluten protein in whole-grain black wheat flour subjected to different thermal treatments was analyzed by in vitro digestion using previously described methods [42]. Firstly, the flour samples were subjected to in vitro digestion. A total of 5 g of the sample was mixed with 30 mL of deionized water. The sample mixture was kept at 37°C with continuous stirring throughout the whole digestion process. The sample mixture was incubated at 37°C with continuous stirring throughout the digestion. For the gastric phase, 2 mL of pepsin solution (10 mg pepsin, CAS: 9001-75-6, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added to the sample mixture. The mixture was incubated with the pH maintained at 1.2. After digestion for 120 min, the digestion mixture was adjusted to 6.8 using 1 mol/L NaOH solution to end the gastric digestion phase. The intestinal digestion was initiated by adding 3 mL of pancreatin solution (12 mg pancreatin, CAS: 8049-47-6, Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) to the digestion mixture. The pH was maintained at 6.8 throughout the 120 min intestinal digestion phase. The digestion was then terminated by heating the mixture at 95°C for 5 min. The sample solutions were collected before digestion, after gastric digestion, and after intestinal digestion. The mixed solutions were centrifuged at 4000 rpm for 10 min, at 4°C. The supernatant was collected for subsequent experiments. Secondly, the SDS-PAGE electropherogram and free amino nitrogen (FAN) content of the digested samples were investigated. The FAN content of the digestive solution was determined by the ninhydrin reaction (ninhydrin, CAS: 485-47-2, Sinopharm Chemical Reagent Co., Ltd., China) according to previous methods [43]. SDS-PAGE under reducing conditions was performed using the same steps as Section 2.9 to analyze the in vitro digestion of gluten protein distribution.

2.13. Statistical Analysis

All the experiments were performed in biological triplicate unless otherwise specified. The data were subjected to one-way analysis of variance (ANOVA), and significant differences (p < 0.05) were determined by Tukey’s Honestly Significant Difference (HSD) test using IBM SPSS Statistics 25.0 (SPSS Inc., Chicago, IL, USA). If data homogeneity of variance was violated, Dunnett’s T3 test was performed. Result analysis and graphing were performed using Origin 2022 (OriginLab, Northampton, MA, USA).

3. Results and Discussion

3.1. Proximate Composition Analysis of Whole-Grain Black Wheat

As shown in Table 1, the proximate composition was analyzed for differently thermally treated whole-grain black wheat flour. The moisture content of the samples ranged from 5.83% to 9.07%. Compared to BW, thermal treatment significantly (p < 0.05) reduced the moisture of the samples. Similarly, Albayrak et al. reported that the moisture content of low-quality wheat flour fell significantly following infrared-assisted thermal and infrared-assisted hydrothermal treatments [44]. Lower moisture content is beneficial for the long shelf life of wheat flour [45]. For the protein content, the three thermal treatments exerted different effects. Compared to the protein content of BW (14.5%), HFL-BW was higher (16.69%), MW-BW was lower (13.51%), and RST-BW was similar. Research has confirmed that thermal treatments affect the secondary structure of wheat proteins, induce protein aggregation, and decrease the extractability of specific proteins [46,47,48]. However, no research evidence suggests a correlation between the protein conformation and protein content of heat-treated wheat. The crude fat contents indicated that thermal treatment significantly decreased the fat content of the samples. The crude fat content of native BW was 2.22%, whereas the crude fat contents of the thermal-treatment groups ranged from 1.02% to 2.01%. A similar result was observed in a study investigating whole-grain quinoa flour, which found that heat–moisture treatment (HMT) and HMT plus microwave treatment reduced the total fat content of the flour [49]. Previous research has shown that microwave heating inactivates lipase and reduces free fatty acid levels in whole-wheat flour during storage [13]. Another study found that microwave treatment (700 W; 30 s) followed by steaming for 20 min significantly decreased the fat content in reconstituted whole-wheat flour. This decrease may be ascribed to fat oxidation under the thermal conditions [14]. Lipid loss might be influenced by high temperature, oxygen, fatty acid composition, and other factors during thermal treatment. Evaporation of water in the treatment process also exerts an influence on lipid content, owing to the leaching of lipids that occurs concomitantly with water evaporation [50]. There were no significant differences in total starch content among different treatments, which ranged from 59.64% to 63.23%. After thermal treatments, the total dietary fiber content reduced significantly (p < 0.05) for each group. Compared to BW (12.59% total dietary fiber), thermal treatment lowered the content to 10.70%, 10.38%, and 10.76% in HFL-BW, MW-BW, and RST-BW, respectively. Similar results reported that thermal processes (including autoclaving, hot-air oven, microwave, and toasting) on wheat bran significantly decreased its total fiber content, and the authors speculated that thermal treatments break glycoside linkages of fiber, leading to hydrolysis and the loss of arabinoxylans [51]. Some parts of total dietary fiber can be decomposed at higher temperatures. Also, some thermal treatments can break down insoluble fiber into soluble forms, changing the overall composition [52]. Compared to BW (1.38% ash), the ash content increased to 1.47% in HFL-BW but decreased to 1.27% and 1.26% in MW-BW and RST-BW, respectively. Ash, which is primarily located in the wheat bran, represents the total mineral content of whole-grain wheat flour. Ash content in flour is related to the milling process. Commonly, refined flour has less ash content, while whole-wheat flour has higher ash content owing to the presence of intact bran and germ [53]. The anthocyanin content in BW was the lowest, at 38.78 mg/kg. After thermal treatment, the anthocyanin contents of HFL-BW, MW-BW, and RST-BW rose to 39.57, 46.06, and 42.12 mg/kg, respectively. Although anthocyanins are less stable at higher solution temperatures, mild heat treatment of food materials can prevent anthocyanin oxidation by polyphenol oxidase [54]. While processing methods (such as heating, ultrasound, micronization, and microfluidization) can degrade some compounds, they can also release bound phenolics (including anthocyanins) by disrupting the cell wall matrix of the bran and endosperm of cereal grain, increasing their bioavailability and antioxidant activity [19]. Overall, the proximate composition results indicated that while different thermal treatments altered the nutritional values of whole-grain black wheat to some extent, they did not cause excessive nutrient loss. The antioxidant component, anthocyanin, increased after thermal treatments.

Table 1.

Proximate composition, physical properties, and color values of whole-grain black wheat (flour) subjected to different thermal treatments.

	BW	HFL-BW	MW-BW	RST-BW
Proximate composition
Moisture (%)	9.07 ± 0.52 a	5.83 ± 0.73 b	6.31 ± 0.14 b	6.36 ± 0.27 b
Crude protein (%)	14.50 ± 0.17 c	16.69 ± 0.16 a	13.51 ± 0.16 d	14.95 ± 0.12 b
Crude fat (%)	2.22 ± 0.02 a	1.02 ± 0.02 d	2.01 ± 0.01 b	1.79 ± 0.01 c
Total starch (%)	59.64 ± 1.46 a	63.23 ± 1.92 a	60.92 ± 0.73 a	62.70 ± 2.26 a
Total dietary fiber (%)	12.59 ± 0.23 a	10.70 ± 0.33 b	10.38 ± 0.41 b	10.76 ± 0.19 b
Ash (%)	1.38 ± 0.01 b	1.47 ± 0.01 a	1.27 ± 0.03 c	1.26 ± 0.02 c
Anthocyanin (mg/kg)	38.78 ± 0.49 c	39.57 ± 0.11 c	46.06 ± 0.32 a	42.12 ± 0.68 b
Physical properties
Thousand-seed weight (g)	35.74 ± 0.59 a	32.84 ± 0.60 b	32.86 ± 0.91 b	33.32 ± 0.62 b
Bulk density (g/L)	756.90 ± 13.02 a	498.01 ± 4.51 c	598.01 ± 2.01 b	605.41 ± 16.40 b
Puffing index	1.00 ± 0.00 c	1.52 ± 0.04 a	1.27 ± 0.02 b	1.25 ± 0.05 b
Hardness/N	29.29 ± 3.48 a	23.08 ± 4.31 b	23.92 ± 2.85 a,b	21.18 ± 4.34 b
Color values
L*	86.50 ± 0.32 a	77.85 ± 0.33 b	75.08 ± 0.31 d	76.44 ± 0.30 c
a*	2.30 ± 0.08 c	4.97 ± 0.20 b	5.76 ± 0.11 a	5.70 ± 0.15 a
b*	7.51 ± 0.10 d	12.42 ± 0.14 c	13.34 ± 0.06 b	13.82 ± 0.15 a
ΔE	0.00	10.30 ± 0.45 c	13.28 ± 0.38 a	12.35 ± 0.16 b

Note: BW represents the whole-grain black wheat (flour) without any thermal treatment. HFL-BW, MW-BW, and RST-BW denote whole-grain black wheat (flour) subjected to heat fluidization, microwave, and roasting treatments, respectively. Different superscript letters in the same row represent significant differences (p < 0.05).

3.2. Physical Properties Analysis of Whole-Grain Black Wheat

To understand the effect of thermal treatment on the whole-grain black wheat kernel, the thousand-seed weight, bulk density, puffing index, and hardness were analyzed. As shown in Table 1, compared to the BW weight of 35.74 g, the thousand-seed weight was significantly (p < 0.05) decreased in samples after thermal treatments, ranging from 32.84 g to 33.32 g. There was no significant difference among the three thermal-treatment groups. Thousand-seed weight, expressed in grams, is the weight of 1000 seeds. It reflects seed size and plumpness and is used to predict field yield [55]. In this research, the thousand-seed weight data suggested that thermal treatment reduced seed weight, likely due to a decrease in moisture content. The bulk density and puffing index indicated that thermal treatment caused significant (p < 0.05) grain expansion, particularly in the HFL-BW group. In addition, Figure 1 also visually shows the whole-grain black wheat under different thermal treatments. Compared to the BW group, the thermal-treated kernels appeared fuller. Cracks in kernels due to expansion were observed. A similar phenomenon was observed in a study on whole-grain highland barley subjected to thermal treatments [21]. The hardness of whole-grain black wheat was decreased appreciably after thermal treatment. According to the results of bulk density, puffing index, and hardness, it could be speculated that, under the thermal treatment, the moisture within the black wheat kernel was converted to a vapor state and the kernel’s dense structure increased vapor pressure, which generated steam and ultimately resulted in the structure expanding. Simultaneously, heating could cause internal cracks and cavities in the endosperm. Roasting induced the formation of large, irregularly distributed, and partially interconnected cavities and cracks in wheat kernels, thereby generating a porous structure [17]. These findings on physical properties suggest that thermal treatment, particularly heat fluidization, greatly promoted the expansion of whole-grain black wheat, likely by disrupting the kernel’s internal structure.

Figure 1.

Appearance of whole-grain black wheat (flour) subjected to different thermal treatments. BW represents the whole-grain black wheat (flour) without any thermal treatment. HFL-BW, MW-BW, and RST-BW denote whole-grain black wheat (flour) subjected to heat fluidization, microwave, and roasting treatments, respectively. The upper panel represents whole-grain black wheat kernel, and the lower panel represents whole-grain black wheat flour.

3.3. Color Characteristics of Whole-Grain Black Wheat Flour

Food color is closely associated with perceived flavor and consumer appetite [56]. Thermal processing of food can significantly impact its color. Figure 1 intuitively shows the color changes in whole-grain black wheat flour after different heat treatments. Thermal treatment resulted in darker flour compared to the untreated sample. The color of whole-grain black wheat flour was characterized by measuring L*, a*, and b* values with a colorimeter. As shown in Table 1, compared to the BW, thermal treatment significantly changed the color of the whole-grain black wheat flour. Thermal treatment decreased the flour lightness from 86.50 (BW) to 77.85 (HFL-BW), 75.08 (MW-BW), and 76.44 (RST-BW), respectively. Conversely, the a* value (redness) and b* value (yellowness) were significantly increased after thermal treatment. The MW-BW group had the highest a* value, and the RST-BW group had the highest b* value. The total color difference (ΔE) of the treated groups differed significantly from that of the control group, with microwave treatment exhibiting the highest ΔE value. The HFL-BW showed the lowest ΔE value among the thermal-treatment groups. The color changes were generally ascribed to the browning and Maillard reaction [57]. Similarly, Albayrak et al. reported that thermal treatment darkened and browned low-quality, unprocessed wheat flour [44]. In conclusion, thermal treatment greatly altered the color of the whole black wheat flour, thereby affecting its appearance. In brief, among the three thermal treatments, microwave treatment had the strongest and most significant effect on the color values of whole-grain black wheat flour. Heat fluidization treatment exhibited the smallest changes in color values.

3.4. Microstructural Characterization of Whole-Grain Black Wheat Flour

The microstructure of the whole-grain black wheat flour was observed using the SEM. Micrographs (Figure 2) revealed flour particles with irregular shapes and a range of sizes. The starches were attached or encapsulated with protein matrices. At 500× magnification, the BW group contained some round, intact starch granules, whereas the thermally treated groups had fewer intact granules. The BW group exhibited larger aggregates of the starch–protein matrix, with intact, smooth oval starch granules embedded in the endosperm (1500× magnification). Similar flour surface morphology was observed in previous research [58]. After thermal treatment, the flour was susceptible to damage. At 500× magnification, images revealed more tiny fragments and many small particles adhering to the starch granules compared to the BW group. The shapes of flour particles tended to be polygonal and angular, rather than round and oval. Both the 500× and 1500× images showed that the aggregations were ground into smaller, more uniform-sized particles. The 1500× images showed that the starches were broken and deformed after thermal treatment. The HFL-BW group image showed a honeycomb-like starch (upper right quarter of the 1500× image). The RST-BW group exhibited damaged, incomplete starch particles. Furthermore, pits or pores were visible on some particles in the middle left of the 1500× image. Research has confirmed that thermal treatment causes significant damage to starch in cereal grains. The changes in the morphology and microstructure of whole-grain black wheat flour can be attributed to starch gelatinization, protein denaturation, and starch–protein interactions during thermal processing [14,59,60].

Figure 2.

SEM images of the whole-grain black wheat flour subjected to different thermal treatments. BW represents the whole-grain black wheat flour without any thermal treatment. HFL-BW, MW-BW, and RST-BW denote whole-grain black wheat flour subjected to heat fluidization, microwave, and roasting treatments, respectively. The upper panel represents a magnification of 500, and the lower panel represents a magnification of 1500.

3.5. Starch Short-Range-Ordered Structures of Whole-Grain Black Wheat Flour

The ATR-FTIR spectroscopy was used to analyze the functional groups and short-range-ordered structures in the whole-grain black wheat flour. As shown in Figure 3A, the spectra of the thermally treated groups were similar to those of the BW group. The infrared spectrum showed neither new absorption peaks nor the loss of characteristic peaks after thermal treatment, indicating that no new chemical substances were formed in the whole-grain black wheat flour. Spectral analysis showed that the broad absorption band at about 3285 cm⁻¹ was attributed to the O-H stretching vibration related to bound water and the interactions between water molecules and the whole-grain black wheat flour components. After thermal treatment, the band intensity decreased, suggesting the disruption of hydroxyl groups and intermolecular interactions [61]. The peak at 2927 cm⁻¹ was C-H stretching vibrations of the aliphatic groups, which indicates the presence of lipids in the whole-grain black wheat flour.

Figure 3.

ATR-FTIR spectra at the wavenumbers 4000 to 400 cm⁻¹ (A), deconvolution spectra at the wavenumbers 1200 to 900 cm⁻¹ (B), and XRD diffractograms (C) of the whole-grain black wheat flour subjected to different thermal treatments. BW represents the whole-grain black wheat flour without any thermal treatment. HFL-BW, MW-BW, and RST-BW denote whole-grain black wheat flour subjected to heat fluidization, microwave, and roasting treatments, respectively.

The wavenumber range of 900–1200 cm⁻¹ corresponds to the starch fingerprint region, which reflects the short-range-ordered structure of starch. The sharp, intense absorption band at 992 cm⁻¹ corresponds to the stretching vibrations of C-O-C of the glycosidic bond and the hydrogen bonding of the glucose ring in the starch component. The absorbance at 1045 cm⁻¹ and 1016 cm⁻¹ indicates the ordered/crystalline and amorphous structures of starch, respectively. The ratio of 1045/1016 cm⁻¹ (R_1045/1016) is related to the ordered degree of starch [34,62]. Figure 3B shows the deconvolution spectrum of the short-range order in starch. The peak intensity at 1045 cm⁻¹ was significantly reduced after thermal treatment. Also, the absorbance ratio R_1045/1016 decreased significantly, especially in the HFL-BW group (Table 2). The results suggest that thermal treatment may attenuate interactions between starch molecules and significantly disrupt the starch crystalline structure. The starch structure underwent a significant transition from short-range-ordered to amorphous. Among the three thermal treatments, heat fluidization treatment exerted the most pronounced disruptive effect on the starch in whole-grain black wheat flour.

Table 2.

Absorbance ratio, protein secondary structure, and relative crystallinity of whole-grain black wheat flour subjected to different thermal treatments.

	BW	HFL-BW	MW-BW	RST-BW
R1045/1016	1.17 ± 0.04 a	0.31 ± 0.02 c	0.52 ± 0.05 b	0.54 ± 0.01 b
β-sheet (%)	36.11 ± 0.47 a	39.66 ± 1.45 a	37.48 ± 0.08 a	37.28 ± 0.13 a
random coil (%)	17.08 ± 0.61 a	18.17 ± 2.92 a	14.66 ± 0.10 a	14.72 ± 0.17 a
α-helix (%)	17.40 ± 0.21 a	15.95 ± 0.03 c	16.74 ± 0.14 b	17.10 ± 0.17 a,b
β-turn (%)	29.40 ± 0.68 a	26.22 ± 4.34 a	31.12 ± 0.06 a	30.90 ± 0.21 a
RC (%)	26.75 ± 1.37 a	2.56 ± 0.19 c	12.52 ± 1.28 b	15.74 ± 1.95 b

Note: BW represents the whole-grain black wheat flour without any thermal treatment. HFL-BW, MW-BW, and RST-BW denote whole-grain black wheat flour subjected to heat fluidization, microwave, and roasting treatments, respectively. R_1045/1016 represents the ratio of absorbance at 1045 to 1016 cm⁻¹. RC represents the relative crystallinity of the whole-grain black wheat flour. Because the assumption of homogeneity of variance was violated, Dunnett’s T3 test was used to analyze the β-sheet, random coil, and β-turn content. Different superscript letters in the same row represent significant differences (p < 0.05).

3.6. Starch Crystalline Structure of Whole-Grain Black Wheat Flour

XRD was used to analyze the effect of thermal treatment on the crystalline structure of starch in the whole-grain black wheat flour. As presented in Figure 3C, diffraction peaks were observed at 15.3°, 17.3°, 18.3°, and 23.2° in the BW group, indicating a typical A-type crystalline structure of starch. After thermal treatment, the diffraction peaks weakened in the MW-BW and RST-BW groups, while those in the HFL-BW group changed more substantially. The HFL-BW pattern showed the disappearance of the typical A-type diffraction peaks, indicating intensive disruption of starch crystallinity. All the diffraction patterns from thermal treatment exhibited a new peak at 19.8°, indicating that the starch structure tended to transform from A-type to V-type, accompanied by the formation of starch–lipid complexes. Previous research found similar results: wheat flour formed V-type starch–lipid complexes during cooking. Extrusion cooking transformed the A-type crystalline structure of native sorghum starch into a V-type structure [63,64]. Furthermore, Table 2 suggests that thermal treatment significantly reduced the relative crystallinity (RC) of starch. Notably, the relative crystallinity decreased from 26.75% to 2.56% (p < 0.05) after heat fluidization. Interestingly, the XRD relative crystallinity results were consistent with the FTIR R_1045/1016 ratio. Both tests demonstrated that thermal treatments, particularly heat fluidization, significantly disrupted the ordered structure of starch in whole-grain black wheat flour, resulting in a decrease in its crystallinity.

3.7. Gluten Protein Distribution and Protein Secondary Structure Alteration of Whole-Grain Black Wheat Flour

Wheat gluten, the primary protein component of wheat flour, consists mainly of gliadins and glutenins. Gluten protein plays an important role in the viscoelastic properties of dough and the quality of flour products. The molecular weight range of gliadins (including ω5-, ω1-, 2-, α/β-, and γ-gliadin) is ~30–75 kDa. The molecular weight range of glutenins is ~30–140 kDa. Glutenins consist of high-molecular-weight glutenin subunits (HMW-GSs; ~90–140 kDa) and low-molecular-weight glutenin subunits (LMW-GSs; ~30–75 kDa) [65,66].

This study employed SDS-PAGE to analyze how thermal treatments affect gluten proteins in whole-grain black wheat flour. The electrophoresis results are shown in Figure 4. Under non-reducing conditions, the protein aggregation observed near the sample wells (within the red box) in the BW lane of the gel may be attributed to high-molecular-weight glutenin polymers. The reducing pattern (within the red box) shows the BW lane resolved into subunits, indicating that disulfide bonds were the cause of this aggregation. For the entire lane (10–200 kDa), compared to BW, the thermal-treatment groups showed very faint bands under non-reducing conditions, suggesting that the proteins became less soluble in SDS. Under reducing conditions, the appearance of HMW-GS bands (~90–140 kDa) indicated that the intermolecular disulfide bonds of glutenin polypeptides were cleaved by β-mercaptoethanol. However, the thermally treated groups still exhibited large protein aggregates near the sample wells (within the red box). These aggregates suggest that non-covalent intermolecular interactions may also exist in thermally treated flour proteins. Notably, both the non-reducing and reducing patterns showed the weakest protein bands (10–200 kDa) in the HFL-BW groups in comparison with other treatments. The diminishment or disappearance of the bands in HFL-BW may be attributed to the decrease in the extractability of gluten protein. Overall, the intensity of protein bands (10–200 kDa) in the thermally treated groups was weaker compared to the BW group. Thermal treatments, especially heat fluidization treatment, likely caused the whole-grain black wheat flour gluten proteins to denature and become less extractable due to aggregation and the formation of new cross-links [67,68,69].

Figure 4.

Non-reducing and reducing SDS-PAGE electropherogram patterns of the whole-grain black wheat flour subjected to different thermal treatments. Mw represents molecular weight. Lane M represents the protein standard marker. BW represents the whole-grain black wheat flour without any thermal treatment. HFL-BW, MW-BW, and RST-BW denote whole-grain black wheat flour subjected to heat fluidization, microwave, and roasting treatments, respectively. The red box represents the sample wells of the gel.

Furthermore, the protein secondary structure of whole-grain black wheat flour was analyzed using FTIR. The amide I band (1700–1600 cm⁻¹) involves the coupling of C=O stretching vibrations and N-H bending vibrations. The amide II band is more complex than amide I, located between 1580 cm⁻¹ and 1480 cm⁻¹, primarily due to N-H bending, with a secondary contribution from C-N stretching. Therefore, the absorption peaks at 1646 cm⁻¹ and 1540 cm⁻¹ were assigned to the amide I and amide II bands, respectively. Interestingly, the spectrum showed that thermal treatment reduced the intensity of these two bands, indicating that thermal treatment disrupted the secondary structure of the protein in whole-grain black wheat flour. The amide I band (1700–1600 cm⁻¹) of infrared spectra reflects the protein secondary structures, which include β-sheet (1640–1600 cm⁻¹), random coil (1650–1640 cm⁻¹), α-helix (1660–1650 cm⁻¹), and β-turn (1700–1660 cm⁻¹) structures [70]. Figure S1 shows the FTIR deconvolution spectra in the amide I region. The proportions of protein secondary structures were calculated based on the FTIR deconvolution spectra. As shown in Table 2, the proportions of protein secondary structures, including β-sheets, random coils, and β-turns, did not change significantly among the different treatment groups. Compared with the BW group, the α-helix content was significantly decreased (from 17.40% to 15.95% and 16.74%) in the thermally treated groups (HFL-BW and MW-BW). Additionally, the β-sheet content increased, though the differences were not statistically significant. Similarly, Fan et al. reported that atmospheric steam treatment induced conformational unfolding, triggering a transition from α-helix to β-sheet [15]. The changes in this study suggest that thermal treatment may exert a slight disruptive effect on the stable conformations of proteins in whole-grain black wheat flour. In short, secondary structure analysis confirmed that the thermal treatment altered the proteins, consistent with the SDS-PAGE results.

3.8. Thermal Properties

The thermal properties of whole-grain black wheat flour were determined using DSC. As shown in Figure 5A, the BW group exhibited a conspicuous endothermic peak between about 60°C and 75°C. After thermal treatment, the endothermic peak intensity of the HFL-BW and MW-BW groups was significantly reduced, whereas that of the RST-BW group showed no significant change. Thermal curves visually demonstrate that thermal treatments (HFL-BW and MW-BW) considerably gelatinized the starch in whole-grain black wheat flour. Table 3 shows that the thermal transition was most significant in the HFL-BW group compared to the BW group. After heat fluidization, the T_o increased from 60.11°C to 62.67°C, the T_p increased from 65.15°C to 71.35°C, and the T_c increased from 75.33°C to 80.17°C. Conversely, the ΔH significantly reduced from 4.76 J/g to 0.59 J/g. Except for the increased T_p in the MW-BW group, the gelatinization temperature changes in the other thermal-treatment groups did not differ significantly from the BW group. In addition, compared with the BW group, the ΔH of MW-BW decreased significantly and that of RST-BW decreased slightly. Similar results have been reported in previous research. Microwave pretreatment of sorghum grains increased the gelatinization temperature (T_o, T_p, and T_c), while ΔH decreased significantly [71]. The gelatinization temperatures of proso millet flour increased significantly after heat–moisture treatment, while the ΔH decreased significantly [72]. The DSC results in the present study suggested that thermal treatments (heat fluidization and microwave treatment) induced starch gelatinization in the whole-grain black wheat flour, disrupting the ordered helical structure and changing the starch from a crystalline to an amorphous state [16]. Furthermore, thermal treatment can also cause protein denaturation and the formation of complexes containing starch, protein, and lipids. All these changes will affect the gelatinization temperature and enthalpy. Previous research has reported that thermal treatment induces the denaturation and structure alteration of gluten protein. The research results revealed that gliadin shows minimal aggregation during heating, and it has better thermal stability than glutenin due to the higher denaturation temperature, thereby impeding glutenin aggregation and overall gluten network formation [73]. Future research is needed to explore the denaturation behavior, via DSC, of gluten protein isolated from thermally treated whole-grain black wheat flour.

Figure 5.

Thermal properties (A) and pasting properties (B) of whole-grain black wheat flour subjected to different thermal treatments. BW represents the whole-grain black wheat flour without any thermal treatment. HFL-BW, MW-BW, and RST-BW denote whole-grain black wheat flour subjected to heat fluidization, microwave, and roasting treatments, respectively.

Table 3.

Thermal properties and pasting properties of whole-grain black wheat flour subjected to different thermal treatments.

	BW	HFL-BW	MW-BW	RST-BW
Thermal properties
To (°C)	60.11 ± 0.31 b	62.67 ± 1.36 a	59.87 ± 0.19 b	59.32 ± 0.68 b
Tp (°C)	65.15 ± 0.22 c	71.35 ± 0.68 a	66.57 ± 0.22 b	65.03 ± 0.42 c
Tc (°C)	75.33 ± 0.64 b	80.17 ± 0.36 a	75.70 ± 1.60 b	75.62 ± 1.74 b
ΔH (J/g)	4.76 ± 0.23 a	0.59 ± 0.17 c	2.16 ± 0.37 b	4.44 ± 0.29 a
Pasting properties
PT (°C)	67.77 ± 1.44 a	67.38 ± 1.46 a	67.07 ± 1.95 a	68.17 ± 3.10 a
PV (cP)	976.00 ± 50.32 a	–	–	992.67 ± 39.80 a
TV (cP)	458.67 ± 51.62	–	–	–
BV (cP)	517.33 ± 13.05	–	–	–
FV (cP)	1201.67 ± 85.03 b	593.67 ± 61.26 d	990.67 ± 80.48 c	1723.33 ± 89.37 a
SV (cP)	743.00 ± 36.86	–	–	–

Note: BW represents the whole-grain black wheat flour without any thermal treatment. HFL-BW, MW-BW, and RST-BW denote whole-grain black wheat flour subjected to heat fluidization, microwave, and roasting treatments, respectively. T_o, onset temperature; T_p, peak temperature; T_c, conclusion temperature; ΔH, enthalpy change; PT, pasting temperature; PV, peak viscosity; TV, trough viscosity; BV, breakdown viscosity; FV, final viscosity; SV, setback viscosity. “–”, not applicable. The independent-samples t-test was used to analyze the PV values. Different superscript letters in the same row represent significant differences (p < 0.05).

3.9. Pasting Properties and Viscoelastic Properties

The pasting properties of whole-grain black wheat flour are shown in Figure 5B and Table 3. RVA curves clearly indicate significant differences in viscosity development among the tested groups. At the beginning, the RVA results showed no significant differences in pasting temperature (67.07–68.17°C) among the groups. As defined by the National Standards of the People’s Republic of China [38], pasting temperature is the temperature at which a sample’s viscosity begins to increase upon heating. However, once pasting initiated, viscosity development among the four groups exhibited distinct trends. The BW group exhibited the complete pasting profile, whose viscosity values are listed in Table 3. Compared to the BW group, thermal-treatment groups showed incomplete pasting profiles, with no discernible peak or trough in viscosity. The highest peak viscosity (992.67 cP) was observed in the RST-BW group. Unlike the BW group, which exhibited a trough viscosity, the thermally treated groups showed continuous viscosity increase until the end of the test. Across the entire RVA profile, the RST-BW group exhibited the highest viscosity, indicating a strong starch granule swelling capacity [74,75]. It is speculated that roasting treatment may induce the formation of amylose–lipid or amylose–protein complexes. Also, roasting may unfold polar groups of proteins, making them more likely to bind water. Structural changes from roasting—including damaged starch granules and an altered protein matrix—resulted in higher viscosity for the RST-BW group [18,72]. A study performed RVA tests on wheat starch that had undergone pre-gelatinization. The authors found that starch with a low gelatinization degree exhibited higher pasting viscosities than native starch, whereas starch with a high gelatinization degree showed lower viscosities [76]. Therefore, it can be speculated that the roasting treatment led to a mild gelatinization of the whole-grain black wheat flour in the RST-BW group. Accordingly, microwave and fluidization treatment caused the greater gelatinization. This conjecture echoes the DSC results of Section 3.8. The viscosity of the microwave-treated group was intermediate between the heat-fluidized and roasted groups, indicating a moderate effect on starch pasting. Conversely, heat fluidization significantly reduced the viscosity throughout the RVA test, suggesting that it disrupted starch granules and thus impaired their pasting capacity [16]. It is presumed that heat fluidization treatment induced the pre-gelatinization of most starch granules in the whole-grain black wheat flour, which corresponds to the DSC results in Section 3.8. Furthermore, heat fluidization treatment may have severely disrupted the starch network and protein structure [77]. Overall, different thermal treatments altered the starch and protein structures in whole-grain black wheat flour, thereby contributing to differences in viscosity development [77].

As shown in Figure S2, the elastic (G’) and viscous (G’’) modulus in the whole-grain black wheat flour subjected to different thermal treatments were determined. The results showed that thermal treatments significantly increased both elastic and viscous moduli. Both moduli (G’ and G’’) of the different treatments from high to low are: HFL-BW > MW-BW > RST-BW > BW. All groups showed that G′ exceeded G″, indicating that the pastes exhibited greater elastic character than viscous behavior. The flour pastes exhibited solid-like rather than liquid-like behavior. The HFL-BW group paste showed the highest values for both elastic (G′) and viscous (G′′) moduli, indicating its strong potential for food gel applications. The improved viscoelasticity, most pronounced in the HFL-BW paste, is likely attributable to thermal treatment-induced starch pasting, protein denaturation/aggregation, and interactions between starch and protein. Furthermore, the observed discrepancy between the rheological and RVA results stems from their distinct methodologies, which involve different temperature–time profiles and shear conditions. The rheometer also provides higher sensitivity to microstructural changes. The gap sizes in rheometer may yield variations in viscoelastic parameters that affect the rheological characteristics of the flour samples [78].

3.10. In Vitro Digestibility

As shown in Figure 6, the in vitro digestibility of starch in the whole-grain black wheat flour was determined. Except for a slowdown in the hydrolysis rate between 20 and 60 min, the BW group had the highest overall starch hydrolysis rate (Figure 6A). Compared with the BW group, thermal treatments reduced the starch hydrolysis rate, with the HFL-BW group exhibiting the lowest rate after 180 min of digestion. Figure 6B shows that the thermal treatments considerably reduced the RDS content. Conversely, the RS contents increased dramatically from 20.1% (BW group) to 30.9%–39.6% (thermally treated groups). Total SDS + RS content ranked as follows: MW-BW (71.8%) > RST-BW (65.8%) > HFL-BW (64.3%) > BW (57.9%). Obviously, thermal treatments greatly slowed the digestibility of starch in whole-grain black wheat flour. The reduced starch digestibility in the thermally treated groups can be attributed to the formation of V-type starch–lipid complexes, as indicated by the XRD results (Section 3.6) [79]. Meanwhile, thermal treatments may cause the proteins to denature. The denatured protein could adhere to starch granules, thereby hindering the amylase’s hydrolysis [80]. In brief, thermal treatments of whole-grain black wheat flour can decrease starch digestibility and increase the RS contents. These thermal processing technologies for whole-grain black wheat will provide a reference for preparing staple foods for people with chronic diseases, such as diabetes.

Figure 6.

In vitro digestion properties of starches from the whole-grain black wheat flour subjected to different thermal treatments. (A) Starch hydrolysis rate; (B) contents of RDS, SDS, and RS. BW represents the whole-grain black wheat flour without any thermal treatment. HFL-BW, MW-BW, and RST-BW denote whole-grain black wheat flour subjected to heat fluidization, microwave, and roasting treatments, respectively. RDS, SDS, and RS represent the rapidly digesting starch, the slowly digesting starch, and the resistant starch, respectively.

The gluten protein distribution and FAN content in whole-grain black wheat flour subjected to different thermal treatments and in vitro digestion were investigated by SDS-PAGE (Figure S3) and ninhydrin reaction (Figure S4). Before digestion, the BW group clearly showed bands for both gliadin and glutenin, while the thermally treated groups showed only faint bands, except in the low-molecular-weight region (below 17 kDa). Band pattern differences from Section 3.7 may be due to different protein loads and gel percentages. For the gastric digestion phase, the colors of all four lanes were enhanced. Bands (30–75 kDa) of gliadins and LMW-GSs were observed among four groups. It could be speculated that the gastric digestive process enzymatically breaks down large, insoluble proteins. More evidently, distinct accumulative bands appeared below 17 kDa, which is in accordance with previous research [42]. These were likely the result of pepsin hydrolyzing the proteins into low-molecular-weight peptides. In the intestinal digestion phase, the protein molecular bands are mainly observed in the BW groups below 17 kDa. The bands of thermally treated groups almost vanished. These results indicate that gluten proteins were almost fully digested in the thermally treated groups after gastrointestinal digestion, while the untreated BW group still contained indigestible polypeptides. Both before and after digestion, all thermally treated groups showed fainter bands compared to the BW group. This may be due to protein aggregation and denaturation, causing less extractable protein after thermal treatments, as mentioned in Section 3.7. Furthermore, since pepsin and pancreatin are themselves proteins, it cannot be ruled out that they might interfere with the results of the electrophoresis. In addition, the thick bands observed at the BW group (both before digestion and in the gastric digestion phase) may be related to the overlay of different protein fractions [81].

The FAN content is presented in Figure S4. Before and after digestion, the BW group showed the highest FAN content. Thermal treatments resulted in lower FAN content compared to the BW group. The FAN content rose significantly after gastric digestion in all four groups, indicating the strong proteolytic potential of pepsin toward whole-grain black wheat flour proteins. In contrast, intestinal digestion did not produce a further evident increase in FAN compared to the gastric phase. This suggests that gastric pepsin plays the dominant role in digesting the protein in this flour. Previous studies confirmed that thermal processes (such as stewing and roasting) on meat products reduce the susceptibility of proteins during gastrointestinal digestion by causing protein aggregation, cross-linking, or increasing disulfide content [82]. Protein aggregation, induced by higher temperatures, can limit protease accessibility to cleavage sites [83]. Therefore, it can be inferred that the thermal treatments in this study led to a decrease in protein bioavailability of whole-grain black wheat flour.

4. Conclusions

This study systematically evaluated the effects of heat fluidization, microwaving, and roasting on whole-grain black wheat flour. All thermal treatments effectively modified the flour with minimal loss to its proximate composition. They increased anthocyanin content, promoted kernel swelling, and darkened flour color. The treatments induced starch gelatinization and protein denaturation, disrupting starch crystallinity and protein structure. This altered starch viscosity and reduced digestibility while increasing resistant starch content. Among the methods, heat fluidization had the most pronounced effect, causing the greatest structural disruption and reduction in digestibility, with minimal color change. Microwave and roasting treatments were relatively gentle. It is worth mentioning that microwave treatment is most effective at preserving anthocyanins. Roasting, on the other hand, produces the most resistant starch. Overall, heat fluidization emerges as the preferred technique for maximizing structural and functional modifications in whole-grain black wheat flour, while microwaving and roasting offer distinct advantages for specific nutritional targets, providing food processors with versatile, evidence-based options for tailored product development.

Based on the findings of this study, future research is expected to focus on: (i) investigating the effects of thermal treatments on the properties of different fractions (bran, germ, and endosperm) of whole-grain black wheat; (ii) optimizing thermal treatment techniques, particularly for the heat fluidization method, which showed the most pronounced effect, to develop whole-grain black wheat personalized products with slow digestibility (including the staple foods of bread, pasta, and Chinese steamed bread); (iii) investigating wet heat treatment (like boiling and steaming), which can be selected for research and compared with the dry heat treatment, to evaluate the effect of thermal treatment on whole-grain black wheat from multiple perspectives and more comprehensively; (iv) conducting in vivo experiments to explore the health benefits and mechanisms of thermal-treated whole-grain black wheat given its reduced digestibility; (v) researching the antioxidant activity and health effects of thermal-treated whole-grain black wheat through in vivo and in vitro studies given the high content of anthocyanins in colored wheat.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods15040791/s1, Figure S1: FTIR deconvolution spectra in the amide I region (1600–1700 cm^–1) of the whole-grain black wheat flour subjected to different thermal treatments; Figure S2: Rheological characteristics of storage modulus (G’) and loss modulus (G’’) in the whole-grain black wheat flour subjected to different thermal treatments; Figure S3: SDS-PAGE electropherogram pattern of gluten protein in whole-grain black wheat flour subjected to different thermal treatments and in vitro digestion; Figure S4: Free amino nitrogen (FAN) content in the in vitro digestates of whole-grain black wheat flour subjected to different thermal treatments.

Author Contributions

S.L.: Conceptualization, Methodology, Investigation, Data curation, Visualization, and Writing—original draft. Y.M.: Methodology and Investigation. J.W.: Methodology and Investigation. M.Z.: Methodology and Investigation. W.Z.: Investigation. Y.X.: Investigation. Z.C.: Supervision, Resources, Conceptualization, and Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.