Study on the Degradation Patterns and Structure–Activity Relationship of Wheat Arabinoxylan Hydrolysate by Wheat Malt β-1,4-Endoxylanase

TL;DR

This study shows how enzymatic treatment changes the structure and properties of wheat arabinoxylan, enabling tailored use in food applications.

Contribution

The study reveals how molecular weight and branching affect arabinoxylan functionality after enzymatic hydrolysis.

Findings

Enzymatic cleavage reduced molecular weight and altered arabinose-to-xylose ratios in wheat arabinoxylan.

High-molecular-weight arabinoxylan showed better water-holding and foam stability due to intact helical structures.

Low-molecular-weight arabinoxylan with high A/X ratios improved emulsifying and antioxidant properties.

Abstract

This study investigates how wheat arabinoxylan (AX) structure influences its functional properties following enzymatic hydrolysis with wheat malt β-1,4-endoxylanase. Using three types of wheat AX with initial molecular weights of 489.42–602.42 kDa, arabinose-to-xylose (A/X) ratios of 0.49–0.55, and average degrees of polymerization (avDP) of 1223.57–1506.05 as substrates, enzymatic cleavage produced four high-purity fractions with reduced molecular weight (98.63–301.42 kDa), increased A/X (0.60–0.65), and lower avDP (246.59–753.56). Enzyme action led to triple-helix unwinding, especially at low avDP, accompanied by reduced storage modulus. Molecular weight was the key factor affecting water-holding capacity and foam stability, with high-molecular-weight AX showing superior performance due to its intact helical structure and higher viscoelasticity. In contrast, low-molecular-weight AX with high A/X ratios exhibited enhanced interfacial adsorption and free radical scavenging, supported by greater hydroxyl exposure and higher negative charge density (−9.23 mV). Its emulsifying activity and hydroxyl radical scavenging rate increased by 32.95% and 32.02%, respectively, compared to the original AX. These findings demonstrate that enzymatic modulation of AX molecular weight and branching can directionally tune its functionality, providing a theoretical basis for targeted applications in food systems.

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1. Introduction

Arabinoxylan (AX) is a key non-starch polysaccharide present in wheat, exerting significant effects on the texture, nutritional value, and health benefits of food products [1]. The molecular structure of AX is complex. Its backbone consists of β-(1,4)-linked D-xylopyranose residues, with side chains composed of L-arabinofuranose units attached to the main chain. The L-arabinofuranose residues are connected to a xylose backbone via O-2 or O-3 positions, generating side chains that can be either mono- or di-substituted [2]. These structural features directly influence its functional characteristics, including the water-holding capacity (WHC), emulsification, and foaming performance. Therefore, the in-depth research of the structure–function relationship of AX holds significant theoretical importance and practical value for the processing and utilization of wheat and its derived products.

Currently, research on AX primarily focuses on extraction, modification, and application; limited studies are focused on the enzymatic degradation patterns of AX and the structure–function relationship of its degradation products. Endo-β-1,4-xylanase is a critical enzyme for degrading AX, capable of catalyzing the cleavage of glycosidic bonds between the xylose units in the AX backbone, thereby altering the molecular weight and structure of AX [3]. AX degradation products with varying molecular weights and structural characteristics can be produced by regulating the enzymatic hydrolysis conditions, which in turn facilitates the study of their structure–function relationships.

In this study, three wheat AX substrates with different molecular weights were enzymatically hydrolyzed using wheat malt β-1,4-endoxylanase, and the hydrolysates were separated and purified by column chromatography to obtain wheat AX fractions with distinct structural characteristics. The structural features of the AX fractions were identified by high-performance liquid chromatography (HPLC) and Fourier transform infrared spectroscopy (FTIR), and their functional properties, such as WHC, emulsifying ability, and foaming properties, were further investigated to elucidate the structure–function relationship of wheat AX. The study revealed the enzymatic degradation patterns of wheat AX and the relationship between AX structural changes and its functional properties, including WHC, foaming stability, and antioxidant activity. This work provides a foundation for a deeper understanding of the structure–function characteristics of AX and for developing high-value-added wheat products. The findings offer theoretical guidance for the targeted enzymatic modification of AX in the food industry and contribute to the comprehensive utilization of wheat resources.

2. Materials and Methods

2.1. Materials

Wheat malt was ordered from Yuehai Yongshentai Malt Co., Ltd. (Shandong, China). Three wheat AX samples with different molecular weights (GWEAX, MWEAX, and LWEAX) were provided by Megazyme (Bray, Ireland). All chemical reagents used were of analytical or HPLC grade (Sinopharm Chemical Reagent Co. (Shanghai, China)).

2.2. Enzymatic Hydrolysis of Wheat AX by Wheat Malt β-1,4-Endoxylanase

The extracted wheat malt β-1,4-endoxylanase was purified based on the method described by Fan et al. [4] with slight modifications. Briefly, 200 g of wheat malt powder was suspended in 750 mL of phosphate buffer (0.05 M, pH 7.0). The suspension was stirred for 0.5 h in an ice bath, and the volume was adjusted to 1 L with the same buffer. After centrifugation, the resulting supernatant was harvested as the crude enzyme extract. Solid ammonium sulfate was gradually added to the crude enzyme solution to 40% saturation. After 90-min stirring in an ice bath, the mixture was centrifuged. The precipitate was redissolved in phosphate buffer (pH 5.5) and dialyzed (molecular weight cutoff: 3500 Da) against the same buffer for 48 h. The dialyzed solution was centrifuged to collect the supernatant, which was subjected to SP-Sepharose column chromatography. The column was equilibrated and eluted using 200 mL of elution buffer with a stepwise NaCl gradient (0–0.5 M, with each gradient step of 0.1 M) at a flow rate of 4 mL/min. The enzyme fraction eluted using 0.1 M NaCl was collected, dialyzed, and freeze-dried to obtain the purified wheat malt β-1,4-endoxylanase. For enzymatic hydrolysis, 0.1 g of each wheat AX sample (GWEAX, MWEAX, or LWEAX) was dissolved in phosphate buffer (pH 5.5). Then, 15 mL of 0.05 M wheat malt β-1,4-endoxylanase solution was added, and the total volume was adjusted to 100 mL with the same buffer. The reaction was performed at 40 °C for 1 h and terminated by boiling for 10 min in a water bath. After centrifugation, the precipitate was collected, rinsed, and treated using the Sevag method for protein removal. The final product was freeze-dried for subsequent use. A fixed hydrolysis time of 1 h was selected to ensure controlled backbone cleavage while avoiding excessive depolymerization, thereby enabling the construction of AX fractions with comparable structural gradients for structure–function analysis.

2.3. Separation and Purification of Wheat AX

The three types of enzymatically hydrolyzed AX obtained in Section 2.2 were prepared as 1.0 mg/mL solutions, respectively. Subsequently, 15 mL of each solution was applied onto a DEAE-52 cellulose ion-exchange column (26 mm × 40 cm, Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China)) for separation. Gradient elution was conducted at a flow rate of 5 mL/min with NaCl solutions with concentrations of 0 M, 0.1 M, 0.2 M, and 0.3 M. The eluent was fractionated with a BS-100A automatic fraction collector (Shanghai Qingpu Huxi Instrument Factory (Shanghai, China)) at 5 mL per tube. The absorbance at 480 nm was monitored, and the elution profile was plotted. The different AX fractions obtained from DEAE-52 cellulose column chromatography were dialyzed and freeze-dried. A 1.0 mg/mL solution of each fraction was prepared by redissolving the material in ultrapure water. After filtration through a membrane, 15 mL of each solution was applied onto a Sephacryl S-300 HR gel permeation chromatography column (26 mm × 40 cm, Shanghai Yuanye Bio-Technology Co., Ltd.). The fraction sample was eluted using ultrapure water at a flow rate of 0.2 mL/min. The eluent was harvested in 8-mL fractions per tube, and the absorbance at 480 nm was recorded to plot the elution profile. The eluted fractions were harvested and subsequently freeze-dried for further use.

2.4. Determination of AX Content, A/X Ratio, and Average Degree of Polymerization

Referring to the report of Jiang et al. [5], sample pretreatment was performed as follows: (1) Acid hydrolysis: 2 mL of 1 mg/mL AX solution was added to 0.7 mL of trifluoroacetic acid (TFA) and hydrolyzed for 3 h at 100 °C. The solution was diluted to 5 mL using ultrapure water after cooling,. (2) Reduction: 1 mL of the hydrolysate was mixed with 0.3 mL of sodium borohydride (NaBH_4_) and 0.3 mL of 12 M ammonia solution, and the reaction was allowed to proceed at 40 °C for 1 h. Glacial acetic acid (0.4 mL) was added to terminate the reaction. (3) Derivatization: the reduced solution (1 mL) was combined with 1-methylimidazole (0.5 mL) and acetic anhydride (4.5 mL). The reaction proceeded precisely for 10 min, and it was then quenched by adding 10 mL of ultrapure water, followed by cooling in the ice bath. (4) Extraction: 3 mL of dichloromethane was mixed with the derivatized solution for extraction. After phase separation, the organic layer was harvested. The extraction was repeated twice, and the combined organic phases were diluted to 10 mL with dichloromethane. The final extract was filtered through a 0.45 μm membrane for further analysis.

Parameters of gas chromatography (GC): a DM-2330 capillary column (30 m × 0.32 mm × 0.2 μm, Agilent Technologies, Santa Clara, CA, USA) was connected to the Shimadzu GC-2030 (Kyoto, Japan), with an injection volume of 2.0 μL. Temperature settings: injector was 250 °C, detector was 260 °C, and column oven was 240 °C. High-purity nitrogen was utilized as the carrier gas with a split ratio of 14:1. The column pressure was 50.0 kPa, with a total flow rate of 40 mL/min and a split flow rate of 13.5 mL/min. The total running time was 20 min.

Free monosaccharides in the samples were detected after reduction, derivatization, and extraction. The following sugars were used as the standard: xylose (Xyl), arabinose (Ara), glucose (Glu), galactose (Gal), rhamnose (Rha), mannose (Man), fucose (Fuc), ribose (Rib), and allose (All). The standard curves for the monosaccharides are shown in Table 1.

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2.5. Determination of Polysaccharide Molecular Weight

The molecular weight was measured according to the protocol reported by Jiang et al. [5] with some modifications. The pre-enzymatically hydrolyzed AX and the enzymatically hydrolyzed and purified AX samples were prepared as solutions of 1.0 mg/mL, respectively, which were filtered using 0.45 μm and 0.22 μm membranes sequentially.

Analysis of polysaccharide molecular weight using HPLC was carried out using a TSK guard column (35 mm × 4.6 mm) connected in series with TSK gel G3000/G4000 PWxl analytical columns (300 mm × 7.8 mm) (Tosoh Co., Ltd., Tokyo, Japan) and a refractive index detector (RID-10, Shimadzu, Kyoto, Japan). The analytical parameters were as follows: injection volume was 10 μL, flow rate was 0.3 mL/min, column temperature was 40 °C, mobile phase was 50 mM NaNO_3_, and total running time was 70 min. A calibration curve was constructed using 1 g/L pullulan standards with different molecular weights.

2.6. Determination of AX Triple-Helical Structure

Based on the method described by Lv et al. [6], a slightly modified protocol was developed and employed in this study. Briefly, 1 mL of 2 mg/mL AX solution was mixed with 1 mL of 0.2 mM Congo red solution. The NaOH concentration in the mixture was adjusted to a gradient of 0 to 0.5 M by adding different volumes of 1 M NaOH. After 20 min of incubation, the maximum absorption wavelength was determined by scanning the sample in the 400–700 nm range using ultraviolet spectrophotometry.

2.7. Determination of ζ-Potential and AX Particle Size

A 0.2 mg/mL solution of AX was prepared by dissolving it in ultrapure water. The ζ-potential and particle size of the AX solution were measured using a laser particle size analyzer (Thermo Fisher Scientific (Shanghai, China)). The particle size was monitored using dynamic light scattering (DLS, 633 nm), while the ζ-potential was measured using electrophoretic light scattering (ELS). All measurements were carried out at 25 °C and repeated in triplicate.

2.8. Analysis of AX Surface Morphology

Scanning electron microscopy (SEM) was conducted according to the method described by Li et al. [7]. Briefly, a small amount of the sample was fixed onto a specimen stub using conductive adhesive, followed by sputter-coating with gold for 1 min using a magnetron sputter coater (SSG-50M). The sample was then transferred to a scanning electron microscope (Hitachi (Tokyo, Japan)) and observed under an accelerating voltage of 2 kV.

2.9. Determination of AX Foaming Capacity and Foam Stability

The operation was carried out in light of the protocol developed by Kaushik et al. [8] with slight modifications. In brief, 0.2 g of AX was mixed with 20 mL of a 1.0% (w/v) wheat protein solution. The initial volume of the mixture was labeled as V0. The mixture was then homogenized for 2 min at 12,000 rpm, and the total volume (including both the liquid and foam) was recorded as V1 immediately after homogenization. The sample was allowed to stand at room temperature for 10 min, and then the total volume was recorded as V2.

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2.10. Determination of the WHC of AX

The WHC of AX was determined as follows: 0.2 g of AX powder was blended with 10 mL of deionized water, and the resulting mixture was sheared at 10,000 rpm for 2 min. Subsequently, the suspension was shaken at 100 rpm for 30 min at 37 °C. After standing, the mixture was equilibrated at 25 °C for 30 min and then centrifuged at 10,000× g for 15 min. The resulting supernatant was carefully removed. The WHC (g/g) was obtained using the following formula:

$$[eqn]$$

where WHC: Water-holding capacity (g water per g sample); m_0_: Mass of the sample (g); m_1_: Mass of the centrifuge tube and the sample (g) before centrifugation; m_2_: Mass of the centrifuge tube and the sample (g) after removing the supernatant.

2.11. Determination of Emulsifying Capacity of AX

The operation was carried out as reported by Li et al. [7] with slight modifications. An AX aqueous solution was prepared, with a concentration of 2 mg/mL. Then, 15 mL of the AX solution was mixed with 5 mL of peanut oil. The mixture was homogenized at 20,000 rpm with a high-speed disperser for 3 min and then allowed to stand. Aliquots of 0.05 mL were taken from the emulsion at 5 min and 10 min after standing, and each aliquot was added to 5 mL of 0.1% SDS solution. After thorough mixing, the absorbance at 500 nm was measured for the 5-min (A_0_) and 10-min (A_10_) samples using 0.1% SDS as the blank control.

$$[eqn]$$ $$[eqn]$$

where N: dilution factor; A_0_: absorbance at 0 min; C: AX concentration; θ: oil volume fraction (0.75); ρ: light path length (1 cm); A_10_: absorbance at 10 min

2.12. Determination of the AX Antioxidant Properties

The antioxidant activity was determined using commercial assay kits for hydroxyl radical scavenging activity, DPPH radical scavenging activity, and ferric reducing antioxidant power (FRAP) (Nanjing Jiancheng Bioengineering Institute, China) (Nanjing, China), following the manufacturer’s instructions.

2.13. Determination of AX Rheological Properties

AX solution (2 mg/mL) was prepared and its rheological characteristics were determined with a modular smart rheometer (Anton Paar). The shear viscosity and shear stress were determined under a shear rate sweep from 0.1 to 100 s^−1^. The storage modulus (G′) and loss modulus (G″) were determined within an angular frequency range of 0.1 to 100 rad/s.

2.14. Data Analysis

All experiments were carried out with three independent replicates. Data were analyzed and plotted with software including Microsoft Excel 2018 and Origin 2018. One-way analysis of variance (ANOVA) was performed with IBM SPSS Statistics 26, followed by significance tests for differences among factors. Different letters suggest statistically significant differences at the 0.05 level. Pearson’s two-tailed test was used for correlation analysis. Data are shown as the mean ± standard deviation.

3. Results and Discussion

3.1. Separation and Purification of AX and Its Enzymatic Hydrolysates

Following enzymatic hydrolysis and deproteinization, the GWEAX, MWEAX, and LWEAX samples were separated by gradient elution on a DEAE-52 cellulose ion-exchange column, with the corresponding elution profiles exhibited in Figure 1A–C. Following sequential elution with NaCl solutions of 0, 0.1, 0.2, and 0.3 M, all three enzymatically hydrolyzed AX samples yielded four distinct fractions. In each case, the elution profile displayed a predominant absorption peak under 0.1 M NaCl, corresponding to fractions of A-2, B-2, and C-2, with peak area percentages of 67.95%, 70.28%, and 66.93%, respectively. These major fractions (A-2, B-2, and C-2) were then dialyzed, concentrated by rotary evaporation, and freeze-dried, ultimately yielding three AX components designated as A, B, and C. Fractions A, B, and C were separately subjected to further purification on a Sephacryl S-300 HR column. Fraction A yielded two distinct elution peaks, which were designated as HAX-1 and HAX-2. HAX-1 exhibited a higher absorbance and an earlier elution time compared to HAX-2, suggesting a higher relative molecular weight. Fractions B and C each yielded a single elution peak, which was designated as MAX and LAX, respectively. All collected fractions were subsequently freeze-dried to obtain purified polysaccharides. Only fractions with high abundance and unimodal molecular weight distributions were selected for subsequent functional analysis to ensure structural representativeness and comparability.

3.2. Structural Analysis of AX and Its Enzymatic Hydrolysates

3.2.1. Analysis of AX Properties and Content

The structural analysis results of the seven AX samples are listed in Table 2. The data in Table 2 show that the purities of the enzymatically hydrolyzed and purified fractions HAX-1, HAX-2, MAX, and LAX all exceeded 90%. The A/X ratios of the undegraded GWEAX, MWEAX, and LWEAX samples were 0.53, 0.49, and 0.55, respectively, with average degrees of polymerization (avDP) all above 1200. In contrast, the four enzymatically hydrolyzed and purified AX fractions exhibited significantly increased A/X ratios from 0.60 to 0.67, along with a notable reduction in avDP, with LAX showing the lowest avDP of 246.59.

These changes in avDP and A/X ratio can be due to the specific cleavage mechanism of endo-xylanase targeting the β-1,4-linked xylan backbone [9]. The observed degradation pattern is consistent with the reported specificity of endo-β-1,4-xylanase. During enzymatic hydrolysis, endo-xylanase preferentially acts on the unsubstituted regions of the main chain, resulting in the breakage of glycosidic bonds between xylose units. This process directly reduces avDP while simultaneously increasing the A/X ratio. Unlike previous studies, the aim of this research was not to maximize hydrolysis efficiency, but to generate AX components with unique and stable structural characteristics for structure–function analysis. Moreover, the lower avDP of the initial LWEAX substrate (1223.57) likely facilitated more efficient enzymatic action of endo-1,4-β-xylanase, further promoting a reduction in avDP [2]. Previous studies have indicated that changes in the A/X ratio are closely related to the solubility and functional characteristics of AX, with higher A/X ratios generally correlating with improved solubility and bioactivity [10].

3.2.2. Molecular Weight Analysis of AX

Figure 2A illustrates the molecular weight distribution of the AX samples, with the corresponding specific data provided in Table 3. The three undegraded WEAX samples showed broad, multimodal peaks. Specifically, GWEAX exhibited a primary molecular weight range of 1200–280 kDa, with an average molecular weight (Mw) of 602.42 kDa; MWEAX ranged mainly from 900 to 280 kDa (Mw = 577.62 kDa); and LWEAX spanned 900–240 kDa (Mw = 489.42 kDa). Following enzymatic hydrolysis and purification, the four purified AX fractions displayed single, narrow peaks shifted distinctly to the right. HAX-1 had an Mw of 301.42 kDa; MAX and HAX-2 partially overlapped with molecular weights of 224.80 kDa and 197.73 kDa, respectively; and LAX had an Mw of 98.63 kDa.

The study by Fan et al. showed that wheat malt β-1,4-endoxylanase specifically degrades AX, with the highest degradation efficiency observed for WEAX fractions with Mw > 300 kDa, typically yielding products around 100 kDa [4]. In this study, the molecular weights of the enzymatic hydrolysates were primarily concentrated in the 100–300 kDa range, a result that may be attributed to differences in enzymatic conditions. Previous research indicates that the complete degradation of AX in the 100–300 kDa range generally requires more than 3 h, with hydrolysis time closely related to AX viscosity [4]. Overall, the four isolated AX fractions showed narrow, unimodal molecular weight distributions, confirming their high polysaccharide purity and effective separation.

3.2.3. FTIR Spectroscopic Analysis of AX

The FTIR spectra of the AX samples are shown in Figure 2B. After enzymatic hydrolysis, significant changes were observed in the infrared absorption spectra of the AX samples. The peak at 3292 cm^−1^ corresponded to the O–H stretching vibration of polysaccharides [11]. This region exhibited the most notable difference before and after degradation, with a pronounced increase in absorption intensity after hydrolysis, indicating substantial exposure of O–H groups. Among the samples, LAX showed the most significant hydroxyl group exposure [4]. The peak at 2906 cm^−1^ was attributed to C–H stretching vibrations [12]. The peak at 1532 cm^−1^ was assigned to the characteristic stretching vibration of the phenolic ring in ferulic acid [13]. This peak was clearly visible in all AX samples, indicating that the ferulic acid structural unit was retained before and after enzymatic hydrolysis. The peak at 1653 cm^−1^ corresponded to the C=O asymmetric stretching vibration, suggesting the presence of uronic acids or esterified carboxyl groups in AX. LAX exhibited the lowest transmittance at this position, reflecting greater exposure of carboxyl groups. The weak shoulder peak at 1412 cm^−1^ was closely associated with the presence of arabinose side-chain substituents, with lower transmittance indicating a higher degree of substitution in AX [14]. Among the four purified AX samples, HAX-1 showed the highest transmittance at this wavelength, indicating the lowest degree of substitution, which is consistent with the A/X ratio results discussed earlier. The region around 1050 cm^−1^ was characteristic of xylose [4]. Absorption peaks at 833 cm^−1^ and 891 cm^−1^ were assigned to α-glycosidic and β-glycosidic bonds, respectively [15]. The presence of these peaks in all AX samples indicated consistency in glycosidic bond types across different AX samples. However, variations in peak intensity and shape were observed among the samples. Based on the structural parameters discussed earlier, HAX-1 had the highest molecular weight and the lowest A/X ratio, with longer molecular chains and a lower degree of branching, which may promote the formation of more compact aggregated structures. This could restrict the vibrational freedom of certain functional groups, thereby influencing the intensity and morphology of the absorption peaks.

3.2.4. Congo Red Assay for AX Conformational Analysis

The Congo red assay is often utilized to characterize conformational changes in polysaccharides. Congo red, an azo dye with a conjugated structure, can align in an orderly manner within the grooves of the triple helical polysaccharide structure, forming a stable complex. Under alkaline conditions, polysaccharide chains undergo moderate extension, which facilitates the exposure of binding sites in the triple helical conformation, promoting specific binding with Congo red. This interaction alters the electron delocalization of the dye, leading to a redshift of the maximum absorption wavelength (λmax). Conversely, disruption of the triple helical structure led to decreased stability of the complex, causing a blueshift (hypsochromic shift) in λmax [16].

As shown in Figure 2C, all seven AX samples exhibited a distinct redshift in λmax in 0.15 M NaOH, indicating that AX can maintain a relatively intact triple helical conformation within this alkaline concentration range. Among them, LWEAX showed the highest λmax (510.32 nm), reflecting the strongest binding with Congo red. Nonetheless, when the NaOH concentration was over 0.15 M, AX generally exhibited a blueshift, suggesting that high alkalinity disrupts both intramolecular and intermolecular hydrogen bonds, leading to the gradual dissociation of the triple helical structure.

The extent of blueshift differed significantly among the AX samples. Undegraded AX (e.g., GWEAX) displayed a smaller blueshift, which may be attributed to their lower A/X ratios (0.49–0.55) and longer backbone (avDP > 1223.57) structural features that favor triple helical stability. In contrast, LAX showed the largest blueshift (12.98 nm). Its backbone was severely cleaved (avDP reduced to 246.59–753.56) and the A/X ratio increased (0.60–0.67), which likely damaged the triple helical groove structure, hindering stable Congo red binding.

FTIR results further revealed that LAX possesses a higher hydroxyl group density. Although increased hydroxyl groups can form more hydrogen bonds and thus strengthen the overall hydrogen bonding network, excessive or unevenly distributed hydrogen bonds may alter molecular conformation and disrupt the original symmetrical structure [17], ultimately reducing the stability of the triple helical conformation. This inference is also indirectly supported by rheological data: at 2% concentration, LWEAX exhibited a significantly higher storage modulus (G′) than LAX, displaying more pronounced gel like behavior, indicating that higher avDP contributes to a more stable molecular network. Combined with the Congo red results, it can be further inferred that increased backbone length helps maintain the helical related conformation of AX.

3.2.5. Particle Size Analysis of AX

As shown in Figure 3A, the three undegraded WEAX samples exhibited a broad particle size distribution (106 nm to 1720 nm), with average particle sizes larger than those of the purified samples. Among the purified AX samples, HAX-1 had the highest average particle size (730 ± 27.41 nm), followed by MAX (522.8 ± 38.82 nm) and HAX-2 (350.8 ± 42.01 nm), while LAX showed the smallest average particle size (285.7 ± 31.40 nm). HAX-1, HAX-2, and MAX all displayed a unimodal particle size distribution, whereas LAX exhibited a bimodal distribution, with peak area percentages of 22.53% and 77.47%, respectively. The phenomenon is likely due to its remarkably lower molecular weight, which provides the molecules with greater conformational freedom in solution, leading to the formation of multiple stable or metastable aggregation states [18]. The small-particle-size peak likely corresponds to individual molecules or small molecular aggregates, while the large-particle-size peak may correspond to multi-molecular aggregates or structural heterogeneity [19].

3.2.6. Zeta Potential Analysis of AX

The zeta potentials of different AX samples are shown in Figure 3B. The undegraded samples (GWEAX, MWEAX, LWEAX) exhibited relatively low absolute zeta potential values (−4.21 to −5.14 mV), while the enzymatically hydrolyzed samples (HAX-1, HAX-2, MAX, LAX) showed significantly increased absolute zeta potential values, with LAX and HAX-2 reaching −9.23 mV and −7.27 mV, respectively. This trend is likely the result of two synergistic factors: first, the reduction in molecular weight leads to a greater exposure of functional groups on the molecular surface [20], enhancing charge carrying capacity; second, the higher A/X ratio increases the number of arabinose side chains, thereby raising the proportion of polar groups such as carboxyl and hydroxyl groups. As observed in Figure 2B, LAX had the lowest transmittance at 1653 cm^−1^ (C=O stretching vibration), indicating a markedly higher carboxyl group density than the other samples, further confirming that the more negative zeta potential of LAX is due to increased carboxyl content. Additionally, the AX′ highly branched side chain structure, due to the reduced steric hindrance and increased conformational flexibility [21], facilitates the exposure of negative charges on the molecular surface, enhancing interfacial potential. In summary, AX degradation not only altered its molecular dimensions but also significantly reshaped its surface chemical environment, with low polymerization degree and high A/X ratio samples exhibiting stronger surface electronegativity. This change in zeta potential reflects not only enhanced intermolecular electrostatic repulsion, but also, due to the negative charge, an improved ability to adsorb surrounding positively charged substances, thereby enhancing the dispersibility of AX. It may also influence its dispersibility in solution and its bioactivity [22].

3.2.7. The Morphological Structure Analysis of AX

Figure 4 presents the morphological structure of AX before and after degradation. Figure 4A–C corresponds to the SEM images of undegraded GWEAX, MWEAX, and LWEAX, respectively, while Figure 4D–G shows the SEM images of HAX-1, MAX, HAX-2, and LAX, respectively. SEM images were recorded at magnifications of 2000× (uppercase labels) and 100× (lowercase labels). As demonstrated in Figure 4, the undegraded AX samples exhibited larger, irregular, block like, or sheet like structures with rough surfaces. MWEAX displayed distinct wrinkles and pores, while LWEAX showed small particles adhering to the surface. These features may be associated with their low A/X ratio and relatively high molecular weight. Higher molecular weight increases the likelihood of molecular chain entanglement and stacking, while lower branching (low A/X ratio) may lead to tighter packing of the backbone, promoting the formation of block like structures [23]. Moreover, complex branching structures can cause uneven molecular shrinkage or aggregation during the drying process, resulting in surface particle deposition [24]. These morphological characteristics reflect, to a certain extent, the spatial conformation and aggregation behavior of AX molecules. After degradation and purification, the four AX samples with different molecular weights exhibited more uniform morphology and significantly smaller particle sizes. HAX-1 displayed a relatively smooth surface, likely due to its higher molecular weight and compact structure, which contribute to a flatter surface. In contrast, MAX, HAX-2, and LAX showed more porous and loose surfaces, possibly due to chain scission during degradation, leading to structural loosening. The formation of surface pores in AX enhances its surface properties, which can significantly improve its solubility and dispersibility [25]. Additionally, the increased uniformity and reduced particle size of the degraded samples may further enhance their adsorption capacity at interfaces and emulsifying performance [26]. Such structural changes provide theoretical support for their application in food processing, particularly in improving emulsifying properties and foam stability.

3.3. Functional Analysis of AX and Its Enzymatic Hydrolysis Products

3.3.1. The WHC of AX

The WHC of different AX samples is shown in Figure 5A. As can be observed from Figure 5A, the undegraded GWEAX, MWEAX, and LWEAX exhibited higher WHC (about 5 g/g), which is likely due to the ability of high molecular weight AX molecules to form a more complex three dimensional network, thereby effectively trapping more water molecules [27]. In contrast, the WHC of the degraded and purified HAX-1, HAX-2, MAX, and LAX decreased significantly. The main reason lies in the marked reduction in molecular weight during degradation, which disrupts the intermolecular network and reduces the number of binding sites for water molecules [28]. In particular, LAX, with its lowest molecular weight, showed the lowest WHC (1.95 g/g).

Despite similar Mw and avDP among some AX samples, their WHC still exhibited significant differences. To further investigate the regulatory role of structure on the WHC, considering that molecular size more directly influences macroscopic functional properties and that avDP and Mw exhibit collinearity, this study constructed a linear regression model with A/X ratio and Mw as independent variables and the WHC as the dependent variable. As shown in Table 4, the model equation is: WHC = 0.101 + 1.486 × A/X + 0.008 × Mw. The R^2^ value of the model was 0.964, indicating that A/X and Mw can explain 96.4% of the variation in the WHC. Further analysis of the standardized coefficients (β) revealed that Mw (β = 1.045) was significantly higher than A/X (β = 0.066). Moreover, the p value for Mw was 0.046 (significant), while that for A/X was 0.866 (not significant), demonstrating that the WHC of AX is primarily driven by molecular weight. For example, when the molecular weight decreased from 301.42 kDa to 224.80 kDa under similar A/X conditions, the WHC clearly declined. In contrast, changes in the A/X ratio had a relatively small effect on the WHC. Given the limited number of AX fractions and the structural correlation between Mw and A/X ratio, multicollinearity may influence the stability of regression coefficients. Therefore, the regression analysis was used to identify general association trends rather than to infer independent causal contributions In summary, the results indicate that molecular weight is more closely associated with the WHC than A/X ratio, although their effects cannot be fully separated due to the structural interdependence.

3.3.2. AX Foam Properties of AX

The foam performance of different AX samples is shown in Figure 5B. AX itself lacks foaming ability. In the 1% wheat protein solution system, the foaming capacity of all AX containing systems was similar to that of the wheat protein alone (51.01–54.81%), indicating that AX did not further enhance the foaming ability of the wheat protein. However, AX significantly improved the foam stability of the solution. After adding different AX samples to the protein solution, foam stability increased by 7.86–28.59%, reaching a final value of 67.21–88.68%. The undegraded AX samples (GWEAX, MWEAX, LWEAX) exhibited higher foam stability, which is likely due to their higher molecular weight and lower A/X ratio, allowing them to form a thicker polysaccharide film at the air–liquid interface [29,30], thereby enhancing foam stability. Among the degraded AX samples (HAX-1, HAX-2, MAX, LAX), the high molecular weight fraction (HAX-1) showed advantages in foam stability (77.68%) due to its high viscoelasticity. In contrast, the LAX sample, with a molecular weight of only 98.63 kDa, exhibited lower solution viscosity, resulting in insufficient coverage at the interface and an inability to effectively increase the strength of the interfacial layer. This finding is in agreement with the findings of Coelho et al. [31].

A linear regression model was established with the A/X ratio and Mw as independent variables and foam stability as the dependent variable. As shown in Table 5, the model equation is: Foam stability = 0.298 + 0.502 × A/X + 0.001 × Mw.

The R^2^ value of the model was 0.962, indicating that A/X and Mw can explain 96.2% of the variation in foam stability. Further analysis of the standardized coefficients (β) showed that Mw (β = 1.407) was significantly higher than A/X (β = 0.448). Additionally, the p value for Mw was 0.020 (significant), while that for A/X was 0.300 (not significant), indicating that the foam stability of AX is primarily driven by molecular weight.

3.3.3. Emulsification Properties of AX

Emulsifying properties of different AX samples are shown in Figure 6. Figure 6A illustrates that the emulsifying activity index (EAI) of AX samples increased significantly within the range of 1–2 mg/mL. This is likely due to the denser adsorption of polysaccharide molecules onto the surface of oil droplets, generating a stable interfacial film, as well as the increased system viscosity, which inhibits droplet coalescence [25,26]. At the same concentration, emulsifying performance was notably influenced by molecular structure. AX with high molecular weight and low A/X ratio (0.49–0.55) exhibited a lower emulsifying activity (EAI ranging from 48.28 to 67.93 m^2^/g). In contrast, LAX, with lower molecular weight and higher A/X ratio (98.63 kDa, A/X = 0.67), showed a higher EAI of 88.31 m^2^/g, representing a 32.95% improvement compared to the undegraded samples. The phenomenon may be due to the higher branching degree, which enhances the flexibility of AX molecules, thereby promoting their adsorption and rearrangement at the oil–water interface [32].

Additionally, ζ potential analysis indicated that LAX carries more negative charge, which can inhibit droplet aggregation by strengthening electrostatic repulsion between oil droplets, further improving emulsion stability [33]. The ESI of different AX samples is presented in Figure 6B. Except for LAX, other AX samples showed an initial increase and a subsequent decrease in ESI. LAX exhibited a continuously improving stability even at 2 mg/mL, suggesting that its lower molecular weight and higher branching degree enhance extensibility and solubility, contributing to the emulsion structure stability [34]. In summary, LAX demonstrated the best emulsifying performance due to its low molecular weight, high flexibility, and high negative charge density. Moreover, its performance improved significantly as the molecular weight decreased.

3.3.4. The Rheological Characteristics of AX

The rheological characteristics of undigested and enzymatically hydrolyzed and purified AX are shown in Figure 7. Figure 7A indicates that all AX samples exhibited typical pseudoplastic fluid behavior. Among them, GWEAX and MWEAX showed the highest shear stress, primarily attributed to their higher molecular weights (602.42 and 577.63 kDa) and degrees of polymerization, which favor the formation of dense molecular networks [35]. Additionally, GWEAX had a lower A/X ratio (0.53), as well as a higher degree of branching, which further enhances intermolecular interactions and flow resistance within the system [33]. In contrast, the shear stress of AX samples after enzymatic hydrolysis and purification decreased significantly, suggesting that enzymatic hydrolysis leads to chain scission and weakening of the network structure. Among these, LAX (98.63 kDa) exhibited the lowest shear stress. Consistent with the shear stress results, Figure 7B shows that all AX samples displayed pronounced shear-thinning behavior within the tested range, i.e., viscosity decreased with increasing shear rate. Due to their retained higher molecular weights and more complex molecular structures, GWEAX and MWEAX exhibited significantly higher shear viscosities compared to other samples. Conversely, LAX with shorter molecular chains and a weaker network structure showed the lowest viscosity [36].

From the perspective of the triple-helical structure of AX, the reduction in molecular chain length diminishes the entanglement and association capacity between triple-helical chains, leading to a significant decrease in the storage modulus of LAX and MAX and an enhancement in fluidity. In Figure 7C, the storage modulus (G′) and loss modulus (G″) of GWEAX and MWEAX were notably higher than those of other samples, with G′ consistently exceeding G″, indicating stronger elastic behavior [37]. In summary, AX solutions with different molecular weights and structures exhibited distinct rheological properties. Higher molecular weight and strong intermolecular interactions are key factors contributing to increased shear viscosity and elasticity.

3.3.5. The Antioxidant Characteristics of AX

The antioxidant activity of AX is shown in Figure 8. The antioxidant activity of AX samples was concentration-dependent, with low-molecular-weight AX (such as LAX) exhibiting the best performance. LAX achieved a scavenging rate of 95.32% at 1.0 mg/mL in the DPPH radical scavenging assay, representing an increase of 21.02–32.02% compared to LWEAX. This enhancement is likely due to the lower molecular weight and viscosity of LAX, which facilitates better dispersion in the reaction system, thereby providing more active sites for electron transfer to scavenge radicals [38]. Furthermore, samples with high A/X ratios (such as HAX-2 and LAX) typically possess a greater number of hydroxyl and carboxyl groups derived from phenolic acids due to the higher degree of arabinose side-chain substitution [1,39], which enhances electron transfer capability.

The hydroxyl radical scavenging assay showed a trend consistent with the DPPH assay, with LAX reaching a scavenging rate of 91.32% at 1.0 mg/mL. Similarly, the FRAP assay results indicated that low-molecular-weight AX samples with high A/X ratios exhibited a stronger ferric ion-reducing ability, likely due to their shorter backbone structures, which allow functional groups on the side chains to be more readily exposed and participate in reaction [1].

In addition to the structural parameters, residual ferulic acid may also contribute to the antioxidant activity of AX fractions. FTIR spectra confirmed the presence of phenolic-related signals, indicating that enzymatic hydrolysis did not remove bound ferulic acid. Enzymatic depolymerization likely increased the accessibility of phenolic groups by reducing the steric hindrance and improving the molecular dispersion, thereby facilitating the radical scavenging reactions. In summary, the antioxidant characteristics of AX are synergistically regulated by A/X ratio, molecular weight, and degree of polymerization. AX samples with low molecular weight, low polymerization degree, and high A/X ratio demonstrated significant advantages across all three antioxidant evaluation systems: DPPH, hydroxyl radical scavenging, and FRAP.

4. Conclusions

This study systematically elucidated the enzymatic degradation patterns of wheat AX by wheat malt β-1,4-endoxylanase and the resulting structure–activity relationships. The results indicate that the β-1,4-glycosidic bonds in the unsubstituted regions of the AX backbone were specifically cleaved by the enzyme, leading to significant structural alterations: the molecular weight decreased from an initial 489.42–602.42 kDa to 98.63–301.42 kDa, the A/X ratio enhanced from 0.49–0.55 to 0.60–0.65, and the avDP dropped substantially from 1223.57–1506.05 to 246.59–753.56. These structural changes were closely associated with the functional characteristics of AX. The AX fraction with high A/X ratio and low molecular weight (e.g., LAX), which experienced the most severe backbone cleavage, exhibited unwinding of the triple-helical structure, greater exposure of hydroxyl and carboxyl groups, and a higher negative charge density (−9.23 mV). This was associated with the enhanced interfacial adsorption and electron transfer capability, accompanied by a 32.95% increase in emulsifying activity and a 32.02% increase in hydroxyl radical scavenging rate compared with the original sample. In contrast, the high-molecular-weight native AX samples (e.g., GWEAX), with their more intact triple-helical structure and higher viscoelasticity, formed a more stable molecular network, leading to superior water-holding capacity (approximately 5 g/g) and foam stability (88.68%). Multiple linear regression analysis further clarified that within the investigated range of A/X ratios, molecular weight was the most critical factor determining the WHC and foam stability of AX.

The novelty of this research lies in the systematic engineering of AX variants with distinct backbone lengths and branching degrees, using the degree of polymerization as the core variable, providing a refined framework for studying AX structure–activity relationships. The findings confirm that the functional properties of AX can be directionally modulated by enzymatically controlling its molecular weight and degree of branching. This provides a crucial theoretical basis and practical guidance for the precise application of AX in food systems (e.g., as an emulsifier, antioxidant, or texture modifier). Specifically, AX fractions with higher molecular weight and higher avDP exhibited superior WHC and viscoelastic behavior, indicating their suitability as texture-enhancing or moisture-retaining components in systems such as dough, batter, and foam-based products. In contrast, AX fractions with lower molecular weight and lower avDP, but relatively higher branching degree, demonstrated improved emulsifying capacity and antioxidant activity, suggesting their potential use as functional stabilizers or antioxidant ingredients in beverage and emulsion-based formulations. This structure–function–application linkage provides a clearer framework for the rational utilization of enzymatically modified wheat AX in different food systems, thereby facilitating the high-value utilization of wheat resources.