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Article

Processing Shapes Architecture, Cultivar Dictates Chemistry: A Structural and Functional Paradigm for Yam Polysaccharides

by
Yajuan Bai
1,2,
Bei Fan
1,2,
Jiameng Liu
1,2,
Fengzhong Wang
1,2,* and
Mingwei Zhang
3,*
1
Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, China
2
National Nanfan Research Institute (Sanya), Chinese Academy of Agricultural Sciences, Sanya 572024, China
3
Sericultural & Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, China
*
Authors to whom correspondence should be addressed.
Foods 2026, 15(5), 921; https://doi.org/10.3390/foods15050921
Submission received: 28 January 2026 / Revised: 23 February 2026 / Accepted: 5 March 2026 / Published: 6 March 2026
(This article belongs to the Special Issue Natural Active Compounds in Foods: Screen, Sources and Health Benefits)
Browse Figures
Versions Notes

Abstract

Yam polysaccharides are promising functional food ingredients, but the systematic understanding of how cultivar and processing synergistically determine their structure and functionality is still lacking. This study systematically investigated how hot water extraction, enzymatic hydrolysis, and extrusion puffing affect the structural and functional properties of polysaccharides from two major cultivars (Dioscorea opposite cv. Tiegun and Dioscorea esculenta cv. Gaozhou). Enzymatic extraction increased yield (1.39–1.77-fold) and solubility, while hot water extraction favored purity. The monosaccharide composition was strongly cultivar-dependent, with Tiegun polysaccharides containing higher mannose levels. Extrusion puffing of Gaozhou polysaccharide improved solubility by 33.3% but induced depolymerization and aggregation, modifying colloidal and functional behaviors. Multivariate analysis revealed that processing methods primarily governed macromolecular architecture and colloidal properties, whereas cultivar determined chemical composition. These findings establish a processing–structure–property framework, enabling the tailored production of yam polysaccharides: Tiegun yam with enzymatic extraction for high bioactivity, and Gaozhou yam with extrusion puffing for superior solubility.

1. Introduction

Yam (Dioscorea opposite Thunb.) polysaccharides are increasingly valued as functional food ingredients due to their versatile technological properties, such as solubility, thickening capacity, and emulsion stability [1], as well as their potential health-promoting effects, including immunomodulatory and anti-inflammatory activities [2,3]. These functional outcomes are intrinsically governed by the structural attributes of the polysaccharides, including molecular weight, monosaccharide composition, chain conformation, and supramolecular assembly [4,5]. These structural attributes are in turn shaped by two key factors: the botanical origin (cultivar) of the plant and the processing methods used for extraction and modification [3].
In industrial practice, processing technologies are routinely applied to enhance extraction yield or to tailor physicochemical properties. For instance, enzymatic extraction can significantly improve polysaccharide recovery by breaking down cell-wall matrices and starch networks [6], while intensive physical treatments like extrusion puffing are known to dramatically improve solubility and dispersibility [7]. However, these processing advantages may come with unintended trade-offs. Enzymatic hydrolysis risks introducing protein impurities or indiscriminately cleaving polysaccharide chains, potentially altering their native architecture [8,9]. Similarly, high-shear, high-temperature processes such as extrusion puffing can induce depolymerization and aggregation [7,10], which may compromise bioactive motifs essential for immunomodulation.
Critically, most existing studies have adopted one of two approaches: either they compare polysaccharides from different yam cultivars using a single extraction method [2], or they optimize and compare different extraction techniques for a single cultivar [6]. While valuable, these single-factor investigations cannot address the interactive effects between botanical origin and processing technology. This represents a significant knowledge gap because in industrial practice, both factors are variable and their interplay may produce non-additive effects on final product properties. Consequently, it remains unclear how the cultivar of yam and its processing method interact to shape the final structure and function of its polysaccharides. This uncertainty limits our ability to rationally design yam polysaccharide ingredients with targeted properties for food applications.
To address this gap, we systematically investigated two commercially important cultivars (Tiegun and Gaozhou) using two extraction methods (hot water and enzymatic), and further examined how extrusion puffing, an intensive industrial process, modifies a selected high-yield fraction. Polysaccharides were characterized for their physicochemical properties and evaluated for immunomodulatory and anti-inflammatory activities in RAW 264.7 macrophages. This unified framework enables us to answer a fundamental question: which class of properties is predominantly governed by cultivar, and which by processing? Elucidating this division of roles moves beyond descriptive comparisons and provides a scientific basis for the targeted design of yam-based functional foods.

2. Materials and Methods

2.1. Materials and Chemicals

Fresh tubers of two yam cultivars, Dioscorea opposite cv. Tiegun (from Jiaozuo, Henan Province, China) and Dioscorea esculenta cv. Gaozhou (from Gaozhou, Guangdong Province, China), were used as raw materials. Murine RAW 264.7 macrophages were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).
Enzymes used for extraction included heat-stable α-amylase (from Bacillus licheniformis, ≥20,000 U/mL), alkaline protease (from Bacillus licheniformis, ≥200,000 U/g), and glucoamylase (from Aspergillus niger, ≥100,000 U/mL) (Beijing Solarbio Ltd., Beijing, China). Dialysis bags (MWCO 8 kDa) were from the same supplier. Monosaccharide standards and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dextran standards were purchased from Waters Co. (Milford, MA, USA). Cell culture reagents (Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin solution, nonessential amino acids, penicillin–streptomycin solution) were purchased from Gibco (Grand Island, NY, USA). The kinetic chromogenic Limulus Amebocyte Lysate (LAL) and Cell Counting Kit-8 (CCK-8) assay kits were provided by Beyotime Biotechnology (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-8 were procured from Neobioscience Technology (Shenzhen, China). Nitric oxide (NO) and Bradford protein assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TRIzol reagent and SYBR Green qPCR master mix were acquired from Vazyme Biotech (Nanjing, China). All other chemicals were analytical grade from Sinopharm Chemical Reagent Ltd. (Shanghai, China).

2.2. Preparation of Yam Polysaccharides

2.2.1. Hot Water Extraction

Fresh yam was peeled and homogenized in distilled water (1:2, w/w), and heated at 90 °C for 2 h [2]. After centrifugation, proteins were removed using Sevag reagent. The supernatant was concentrated under reduced pressure, dialyzed (48 h), precipitated with ethanol (final concentration of 80%), washed five times with anhydrous ethanol, and lyophilized to obtain crude polysaccharides (THP for Tiegun yam, GHP for Gaozhou yam).

2.2.2. Enzymatic Extraction

To overcome the low extraction yield caused by starch gelatinization during hot water extraction, an enzymatic pretreatment was employed to remove starch and protein matrices prior to polysaccharide recovery [10]. Fresh yam was peeled, homogenized with distilled water (1:2, w/w), and subjected to sequential enzymatic treatments. The homogenate was first incubated with heat-stable α-amylase at an enzyme-to-sample ratio of 0.03:1 (v/w) at 90 °C for 30 min to liquefy starch. After cooling, the pH was adjusted to 9.5, and alkaline protease was added at 0.005:1 (v/w) and incubated at 60 °C for 1.5 h to hydrolyze proteins. The pH was then adjusted to 4.5, followed by addition of glucoamylase at 0.02:1 (v/w) and incubation at 60 °C for 30 min to further degrade starch fragments. After enzymatic treatment, the mixture was centrifuged (4000× g, 15 min). The supernatant was collected and subjected to Sevag deproteination (chloroform:butanol, 4:1 v/v, repeated three times). The aqueous phase was concentrated under reduced pressure at 50 °C, dialyzed against distilled water (MWCO 8 kDa, 48 h), and precipitated with ethanol (final concentration 80%, v/v) at 4 °C overnight. The precipitate was washed five times with anhydrous ethanol and lyophilized to obtain crude polysaccharides, designated as TEP (Tiegun yam, enzymatic extraction) and GEP (Gaozhou yam, enzymatic extraction).

2.2.3. Preparation of Polysaccharides from Extrusion-Puffed Yam

Fresh peeled Gaozhou yam slices were dried at 50 °C, extrusion-puffed, and ground into powder. The extrudate was then extracted enzymatically as described in Section 2.2.2 to obtain extrusion-puffed Gaozhou yam polysaccharides (PGEP).

2.3. Basic Physicochemical Characterization of Yam Polysaccharides

2.3.1. Chemical Composition Analysis

The extraction yield of yam polysaccharide was calculated as the percentage of dry polysaccharide weight relative to the initial dry yam powder weight. Total carbohydrate, uronic acid, reducing sugar, and protein contents were determined using the phenol-sulfuric acid method (with glucose as standard) [11], m-hydroxybiphenyl method (with glucuronic acid as standard) [12], 3,5-dinitrosalicylic acid (DNS) method (with glucose as standard) [13], and Bradford method (with BSA as standard), respectively. Monosaccharide composition was analyzed by GC-MS after hydrolysis and derivatization to alditol acetates, following a previously described method [14].

2.3.2. Molecular Weight Distribution

Polysaccharide solutions (2 mg/mL) were filtered (0.45 μm membrane) and analyzed using an ultra-high performance polymer chromatography system (Waters Co., Milford, MA, USA) equipped with serially connected columns (ACQUITY APCTM AQ 450, AQ 200, AQ 125) referenced by the method of Bai [15]. Dextran standards were used for calibration.

2.3.3. Particle Size, Zeta Potential, and Solubility

Particle size (dynamic light scattering) and zeta potential were measured using a ZetaSizer Nano instrument on 2 mg/mL polysaccharide solutions [16]. Solubility was determined by vortexing a supersaturated solution (50 mg/mL) for 30 min, centrifuging, and lyophilizing an aliquot of the supernatant to determine the dissolved solid mass.

2.3.4. Spectroscopic and Microscopic Characterization

Three complementary techniques were employed to characterize the structural features of the polysaccharides.
Iodine-Potassium Iodide (I2-KI) reaction: 2 mL of polysaccharide solution (1 mg/mL) was mixed with 1.2 mL of iodine reagent (0.02% I2 in 0.2% KI solution) and incubated at room temperature for 15 min in the dark [17]. Absorbance spectra from 300 to 700 nm were recorded using a microplate reader (SpectraMax M5, Molecular Devices, San Jose, CA, USA)
Fourier-Transform Infrared (FT-IR) spectroscopy: Lyophilized polysaccharide samples (1 mg) were ground with 100 mg KBr powder and pressed into transparent pellets. FT-IR spectra were recorded using a Bruker VERTEX 70 spectrometer (Bruker, Mannheim, Germany) in transmission mode over the range 4000–400 cm−1.
Field-Emission Scanning Electron Microscopy (FE-SEM): Lyophilized polysaccharide powders were mounted on aluminum stubs using double-sided carbon tape and sputter-coated with gold for 60 s at 20 mA (Quorum Q150R S, East Sussex, UK). Samples were examined under a FE-SEM (Merlin, Carl Zeiss AG, Oberkochen, Germany) at an accelerating voltage of 5.0 kV, working distance of 8.5 mm, using a secondary electron detector. Images were captured at 1000× and 5000× magnification from at least five randomly selected fields per sample.

2.4. Cell Culture and Bioactivity Assays

RAW 264.7 macrophages were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C in a 5% CO2 atmosphere. Cells within ten passages were used.

2.4.1. Endotoxin Level Assessment

To exclude potential interference from bacterial endotoxin contamination, the endotoxin levels in all polysaccharide samples were quantified using a kinetic chromogenic LAL assay kit according to the manufacturer’s instructions. The measured endotoxin concentrations were below 1.0 EU/mg for all samples, a level considered negligible at the working concentrations used (1–200 μg/mL) [18]. This confirms that the observed bioactivities originated from the polysaccharides themselves.

2.4.2. Cytotoxicity Assay

Cell viability was assessed using a CCK-8 assay kit according to the manufacturer’s instructions [19]. RAW 264.7 macrophages at a density of 5 × 105 cells/mL were seeded in a 96-well plate at 100 μL per well and treated with polysaccharide solutions at final concentrations of 0, 25, 50, 100, 150, and 200 μg/mL for 24 h. Cells treated with DMEM complete medium served as the blank control. Six replicate wells were set up for each group. After incubation, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for an additional 2 h. The absorbance at 450 nm was measured using a microplate reader. Based on the results confirming non-cytotoxicity of yam polysaccharides (THP, TEP, GHP, GEP, and PGEP) up to 200 μg/mL, subsequent immunomodulatory and anti-inflammatory assays were conducted within a safe concentration range of 1–100 μg/mL.

2.4.3. Immunomodulatory Activity

Macrophages at a density of 4 × 105 cells/mL were seeded in 24-well plates at 400 μL per well and allowed to adhere for 24 h. Cells were then treated with polysaccharide solutions at final concentrations of 1, 10, and 100 μg/mL in complete DMEM. Cells treated with 0.1 μg/mL LPS and complete DMEM alone served as the positive and blank controls, respectively. Following a 12 h incubation period, the culture supernatants were collected for the quantification of NO and proinflammatory cytokines (IL-1β, IL-6, and TNF-α) using ELISA kits  [19]. Concurrently, cellular pellets were harvested for real-time quantitative polymerase chain reaction (RT-qPCR) assessment of target gene expression. Each group was assigned six replicate wells.

2.4.4. Anti-Inflammatory Activity

In a parallel experimental setup, macrophages were cultured at 4 × 105 cells/mL in 24-well plates under humidified conditions for 24 h. Polysaccharide treatments (1, 10, 100 μg/mL) were administered in DMEM supplemented with 0.1 μg/mL LPS to simulate inflammatory conditions, with unstimulated DMEM-treated cells serving as the blank control and LPS-only treatment as the positive control [20]. After 12 h of stimulation, supernatants were collected for analysis of NO content by commercial kits. Total RNA was isolated from cell pellets for RT-qPCR evaluation of inflammation-related gene transcription. For each group, six replicate wells were prepared.

2.5. RT-qPCR

Total RNA was extracted, reverse transcribed into cDNA, and subjected to qPCR using a SYBR Green Master Mix. Gene expression was normalized to 18S rRNA and calculated via the 2−ΔΔCT method. Primer sequences are listed in Supplementary Table S1.

2.6. Statistical Analysis

Data are presented as mean ± standard error (SEM) from at least three independent experiments. Multiple group comparisons were analyzed by one-way ANOVA followed by Tukey’s HSD test. Comparisons between two groups used Student’s t-test, with significance denoted as asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001).

3. Results and Discussion

3.1. Physicochemical Characteristics of Yam Polysaccharides

3.1.1. Yield and Chemical Composition: Processing Dictates Yield and Purity, Cultivar Defines Signature Composition

The extraction method exerted a profound influence on both the yield and chemical profile of the polysaccharides (Table 1). Enzymatic extraction markedly enhanced the yield regardless of the yam cultivar. The yield from Gaozhou yam via enzymatic extraction (GEP) was 1.77-fold and 1.39-fold higher than that via hot-water extraction (GHP, p < 0.05) and from Tiegun yam via hot-water extraction (THP, p < 0.05), respectively, and was comparable to the yield from Tiegun enzymatic extraction (TEP). This substantial improvement is attributed to the multi-step enzymatic hydrolysis, which effectively disrupts the starch gel network formed during processing, thereby liberating entrapped polysaccharides and increasing extractability [6,21], a key advantage for industrial scale-up where yield is paramount.
In contrast, while hot water extraction resulted in lower yields, it produced polysaccharides with higher purity. This was evidenced by their significantly higher contents of neutral sugar and uronic acid, coupled with concomitantly lower levels of reducing sugars and protein (Table 1). The elevated reducing sugar content in the enzymatic extracts (TEP and GEP) is a direct consequence of starch breakdown by amylase [22]. The higher protein content in these samples likely stems from either residual enzymes or the co-extraction of glycoprotein complexes. Collectively, these findings demonstrate that the extraction method is the predominant factor determining the yield and general chemical profile of polysaccharides, presenting a clear trade-off between extraction efficiency and ingredient purity.
Notably, the monosaccharide profile, a critical determinant of biofunctional specificity, was predominantly governed by the botanical cultivar (Table 1). Polysaccharides from Tiegun yam (THP and TEP) were rich in glucose and mannose, whereas those from Gaozhou yam (GHP and GEP) were overwhelmingly dominated by glucose. The presence of minor amounts of rhamnose and arabinose exclusively in GEP served as a varietal marker. This strong cultivar-dependent chemical signature highlights that the fine chemical structure is an intrinsic property of the raw material, which processing can modify in quantity but not fundamentally alter in its core compositional identity.

3.1.2. Molecular Weight: Cultivar-Specific Architecture Leads to Divergent Responses to Processing

The molecular weight profiles revealed complex, cultivar-dependent responses to extraction, highlighting fundamental differences in native polysaccharide architecture (Table 2). For Gaozhou yam, the number-average molecular weight (Mn) of the hot-water extracted polysaccharide (GHP, 4.85 × 104 Da) was significantly larger than that of its enzymatic counterpart (GEP, 3.02 × 104 Da). This conventional pattern, where enzymatic hydrolysis reduces molecular size, suggests that the native polysaccharides in Gaozhou yam exist as large networks that are physically liberated by hot water but are efficiently cleaved by the specific actions of amylolytic and proteolytic enzymes [23].
In contrast, the opposite trend was observed for Tiegun yam. The enzymatic extract (TEP) possessed a higher Mn (3.31 × 104 Da) than the hot-water extract (THP, 1.71 × 104 Da). This counterintuitive result implies that native Tiegun polysaccharides may not exist as large, continuous networks susceptible to simple thermal liberation. Instead, we propose they exist as core polysaccharide units entangled within or non-covalently associated with a protein–starch matrix. In this context, thermal energy during hot water extraction may induce random thermal scission of these complexes, yielding smaller fragments (THP) [24,25]. Conversely, the multi-enzyme system acts as a precision liberator by specifically targeting and removing the surrounding starch and protein components, thereby revealing the intrinsic, larger polysaccharide cores in a more intact state (TEP) [26].
This structural divergence is corroborated by the polydispersity index (PID). The exceptionally high PID of THP indicates a broad, fragmented mixture resulting from non-specific thermal degradation. The more homogeneous profile of GHP aligns with the concerted release of a large, structured polymer. Collectively, these divergent molecular weight outcomes directly translate to differences in solution behavior and potential bioactivity, which are explored in subsequent sections.

3.1.3. Solution Properties: Chain Conformation and Aggregation State Govern Functional Performance

The hydrodynamic properties of polysaccharides in solution are crucial for their application as food ingredients. THP, despite having the lowest Mw, formed the largest aggregates (Table 3), suggesting an extended chain conformation that promotes extensive hydration and intermolecular association [27]. Conversely, GEP exhibited the smallest particle size despite a higher Mw, suggestive of a more compact or stable conformation that limits aggregation. This divergence underscores that hydrodynamic volume is a function of supra-molecular assembly, not just polymer chain length [28].
Further complexity was revealed by comparing molecular weight distribution (PID) and particle size dispersity (PdI). THP, with the broadest molecular weight distribution (highest PID), formed the most homogeneous aggregates (lowest PdI). This implies that its extended chains foster a consistent intermolecular network, overriding the inherent chain length variability. In contrast, TEP displayed a narrow molecular weight profile (low PID), yet the most heterogeneous particle size distribution (highest PdI). This suggests that enzymatic extraction, while homogenizing chain lengths, likely altered chain conformation or surface properties, resulting in disparate aggregation pathways [29]. The behaviors of GHP and GEP further corroborate this complexity. GHP showed intermediate PdI with the narrowest molecular weight distribution, while GEP presented an opposite case with intermediate PID and PdI.
All polysaccharides exhibited negative zeta potentials (approximately −33 to −47 mV), attributed to ionization of uronic acid residues. THP showed a moderate value of approximately −33 mV, while TEP, GHP, and GEP displayed higher absolute values (−41 to −47 mV), indicating stronger electrostatic repulsion and thus greater colloidal stability in aqueous dispersion [1,16].
The iodine-binding profiles (I2-KI, Figure 1A) provide additional insight into chain topology. GHP exhibited an absorption band at 565 nm, consistent with linear or lightly branched segments, while THP, TEP, and GEP exhibited a peak at 350 nm, characteristic of highly branched structures [30]. We acknowledge that this method has limitations: it provides qualitative rather than quantitative information, is most informative for glucose-rich polysaccharides, and cannot identify specific structural features such as branch points. Therefore, these results are interpreted as preliminary evidence for topological differences that warrant confirmation through techniques such as NMR in future studies.
Integrating these data yields a coherent mechanistic framework. For THP, the highly branched structure promotes an expanded chain conformation, leading to extensive inter-chain entanglement and large aggregate formation. The moderate zeta-potential (−33.30 mV) provides insufficient electrostatic repulsion to overcome this attraction, explaining its low solubility (3.18%). For TEP, high branching combined with strong electrostatic repulsion (−43.67 mV) creates heterogeneous aggregates (PdI 0.93) and intermediate solubility (4.57%). For GHP, linear segments may facilitate ordered associations, resulting in intermediate particle size (422.57 nm) and moderate solubility (4.33%). For GEP, high branching and strong repulsion (−41.17 mV) favor a compact conformation that minimizes aggregation, yielding the smallest particles (132.25 nm) and highest solubility (5.98%).
Correlation analysis revealed that solubility correlates strongly with aggregate size (R2 = 0.97) but not with molecular weight (Figure S1), confirming that aggregation state governs water accessibility. The solubility hierarchy (GEP > TEP ≈ GHP > THP) clearly demonstrates this relationship. Thus, for industrial applications requiring high solubility (e.g., beverages), GEP presents as the most favorable candidate based on its combined high yield and superior solubility.

3.1.4. FT-IR Spectroscopy: Confirmation of Polysaccharide Identity and Degree of Esterification

FT-IR analysis confirmed the typical polysaccharide structure for all samples (Figure 1B), featuring characteristic O-H (~3420 cm−1) and C-H (~2930 cm−1) stretching vibrations [31]. A key difference among them lay in the uronic acid carbonyl region. The comparable intensity of the band at ~1734 cm−1 (attributed to esterified carboxyl groups) [32,33] and at ~1629 cm−1 (attributed to free carboxylate groups) [34] in THP suggested a balanced ratio. In contrast, the other three polysaccharides (TEP, GHP, GEP) showed a clear dominance of the free carboxylate peak, indicating a lower degree of esterification. The degree of uronic acid esterification influences charge density, calcium-binding capacity, and thereby gelation potential—properties critical for texture modification in food systems. Absorptions between 1200 and 1000 cm−1 are associated with pyranose ring vibrations [35]. The peak at 776 cm−1 is suggestive of α-glycosidic linkages [36,37]. This confirms the fundamental structural similarities across the polysaccharide samples, with the degree of uronic acid esterification being the primary differentiating factor.

3.1.5. Morphological Analysis (FE-SEM): Processing Method Overrides Cultivar in Determining Supramolecular Morphology

FE-SEM analysis demonstrated that the extraction method was the primary determinant of polysaccharide morphology, overriding the influence of the cultivar (Figure 1C). Polysaccharides isolated via hot water extraction (THP, GHP) displayed a characteristic porous, lamellar network with interconnected sheets. This coherent, gel-like microstructure is typical of polysaccharides that have undergone slow reassembly from an aqueous state during drying, preserving a memory of their hydrated network structure. Such a morphology often correlates with high water-holding capacity and may form viscous dispersions. Conversely, enzymatic extraction (TEP, GEP) completely disrupted this organization, yielding only dense, amorphous aggregates with no discernible porosity. This morphological transformation is a direct physical manifestation of the enzymatic process. The specific cleavage of surrounding structural matrices (starch, protein) disrupts the native architecture and prevents the reformation of an organized polysaccharide network during recovery. This stark morphological contrast has direct practical implications: the porous networks of hot-water extracts may be advantageous in applications requiring hydration and swelling (e.g., fat replacers, thickeners), while the dense aggregates of enzymatic extracts might offer higher bulk density and different flow properties as powdered ingredients.

3.2. Functional Performance: Correlating Structural Profiles with Bioactivity as a Quality Parameter

The immunomodulatory and anti-inflammatory capacities were evaluated as critical functional properties for potential health-oriented applications. This section aims to correlate the multivariate structural profiles of the polysaccharides with their functional bioactivity outcomes. This integrative analysis completes the “processing–structure–function” pipeline, demonstrating how upstream decisions in raw material selection and processing technology manifest in downstream biological functionality.

3.2.1. Immunomodulatory Activity: A Cultivar-Driven Potency Modulated by Processing

The immunomodulatory potential of the polysaccharides was assessed by measuring the secretion and gene expression of pro-inflammatory cytokines in RAW 264.7 macrophages in the absence of LPS [38]. A dominant influence of the botanical cultivar on the stimulation intensity was observed (Figure 2A–C). Polysaccharides from the Tiegun cultivar (THP, TEP) consistently induced significantly higher secretion of TNF-α, IL-6, and IL-1β compared to those from the Gaozhou cultivar (GHP, GEP) at equivalent concentrations. Notably, Tiegun polysaccharides contained substantially higher mannose levels (17.06–24.33%) than Gaozhou polysaccharides (3.62–4.93%) (Table 1). Given that mannose is known to serve as a ligand for macrophage receptors such as CD206, this compositional difference may contribute to the enhanced immunomodulatory activity observed for Tiegun samples [39].
The extraction method fine-tuned the immunomodulatory response in a cytokine-specific manner. Enzymatic extraction enhanced TNF-α secretion compared to hot-water extraction within each cultivar (TEP > THP; GEP > GHP) (Figure 2A). For IL-6, both cultivars showed enhanced secretion with enzymatic extraction, though the magnitude of increase was greater for Tiegun samples (Figure 2B). However, the effect on IL-1β differed between cultivars: enzymatic extraction increased IL-1β for Tiegun (TEP > THP) but decreased it for Gaozhou (GEP < GHP) (Figure 2C).
These protein-level patterns were reflected in the transcriptional responses. Initial analysis including the LPS control showed that the potent induction of IL-1β and TNF-α mRNA by LPS dominated the statistical model, masking the subtler effects of the polysaccharides (Figure S2A–C). After excluding the LPS group to better evaluate intrinsic polysaccharide activity, all samples significantly upregulated mRNA expression of IL-1β, IL-6, and TNF-α compared to the unstimulated control (Figure 2D–F). The transcriptional patterns mirrored the protein data: enzymatic extraction consistently enhanced TNF-α mRNA for both cultivars, while its effect on IL-1β mRNA was cultivar-dependent (increased for Tiegun, not for Gaozhou). These concordant patterns across transcriptional and translational levels confirm that the polysaccharides activate macrophages through regulation at the gene expression level.
The differential effects of the extraction method on specific cytokines suggest that processing-induced structural modifications may selectively modulate distinct immune activation pathways. The consistent enhancement of TNF-α by enzymatic extraction across both cultivars points to structural features common to both Tiegun and Gaozhou polysaccharides that influence TNF-α production, such as increased accessibility due to starch removal. In contrast, the cultivar-dependent effects on IL-1β indicate that processing outcomes may depend on the native polysaccharide architecture, with the same enzymatic treatment having opposite consequences depending on the starting material. These observations highlight that processing not only affects overall immunomodulatory potency but can also shape the qualitative profile of the immune response.

3.2.2. Anti-Inflammatory Function: Efficacy Linked to Specific Structural Ensembles

The anti-inflammatory potential of the polysaccharides was evaluated by assessing their capacity to suppress the inflammatory response in macrophages pre-stimulated with LPS [20]. All polysaccharides (THP, TEP, GHP, GEP) demonstrated a significant capacity to suppress inflammation, attenuating LPS-induced NO production and downregulating key inflammatory genes (iNOS, TNF-α), though the efficacy varied among samples (Figure 2G–I).
Polysaccharides from the Tiegun cultivar (THP, TEP) again exhibited superior functional performance, with THP showing the most potent inhibition across all tested concentrations and maximal effect at 50 μg/mL. The high efficacy of THP coincides with a specific structural ensemble: its mid-range molecular weight profile, balanced uronic acid esterification, and porous lamellar morphology (Table 1 and Table 2; Figure 1). In contrast to THP, which maintained potent inhibition across all concentrations, its enzymatic counterpart TEP showed diminished activity at higher concentrations. Gaozhou polysaccharides (GHP, GEP) were consistently less effective across the tested concentration range. This structure–function correlation highlights that the functional outcome is a product of the entire structural package, where altering one parameter (e.g., via enzymatic extraction) can shift the functional efficacy, even within the same cultivar.
In summary, the structure–activity relationship analysis delineates two promising paths for the commercial development of yam polysaccharides. Polysaccharides from the premium Tiegun cultivar, particularly TEP, demonstrated superior bioactivity, positioning them as high-value ingredients for pharmaceutical or nutraceutical applications where efficacy is paramount. Conversely, for broad-based applications in the functional food industry, cost-effectiveness and scalability are critical. In this regard, the enzymatic extract from the more affordable Gaozhou yam (GEP) presents a compelling candidate due to its highest extraction yield. The above results establish that extraction methods can fine-tune bioactivity. To further explore the boundaries of this principle and its industrial relevance, we next investigated the impact of an intensive physical process, extrusion puffing, on a high-yield candidate, examining the potential trade-offs between enhanced technological properties and preserved biological function.

3.3. Engineering Functionality Through Physical Processing: The Trade-Off of Extrusion Puffing

To explore the impact of an intensive physical process on functionality, the high-yield GEP fraction was subjected to extrusion puffing (PGEP). This investigation aimed to quantify the trade-off between enhanced technological property (solubility) and preserved bioactivity.
Extrusion puffing induced a drastic structural reorganization (Table 4). The process achieved its primary technological goal, enhancing solubility by 33.3%. This enhancement was accompanied by severe depolymerization, as evidenced by a drastic reduction in Mw and lower polydispersity (Table 4). Despite this molecular downsizing, the hydrodynamic particle size increased 4.2-fold, and PdI became exceptionally uniform. FE-SEM imaging revealed that the dense, amorphous aggregates of GEP were transformed into a loose, porous morphology after puffing (Figure 3B). These observations suggest that shear-induced chain scission during extrusion, followed by the reassembly of the resulting shorter chains into larger, highly uniform, and hydrated aggregates upon expansion, directly explains the concurrent increase in solubility. FT-IR analysis confirmed that the fundamental chemical groups and glycosidic linkages were preserved (Figure 3A), indicating that the modifications were primarily physical (depolymerization and aggregation) rather than chemical.
The structural alterations induced by extrusion puffing substantially reshaped the immunomodulatory activity. In the immunomodulatory assay conducted under resting conditions (without LPS), native GEP exhibited broad-spectrum immunostimulatory activity, significantly elevating the mRNA expression of iNOS, TNF-α, and IL-1β compared to the blank control across multiple concentrations (Figure 3C–E). In contrast, the puffed polysaccharide (PGEP) exhibited a narrowed and weakened immunomodulatory profile. It stimulated iNOS in a dose-dependent manner but failed to significantly induce TNF-α or IL-1β (Figure 3C–E). This shift from a broad to a more specific immune signal suggests that the loss of activity results from both depolymerization and aggregation. Depolymerization likely plays a primary role: the 96.4% reduction in Mw probably destroyed the higher-order structural motifs required for TNF-α and IL-1β induction, which depend on longer chains for multivalent receptor engagement [40]. Aggregation then further contributes: the 4.2-fold increase in particle size may bury any surviving motifs inside large aggregates, making them inaccessible to macrophages [41]. The preserved iNOS-inducing capacity suggests that this particular activity may require only shorter recognition motifs that survive both depolymerization and aggregation [42]. These observations demonstrate that different immunomodulatory activities have distinct structural requirements and can be differentially affected by processing [25,42,43].
While the precise molecular mechanism underlying the selective loss of immunomodulatory breadth warrants further investigation, our current findings deliver immediate practical implications for product formulation. PGEP is optimal where high solubility is paramount and specific immunomodulation is acceptable. For applications requiring anti-inflammatory function, direct validation in appropriate inflammatory models would be necessary before use. This study underscores a central tenet of food ingredient science: physical processing is a powerful tool for achieving desired technological functionalities, but it can differentially modulate biological activities [25]. The choice of process must balance technological properties with intended functional outcomes.

4. Conclusions

This study establishes that cultivar dictates chemical composition, while processing governs macromolecular architecture, providing a framework for evidence-based design of yam polysaccharide ingredients. A central finding is the inevitable trade-off with intense processes like extrusion puffing, which enhances solubility but compromises bioactivity breadth. For industrial applications requiring a single versatile ingredient, we recommend the Tiegun cultivar with enzymatic extraction (TEP) as the optimal choice, offering the highest immunomodulatory potency, good yield, and flexibility for further processing. For more specific applications, Gaozhou with enzymatic extraction (GEP) provides maximum solubility ideal for beverages, while extrusion-puffed Gaozhou (PGEP) offers extreme solubility when rapid dispersion is critical and selective immunomodulation is sufficient. In summary, this study provides an actionable framework for engineering yam polysaccharides: select a cultivar for the desired bioactivity profile, and choose processing methods according to the target technological properties. Future work should identify the specific structural motifs responsible for distinct bioactivities and design precision processing methods to preserve them.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods15050921/s1, Table S1: Gene primer sequences detected by RT-qPCR. Figure S1: Correlation analysis of solubility with particle size (A) and molecular weight (B). Data points represent mean values from three independent experiments for each sample. Figure S2: Immunomodulatory and anti-inflammatory effects of yam polysaccharides in RAW264.7 macrophages. (A–C) Cytokine mRNA expression levels under immunomodulatory conditions. Data are presented as mean ± SEM (n = 6). Statistical significance was determined by one-way ANOVA followed by Tukey’s HSD test. Different lowercase letters above bars indicate significant differences among treatment groups at the same concentration (p < 0.05). LPS, lipopolysaccharide.

Author Contributions

Y.B.: Conceptualization, Writing—original draft, Methodology, Data curation, Investigation, Formal analysis, Funding acquisition. B.F.: Investigation, Visualization, Methodology, Resources. J.L.: Validation, Funding acquisition. F.W.: Supervision, Writing—review & editing, Funding acquisition. M.Z.: Supervision, Conceptualization, Writing—review & editing, Project administration. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key Research and Development Program project (2021YFD1600100), Nanfan special project of Chinese Academy of Agricultural Sciences (YYLH2307, YBXM2518).

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/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Acknowledgments

We thank Qin Ma for language help and proofreading.

Conflicts of Interest

The authors declare no conflicts of interest.

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Foods 15 00921 g001
Figure 1. Structure characteristics of polysaccharides from different yams. (A) I2-KI reaction curve. (B) FT-IR spectra. (C) FESEM images. Images in (C) are representative of observations from multiple fields of view.
Figure 1. Structure characteristics of polysaccharides from different yams. (A) I2-KI reaction curve. (B) FT-IR spectra. (C) FESEM images. Images in (C) are representative of observations from multiple fields of view.
Foods 15 00921 g001
Foods 15 00921 g002
Figure 2. Immunomodulatory and anti-inflammatory effects of yam polysaccharides in RAW264.7 macrophages. (A–C) Cytokine secretion levels under immunomodulatory conditions. (D–F) Corresponding mRNA expression levels under immunomodulatory conditions from panels (Figure S2) re-plotted after the exclusion of LPS-treated groups. (G–I) Anti-inflammatory activity assessed by NO production and cytokine mRNA expression in LPS-stimulated macrophages. Data are presented as mean ± SEM (n = 6). Statistical significance was determined by one-way ANOVA followed by Tukey’s HSD test. Different lowercase letters above bars indicate significant differences among treatment groups at the same concentration (p < 0.05). LPS, lipopolysaccharide.
Figure 2. Immunomodulatory and anti-inflammatory effects of yam polysaccharides in RAW264.7 macrophages. (A–C) Cytokine secretion levels under immunomodulatory conditions. (D–F) Corresponding mRNA expression levels under immunomodulatory conditions from panels (Figure S2) re-plotted after the exclusion of LPS-treated groups. (G–I) Anti-inflammatory activity assessed by NO production and cytokine mRNA expression in LPS-stimulated macrophages. Data are presented as mean ± SEM (n = 6). Statistical significance was determined by one-way ANOVA followed by Tukey’s HSD test. Different lowercase letters above bars indicate significant differences among treatment groups at the same concentration (p < 0.05). LPS, lipopolysaccharide.
Foods 15 00921 g002
Foods 15 00921 g003
Figure 3. Structure characteristics and immunological activity of Gaozhou yam polysaccharides before and after extrusion puffing. (A) FT-IR spectra. (B) SEM. (C) INOS. (D) TNF-α. (E) IL-1β. Statistical significance was determined by one-way ANOVA followed by Tukey’s HSD test. Different lowercase letters above bars indicate significant differences among treatment groups at the same concentration (p < 0.05). LPS, lipopolysaccharide.
Figure 3. Structure characteristics and immunological activity of Gaozhou yam polysaccharides before and after extrusion puffing. (A) FT-IR spectra. (B) SEM. (C) INOS. (D) TNF-α. (E) IL-1β. Statistical significance was determined by one-way ANOVA followed by Tukey’s HSD test. Different lowercase letters above bars indicate significant differences among treatment groups at the same concentration (p < 0.05). LPS, lipopolysaccharide.
Foods 15 00921 g003
Table 1. Comparison of the chemical composition of yam polysaccharides extracted by different methods.
Table 1. Comparison of the chemical composition of yam polysaccharides extracted by different methods.
Composition (%)THPTEPGHPGEP
Yield1.52 ± 0.29 a2.10 ± 0.41 b1.20 ± 0.21 a2.12 ± 0.38 b
Neutral sugar74.12 ± 5.12 c60.00 ± 5.44 a69.25 ± 1.51 b61.10 ± 3.37 a
Reducing sugar6.57 ± 0.02 a17.57 ± 0.77 c4.62 ± 2.06 a12.18 ± 0.74 b
Uronic acid10.91 ± 0.19 c9.18 ± 0.21 b12.25 ± 0.04 d7.33 ± 0.79 a
Protein6.66 ± 1.58 a15.30 ± 3.71 c5.00 ± 1.17 a10.87 ± 0.14 b
Monosaccharide composition (%)
Rhamnose---0.86 ± 0.25
Arabinose---0.61 ± 0.04
Mannose24.33 ± 3.95 c17.06 ± 0.90 b3.62 ± 0.12 a4.93 ± 0.09 a
Glucose72.04 ± 3.80 a78.75 ± 0.79 b92.90 ± 0.27 c91.68 ± 0.18 c
Galactose3.62 ± 0.26 b4.19 ± 0.10 c3.47 ± 0.15 b1.93 ± 0.07 a
Values are expressed as mean ± SEM (n = 3). “-” Indicates absence of detection. Data with different lowercase letters within the same row represent significant differences among samples as determined by one-way ANOVA followed by Tukey’s HSD test (p < 0.05).
Table 2. Molecular weight of CYPs extracted by different methods.
Table 2. Molecular weight of CYPs extracted by different methods.
GroupMn (×104 Da)Mw (×104 Da)Mp (×104 Da)PID (Mw/Mn)
THP1.71 ± 0.14 a2.58 ± 0.21 a1.62 ± 0.13 a1.51 ± 0.07 c
TEP3.31 ± 0.20 c4.25 ± 0.26 b4.14 ± 0.25 b1.29 ± 0.05 ab
GHP4.85 ± 0.26 d6.12 ± 0.33 c7.12 ± 0.38 c1.26 ± 0.04 a
GEP3.02 ± 0.22 b4.40 ± 0.32 b4.48 ± 0.33 b1.46 ± 0.07 bc
Values are expressed as mean ± SEM (n = 3). Data with different lowercase letters within the same row represent significant differences among samples as determined by one-way ANOVA followed by Tukey’s HSD test (p < 0.05).
Table 3. Solution properties of CYPs extracted by different methods.
Table 3. Solution properties of CYPs extracted by different methods.
GroupParticle SizePdIZeta PotentialSolubility
THP585.95 ± 5.59 d0.56 ± 0.01 a−33.30 ± 3.16 c3.18 ± 0.42 a
TEP299.87 ± 16.08 b0.93 ± 0.06 c−43.67 ± 1.55 ab4.57 ± 0.72 b
GHP422.57 ± 8.95 c0.79 ± 0.09 b−46.77 ± 0.90 a4.33 ± 0.25 b
GEP132.25 ± 10.96 a0.70 ± 0.04 b−41.17 ± 2.83 b5.98 ± 0.17 c
Values are expressed as mean ± SEM (n = 3). Different lowercase letters within the same column indicate significant differences as determined by one-way ANOVA followed by Tukey’s HSD test (p < 0.05).
Table 4. Effects of extrusion puffing on molecular weight, solubility, particle size, and Zeta potential of Gaozhou yam polysaccharides.
Table 4. Effects of extrusion puffing on molecular weight, solubility, particle size, and Zeta potential of Gaozhou yam polysaccharides.
GroupSolubilityParticle SizePdIZeta PotentialMw (1 × 104 Da)
GEP5.98 ± 0.17132.25 ± 10.960.70 ± 0.04−41.17 ± 2.834.40
PGEP7.97 ± 0.75 *536.37 ± 15.79 *0.09 ± 0.07 *−22.23 ± 2.85 *0.16
Values are expressed as mean ± SEM (n = 3). Values are expressed as mean ± SEM (n = 3). Significant differences between the GEP and PGEP groups, as determined by Student’s t-test, are denoted by asterisks (* p < 0.05).
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Bai, Y.; Fan, B.; Liu, J.; Wang, F.; Zhang, M. Processing Shapes Architecture, Cultivar Dictates Chemistry: A Structural and Functional Paradigm for Yam Polysaccharides. Foods 2026, 15, 921. https://doi.org/10.3390/foods15050921

AMA Style

Bai Y, Fan B, Liu J, Wang F, Zhang M. Processing Shapes Architecture, Cultivar Dictates Chemistry: A Structural and Functional Paradigm for Yam Polysaccharides. Foods. 2026; 15(5):921. https://doi.org/10.3390/foods15050921

Chicago/Turabian Style

Bai, Yajuan, Bei Fan, Jiameng Liu, Fengzhong Wang, and Mingwei Zhang. 2026. "Processing Shapes Architecture, Cultivar Dictates Chemistry: A Structural and Functional Paradigm for Yam Polysaccharides" Foods 15, no. 5: 921. https://doi.org/10.3390/foods15050921

APA Style

Bai, Y., Fan, B., Liu, J., Wang, F., & Zhang, M. (2026). Processing Shapes Architecture, Cultivar Dictates Chemistry: A Structural and Functional Paradigm for Yam Polysaccharides. Foods, 15(5), 921. https://doi.org/10.3390/foods15050921

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