Molecular structure and characteristics of phytoglycogen, glycogen and amylopectin subjected to mild acid hydrolysis

Polysaccharides are ubiquitous biopolymers crucial for structural and energy-reserve roles. Starch (amylose and amylopectin) and related highly branched glucans like phytoglycogen (plant origin) and glycogen (animal origin) are key materials in food and biomedical applications. Phytoglycogen is a dendrimer-like, spherical nanoparticle with short chains and high branching, whereas amylopectin forms larger, worm-like molecules with lower branching. Glycogen exists as β (single) and α (aggregated) particles and structurally resembles phytoglycogen. Understanding how mild acid hydrolysis alters these polymers can reveal fine structural features and tune functional properties (e.g., solubility, interfacial behavior, digestibility). The research question is how hydrochloric acid hydrolysis fragments phytoglycogen and glycogen relative to amylopectin, how particulate structure and linkages change over time, and how these changes affect digestibility. The study aims to quantify kinetic depolymerization behavior, molar mass/radius distributions, branching levels (α‑1,6), and digestion fractions to provide guidelines for tailored applications of branched glucans.

Prior work shows acid hydrolysis preferentially attacks amorphous regions of starch granules and α‑1,4 linkages more readily than α‑1,6 linkages; degradation often proceeds in two phases and can reveal residual chain populations (e.g., DP 15 and 25). Acid treatment modifies functional properties, enabling products like thin-boiling starches, nanocrystals, and resistant dextrins. For amylopectin, medium chains (DP 13–33) increase while long and very short chains decrease upon hydrolysis. Phytoglycogen and glycogen nanoparticles have been explored for encapsulation and delivery due to their hyper-branched particulate structures. Models for glycogen structure include tiered and hairy-particle models, with differing density profiles. Limited work has addressed mild acid degradation of phytoglycogen/glycogen nanoparticles and the mechanisms governing their hydrolysis profiles. Kinetic treatments of polysaccharide depolymerization (e.g., dextran) and analyses of molar mass distributions have been used to elucidate linkage cleavage behavior. This study builds on these findings to compare phytoglycogen, glycogen, and amylopectin under identical mild acid conditions.

Materials: Phytoglycogen was extracted from su-1 maize kernels (uniform particle radius ~72 nm, >99% purity, <1% protein). Commercial glycogen (oyster) and maize amylopectin were used. Enzymes for digestion assays included porcine pancreatic α-amylase and amyloglucosidase; standard reagents were sourced from established suppliers. Mild acid hydrolysis: Samples (1.0 g/20 mL) were dissolved in 1.0 M HCl and incubated at 50 °C for varying times (0, 5, 30 min; 2, 6, 12, 24, 48, 72, 120 h). Aliquots were neutralized, precipitated with ethanol (3× volume), centrifuged (5000 g, 10 min), and dried. Degree of hydrolysis was calculated as supernatant carbohydrate (phenol–sulfuric acid assay) relative to initial dry weight. SEC-MALLS-RI: Molar mass (Mn, Mw, Mz) and z-average radius of gyration (Rz) were determined using SEC with MALLS and RI detection (He–Ne laser λ=632.8 nm; dn/dc=0.138; mobile phase water, 0.5 mL/min; columns Shodex OH-pak). ASTRA software (first-order Berry model) was used. Molecular density ρ was computed as Mw/Rz³. Dispersity D = Mw/Mn; combined dispersity ratio (CDR) = Mw²/(Mn·Mz). Kinetic modeling: Depolymerization was modeled with dB/dt = k·Mn^α, with α set to 1/2. A simplified relationship using Mn(t) enabled estimation of the rate constant k from plots of 1000/Mn^1/2 versus time. Hydrodynamic radius (DLS): Samples (0.01% w/w) were boiled (90 °C, 15 min) and measured on a Zetasizer Nano ZS. Number-weighted hydrodynamic radius distributions were obtained via CONTIN analysis. 1H NMR: Spectra were recorded at 500 MHz, 80 °C in D2O after exchange/lyophilization. Peaks near 5.4 ppm (α‑1,4) and 5.0 ppm (α‑1,6) were integrated to quantify linkage percentages. In vitro digestion (Englyst method): Samples (200 mg) in phosphate buffer (0.2 M, pH 5.2) were preheated, then digested with α-amylase (290 U/mL) and amyloglucosidase (6 U/mL) at 37 °C with shaking. Aliquots at 20 and 120 min were ethanol-quenched, and glucose measured (GOPOD). RDS, SDS, and RS were calculated from FG, G20, G120, and TG using standard Englyst equations. Statistics: Experiments were performed in triplicate (or duplicate for DLS); results are mean ± SD; significance at p < 0.05.

- Two-stage acid degradation was observed for all substrates (rapid 0–48 h to >70% hydrolysis, followed by slower phase to ~100% by 120 h). Degree of hydrolysis order under identical conditions: amylopectin > phytoglycogen > glycogen. - Native structural metrics (Table 1): Mw (×10⁷ g/mol) and Rz (nm) were amylopectin 8.37 ± 0.06 and 167.8 ± 2.3; phytoglycogen 2.14 ± 0.01 and 43.1 ± 0.7; glycogen 0.53 ± 0.05 and 29.4 ± 0.6. Dispersity D: amylopectin 2.3, glycogen 1.5, phytoglycogen 1.1. - Molar mass distributions shifted to lower values with time. Phytoglycogen’s initially monomodal narrow peak broadened, developed a shoulder (~30 min), and became bimodal by 12 h. Amylopectin changed from bimodal to monomodal, indicating preferential degradation of larger fragments. Glycogen showed rapid broadening by 5 min, reflecting heterogeneity and fragmentation of larger particles. - Time-dependent Mn, Mw, Mz decreased markedly within the first 5 min, then more gradually. Reduction magnitude: amylopectin > phytoglycogen > glycogen. Early-stage decreases followed Mn > Mw > Mz. - Kinetics: With α = 1/2, rate constants k (s⁻¹) were amylopectin 6.13 × 10⁻⁵, phytoglycogen 3.45 × 10⁻⁵, glycogen 0.96 × 10⁻⁵. Higher flexibility/lower density corresponded to faster depolymerization. - Molecular density ρ (g/mol·nm³) at 0 min (Table 2): phytoglycogen 266.4, amylopectin 17.7, glycogen 209.4. ρ dropped substantially in early hydrolysis (e.g., glycogen to ~107.4 by 5 min), reflecting loosening/fragmentation. - Particle radius distributions (Fig. 5): Native ranges were phytoglycogen 25–122 nm, amylopectin 18–70 nm, glycogen 13–60 nm; after 720 min they shifted to 5–18, 2–10, and 3–15 nm, respectively. - Branching (α‑1,6 linkages, Table 3): Decreased with time. Phytoglycogen: 7.6% → 2.3%; amylopectin: 5.2% → 1.9%; glycogen: 6.5% → 2.8% (0 to 720 min). - Digestibility (Table 4): RDS increased and SDS decreased for all. Amylopectin RDS: 72.4% → 89.0%, SDS: 10.7% → 4.0%, RS: 16.8% → 7.1% (0 to 720 min). Phytoglycogen RDS: 74.7% → 84.1%, SDS: 12.2% → 4.0%, RS: 13.1% → 12.0%. Glycogen RDS: 69.9% → 75.4%, SDS: 17.9% → 4.8%, RS: 12.2% → 19.8%. - Dispersity D increased over time for phytoglycogen and amylopectin, indicating polydispersity growth; glycogen briefly showed decreased D at 5 min due to preferential loss of larger particles. CDR trends were consistent with evolving distributions. - Structural modeling suggests acid preferentially cleaves α‑1,4 near chain ends and in less dense regions first; protein linkages in glycogen α-particles may be more acid-labile than glycosidic bonds under mild conditions, facilitating α→β particle breakdown.

Findings support that polymer architecture and density govern acid susceptibility. Amylopectin, with longer internal chains, lower molecular density, and greater flexibility, hydrolyzed fastest, rapidly losing large fragments and becoming monomodal in molar mass distribution. Phytoglycogen and glycogen, being more highly branched and densely packed, showed slower, two-stage depolymerization with early formation of smaller fractions but sustained denser-core persistence. The kinetic constants quantify this hierarchy (amylopectin > phytoglycogen > glycogen) and align with the notion that linkages near chain ends and in less dense regions are more reactive. Decreases in α‑1,6 linkage percentages and particle radius corroborate progressive debranching/fragmentation. Digestibility shifts (increased RDS, decreased SDS) reflect enhanced enzyme accessibility due to smaller particle sizes and altered branching; amylopectin’s RS decrease is consistent with fewer α‑1,6 linkages and smaller particles, whereas glycogen’s RS increase suggests residual higher branching, larger fragments, or inhibitory products impacting amylolysis. Overall, the results connect acid-induced structural changes with functional outcomes, guiding the design of branched glucans for targeted digestibility and delivery properties.

This study elucidates how mild HCl hydrolysis modifies phytoglycogen, glycogen, and amylopectin at the molecular and particulate levels, establishing a consistent two-stage depolymerization behavior with a hydrolysis susceptibility order of amylopectin > phytoglycogen > glycogen. Quantitative kinetics (k values), evolving molar mass/radius distributions, decreased α‑1,6 branching, and particle downsizing explain observed increases in rapidly digestible fractions and changes in RS. The proposed depolymerization model links branching architecture and density to reactivity, offering principles to tailor branched glucans for food and biomaterial applications. Future work should systematically vary acid concentration and temperature, resolve branch-chain length distributions and linkage positions in greater detail, and relate structural evolution to in vivo digestion and delivery performance.

- Acid concentration and other reaction parameters (e.g., temperature, ionic strength) were fixed; effects of varying these conditions were not explored. - Branch chain-length distributions and precise linkage site susceptibility were not fully resolved beyond α‑1,4 vs α‑1,6 quantitation. - Glycogen protein components implicated in α-to-β particle transitions were inferred; direct proteomic/structural confirmation under these conditions was limited. - Digestibility assessments were in vitro (Englyst method); in vivo relevance requires further validation.