Study on the accumulation patterns of crude polysaccharides in chestnut seeds and their physiological factors

【Objective】Chestnut (Castanea mollissima Bl.) is a traditional woody grain crop in China, valued for its nutritional and functional properties. Polysaccharides, as key bioactive components in chestnut kernels, exhibit significant antioxidant, anti-inflammatory, and immunomodulatory activities. Understanding the accumulation patterns of crude polysaccharides during kernel development and their physiological regulatory mechanisms is crucial for quality improvement and functional food development. This study aimed to systematically investigate the dynamic accumulation of crude polysaccharides in two chestnut cultivars, Yanshanzaofeng (early-maturing) and Yanlong (late-maturing), and to elucidate the key physiological factors involved, including carbohydrate metabolism, cell wall composition, and related enzyme activities.【Methods】The experiment was conducted in 2024 at the Chestnut Resource Nursery of Hebei Normal University of Science and Technology in Qinhuangdao. Fruits from both cultivars were sampled at 70, 80, 90, and 100 days after artificial bagging and pollination. Kernel samples were immediately frozen in liquid nitrogen and stored at -80 ℃ for subsequent analysis. The contents of various carbohydrates, including reducing sugars, soluble solids, starch, and cell wall com-ponents (cellulose, hemicellulose, soluble and insoluble pectin), were measured using standard spectrophotometric and colorimetric methods (e.g., DNS method, anthrone-sulfuric acid method, carbazole-sulfuric acid method). Crude polysaccharides were extracted using hot water extraction methods, and their content was determined by the phenol-sulfuric acid method. Monosaccharide composition of the polysaccharides was analyzed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC- PAD) on a Thermo ICS 5000 + system. The activities of key enzymes involved in sugar metabolism were assayed, including α-amylase, β-amylase, total amylase (using iodinestarch colorimetry), acid invertase (AI), neutral invertase (NI) (using DNS method), and sucrose phosphate synthase (SPS) (using anthrone method). Activities of antioxidant enzymes such as catalase (CAT, UV spectrophotometry), superoxide dismutase (SOD, NBT method), and peroxidase (POD, catechol method), as were the contents of malondialdehyde (MDA, TBA method) and hydrogen peroxide (H2O2, titanium sulfate colorimetry).【Results】The content of reducing sugars increased during development in both cultivars, with Yanlong showing significantly higher levels than Yanshanzaofeng at 90 and 100 DAP. Soluble solids content remained relatively stable during the initial stages but increased slowly from 80 to 100 DAP, with Yanlong exhibiting significantly higher values than Yanshanzaofeng at 90 and 100 DAP. The content of crude polysaccharides in Yanlong peaked at 100 DAP, reaching 34.86%, which was significantly higher than that in Yanshanzaofeng (13.80%). Cell wall components showed cultivar-specific dynamics: soluble pectin in Yanlong increased sharply, peaking at 90 DAP (4.9 mg · g- 1 ), while insoluble pectin peaked earlier at 80 DAP (13.72 mg·g-1 ). Cellulose content in Yanlong was highest at 90 DAP (27.70 mg·g-1 ), whereas hemicellulose content in Yanshanzaofeng surpassed that of Yanlong at 100 DAP, reaching 38.81 mg · g-1 . The crude polysaccharides from both cultivars were primarily composed of glucose (approx. 89.00% ), galactose (approx. 9.70% ), arabinose (approx. 0.80% ), and rhamnose (approx. 0.42%). Fucose, xylose, mannose, fructose, ribose, mannuronic acid, and guluronic acid were not detected. At 70 DAP, the glucose content in Yanshanzaofeng was significantly higher than that in Yanlong; subsequently, it exhibited a downward trend and remained at a lower level. The galactose content in Yanlong followed a unimodal pattern, peaking at 90 DAP. Arabinose content differences were minor except at 70 DAP. Rhamnose content decreased continuously in both cultivars. The activities of total amylase, α-amylase, and β-amylase generally increased then decreased during development, with Yanlong showing higher activities except at 90 DAP. AI activity in Yanlong increased slowly from 70 DAP, peaked at 80 DAP, and then declined. NI activity remained relatively stable but increased slightly when AI decreased after 80 DAP. SPS activity declined overall during development; it was highest in Yanlong at 70 DAP and decreased thereafter, while in Yanshanzaofeng, it increased until 90 DAP before decreasing, with overall higher activity than Yanlong. CAT activity increased slowly initially and more rapidly towards maturity, peaking at 100 DAP in both cultivars (Yanlong: 935.71 U·g-1 ; Yanshanzaofeng: 876.39 U·g-1 ). SOD activity showed a unimodal pattern, with Yanshanzaofeng surpassing Yanlong after 80 DAP and peaking at 100 DAP (356.13 U·g-1 ). POD activity was consistently significantly higher in Yanlong than in Yanshanzaofeng, peaking at 90 DAP (Yanlong: 454.37 U · g- 1 ; Yanshanzaofeng: 292.72 U · g- 1 ). MDA content in Yanshanzaofeng increased then decreased, peaking at 90 DAP (285.86 ng · g-1 ), whereas in Yanlong, it decreased then increased, peaking at 100 DAP (279.96 ng · g-1 ). H2O2 content decreased significantly in Yanshanzaofeng but fluctuated irregularly in Yanlong, reaching 24.91 µg·g-1 at 90 DAP, significantly higher than in Yanshanzaofeng. Correlation analysis revealed that polysaccharide content was positively correlated with NI and H2O2. Reducing sugar content showed significant positive correlations with soluble solids, hemicellulose, SOD, and POD, but a significant nega-tive correlation with SPS. Soluble pectin was positively correlated with insoluble pectin, cellulose, hemicellulose, CAT, and POD, and negatively correlated with SPS. Soluble solids were positively correlated with polysaccharides, soluble pectin, cellulose, hemicellulose, α-amylase, CAT, SOD, and POD, and negatively correlated with SPS. Cellulose showed significant positive correlations with hemicellulose, β-amylase, total amylase, and POD, and a negative correlation with SPS.【Conclusion】The accumulation of crude polysaccharides in chestnut kernels is varietal-dependent and synergistically regulated by key enzymes involved in sucrose metabolism and the dynamic changes in cell wall components. The late-maturing cultivar Yanlong exhibits higher potential for polysaccharide accumulation, reaching its peak later in maturation, associated with its higher reducing sugar, soluble solids, and amylase activities. Polysaccharide accumulation is positively linked to neutral invertase activity. The parallel trends between H2O2 and soluble pectin suggest a role for reactive oxygen species metabolism in carbohydrate transformation. This study provides a theoretical basis for high-polysaccharide chestnut breeding and quality cultivation practices, offering new insights into the targeted regulation of functional polysaccharides.