Drying conditions affected chemical profiles and health promoting properties of Castanopsis piriformis Hickel & A.Camus

Abstract

Castanopsis piriformis, commonly referred to as the native chestnut of Thailand, has traditionally been dried for off-season storage. We investigated the impact of different drying conditions, namely hot air drying (HD) at 40 °C, 60 °C, 80 °C, and traditional sun drying (SD), on the physical and chemical properties of the nut. Scanning electron microscopy revealed that HD better preserved the cellular structure compared to SD. Among the hot air treatments, drying at 40 °C most effectively retained amino acids, with essential and non-essential amino acid of 619.6 and 927.0 μg/g dry basis, respectively. The levels of organic acids, phenolic compounds, flavonoids, and antioxidant activity were influenced by drying temperature. Fourier-transform infrared spectroscopy confirmed alterations in chemical composition associated with different drying conditions. Protein molecular weight analysis indicated structural changes and protein degradation at higher temperatures. HD at 40 °C is optimal for preserving both the chemical compounds and structural integrity of C. piriformis.

Highlights

• •Hot air drying preserved C. piriformis structure better than sun drying.
• •Hot air drying at 40 °C retained the highest amino acid content in the nuts.
• •Drying method affected phenolics, flavonoids, and antioxidant activity.
• •Protein degradation increased with higher drying temperatures.
• •FTIR showed chemical changes linked to drying temperature variations.

1. Introduction

Castanopsis piriformis, also known as the native chestnut, grows in the upper northeastern, eastern, and southeastern regions of Thailand. The tree is indigenous to Southeast Asia and is also found in Cambodia, Vietnam, and Laos (Thang et al., 2016; L. Zhang et al., 2018). The nuts are ovoid-shaped and partially enclosed by a cupule, similar to chestnuts. This tree is a valuable source of timber and is utilized in traditional medicine Fresh fruit are initially harvested and meticulously washed to obtain the nuts. The fruits are subsequently roasted in a baking pan for 20 to 30 min. Until the shells fracture, revealing the nuts inside. The roasted nuts can be consumed as a snack or included into favorite dishes for enhanced flavor and texture. This research evaluated the impact of different drying conditions and temperatures on the structural and chemical properties of the chestnuts to improve processing techniques for nutritional and functional preservation. Chestnuts provide several health benefits, and drying this seasonal product offers a practical strategy for extending its usability. The drying method facilitates the preservation of essential phytochemicals. Upon drying, C. piriformis is ground into a fine powder suitable for use as a healthy and tasty food additive. This powdered form possesses potential applications across several products, including baked foods, beverages, and functional dietary supplements.

Previous reports have shown that chestnuts from different regions vary in their proximate composition. Chinese chestnuts exhibited moisture contents ranging from 46.43 % to 49.75 %, crude protein between 7.54 % and 9.74 %, and soluble protein from 4.86 % to 6.68 % (Yang et al., 2018), Previous reports have shown that chestnuts from different regions vary in their proximate composition. Chinese chestnuts exhibited moisture contents ranging from 46.43 % to 49.75 %, crude protein between 7.54 % and 9.74 %, and soluble protein from 4.86 % to 6.68 % (Yang et al., 2018), while Portuguese chestnuts contained approximately 6.51 g/100 g DW of protein, with higher total dietary fiber (13.70/100 g DW) and fat content (3.20 g/100 g DW),whereas ash content was 2.06 g/100 g DW (Gonçalves et al., 2010). These variations reflect the influence of genotype, environmental conditions, and post-harvest handling, and they provide useful reference values for evaluating the nutritional potential of C. piriformis, a lesser-known native Thai species. In addition, a study has reported the fatty acid composition of Romanian chestnuts, A total of ten fatty acids was identified, consisting of five saturated fatty acids, namely lignoceric acid, phytanic acid, stearic acid, palmitic acid, and behenic acid, and five unsaturated fatty acids, including oleic acid, linoleic acid, farnesoic acid, eicosanoic acid, and pinellic acid. Among them, palmitic acid was found to be the most abundant saturated fatty acid, while oleic, linoleic, and α-linolenic acids were the predominant in unsaturated fatty acid (Ciucure et al., 2022). Moreover, chestnut dry matter consists of carbohydrates ranging from 75 % to 91 %, with starch containing 39 % to 82 %, followed by sucrose. These amounts of these polysaccharides, together with glucose, fructose, and raffinose, are utilized for identifying chestnut cultivars (Santos et al., 2022). Amino acids, phenolic acids, and organic acids are also found in chestnuts (Santos et al., 2022; Suárez et al., 2012). Fruits exhibit significant antioxidant activity associated with their amino acid, organic acid, phenolic acids and flavonoid compounds (Gonçalves et al., 2010; Hu et al., 2021), while drying temperature also impacts the quality of the sample. The conventional drying method relies on solar energy; however, the variable climate renders products susceptible to degradation and quality loss (Massantini et al., 2021). Oven drying is commonly used for drying plant material, with varying temperatures affecting the physical characteristic and chemical composition of the sample (Correia et al., 2009). Sun drying, while a cost-effective traditional method, has disadvantages including nutrient loss, contamination, and prolonged drying durations resulting from uncontrollable temperature and ambient factors (Chan et al., 2009; Z. Zhang et al., 2022). In contrast, hot air drying provides rapid and consistent drying while preserving quality; however, a high temperature may degrade heat-sensitive compounds (Attanasio et al., 2004; Turan, 2018; L. Zhang et al., 2018; Z. Zhang et al., 2022). Previous studies have investigated the effects of different drying conditions on plant-derived food products such as Italian chestnuts (Castanea sativa) (Attanasio et al., 2004), Spanish chickpea (Castellano and Sinaloa varieties) (Martín-Cabrejas et al., 2009), Portuguese chestnut (C. sativa) (Correia et al., 2009; Delgado et al., 2018), Turkish hazelnuts (Corylus avellana L.) (Turan, 2018), Chinese chestnuts (Castanea mollissima Blume) (L. Zhang et al., 2018), and Chinese tiger nuts (Cyperus esculents L.)(Z. Zhang et al., 2022). There are no previous reports on the effects of different constant temperatures during hot air drying and uncontrolled sun drying on its physicochemical properties and chemical composition. in C. piriformis, a nutritionally valuable species with potential applications in food and pharmaceutical industries. Additionally, the previous research commonly applies drying temperatures in the range of 40–80 °C for plant-based materials, which can be classified into low (40 °C), medium (60 °C), and high (80 °C) heat levels. This temperature range is widely used in the drying of herbs and local fruits due to its effects on moisture removal, bioactive compound retention, and tissue structure (Attanasio et al., 2004; Hu et al., 2021; Turan, 2018). Although the low temperature (40 °C) was slower, it can preserve heat-sensitive compounds such as amino acid, phenolic compounds along with antioxidant activity. While moderate temperature (60 °C) provides a balance between reducing moisture and maintaining active substances. Although high temperature (80 °C) reduces the drying time, it increases the risk of biodegradation and changes in tissue structure (Correia et al., 2009). Consequently, the selection of the range of 40–80 °C is suitable for comprehensively assessing the effects of drying on the quality of raw materials (Miranda et al., 2010). Therefore, this study aims to compare the impact of sun drying and hot air drying at different constant temperatures (40 °C, 60 °C, 80 °C) on the morphological characteristics, and chemical composition of C. piriformis, providing insights into the most effective drying method for maintaining its properties and functional components.

2. Materials and methods

2.1. Plant materials and sample preparation

Fresh fruit samples were collected in November 2021 from Phibun Mangsahan District, Ubon Ratchathani Province, in the northeastern region of Thailand. The voucher specimen (number CPHCT021) was deposited in the herbarium. Specifically, mature C. piriformis nuts of uniform size and appearance were harvested at the same ripeness stage with fully ripe fruits that could be peeled off the outer skin, and the seeds inside were extracted for study. The seeds inside were round or oval in shape and had a diameter of 1–2 cm. Nuts with visible defects or immature characteristics were excluded. The fruits were peeled off to remove the outer shells, and the seed samples were dried under different drying conditions. An electric thermostatic dryer (model FED 115, WTB Binder, Germany) was used for drying at 40, 60, and 80 °C, using hot air drying (H40, H60 and H80). For sun drying (HS), the seed samples were exposed to sunlight for three days (19–21 November 2021) at ambient temperatures ranging from 20 to 35 °C as monitored throughout the drying period. Weather conditions were monitored in Kantarawichai District, Maha Sarakham Province, during this period. The daily maximum temperatures remained relatively stable, ranging from 34.0 °C to 34.6 °C, while minimum temperatures varied between 20.5 °C and 23.8 °C. Relative humidity was moderately high, estimated between 60 % and 70 %. The final dried samples were required to have a moisture content below than 7 % for each drying condition. Following each drying process, the materials were ground and sieved through 60 mesh (250 μm), then stored at −20 °C until further analysis.

2.2. Microstructure of fresh and dried C. piriformis

Samples of both the fresh and dried materials were sectioned and examined using scanning electron microscopy (SEM) (Hitachi, TM-4000plus, Japan) with TM 4000plus software. The samples were coated with gold using a sputter coater (Hitachi, MC1000, Japan), and SEM analysis was performed under vacuum mode.

2.3. Free amino acid profile determination

Free amino acids, were analyzed following the method previously published (Chumroenphat et al., 2025) including essential amino acids such as arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), and valine (Val) and non-essential amino acids including alanine (Ala), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), proline (Pro), serine (Ser), and tyrosine (Tyr) were analyzed following the method previously published (Li et al., 2024). The analysis was performed using a Liquid Chromatography-Mass Spectrometer (LC/MS/MS) equipped with a triple quadrupole mass spectrometer (Shimadzu LCMS-8030, Shimadzu, Kyoto, Japan) operating in electrospray ionization (ESI) mode. Amino acid separation was performed using isocratic elution on an InertSustain® C18 column (2.1 mm × 150 mm, 3 μm, GL Sciences Inc., Tokyo, Japan). Results were reported in micrograms of amino acids in 1.0 g of dry basis sample (μg/g db). The calculation of Delicious Amino Acids (DAA) content is typically used in food science to evaluate amino acids contributing to flavor, particularly umami. DAA was calculated as the sum of the Glu, Asp, Ser, Gly, Ala, and Tyr (Chumroenphat et al., 2025).

2.4. Soluble sugar contents

The extraction and analysis of free soluble sugar, including fructooligosaccharide (FOS), stachyose (SCY), raffinose (RFN), sucrose, glucose, and fructose were conducted following previously published protocols (Saensouk et al., 2022). A Shimadzu 20 series HPLC system was used to quantify three soluble sugars: glucose, fructose, and sucrose. Separation was performed using a Sugar-Pak I column (300 mm × 6.5 mm id, 10 μm, Waters, USA) with guard column (Waters, USA), operated under isocratic conditions at a flow rate of 0.5 mL/min. Deionized water (DI) was used as the mobile phase, with a run time of 40 min at 80 °C. A refractive index detector was employed, and sugar concentration were determined using previously established calibration curves with appropriate external standards.

2.5. Organic acids determination

The organic acid extraction procedure was performed according to a previously published method (Chumroenphat & Saensouk, 2022). Freeze-dried samples (0.5 g) were acidified with 10 mL of 3 % phosphoric acid and mixed using a vortex at room temperature for 2 min. The mixture was then subjected to ultrasonic extraction using an ultrasonic cleaner (model 6210HP, KUDOS, Japan) for 10 min, followed by centrifugation (model 2–16 KL, Sigma, Germany) at 12,000 rpm for 20 min. at 25 °C. The supernatant was filtrated through a 0.22 μm nylon membrane filter, and the filtrate was analyzed using an HPLC system (model 20 Series, Shimadzu, Kyoto Japan). HPLC analysis was carried out using 0.05 M sulfuric acid as the mobile phase under isocratic at a flow rate of 0.5 mL/min. Separation was achieved using an Aminex® HPX-87H column (300 mm × 7.8 mm id, 5.0 μm, Bio-Rad Lab., Calif., USA). The column oven temperature was 80 °C, with an injection volume of 20 μL. A refractive index detector was used, set at a wavelength of 210 nm. Individual organic acids were identified by comparing their retention times to those of authentic standards (oxalic acid, citric acid, malic acid, quinic acid, and fumaric acid). All analyses were performed in triplicate, and results were reported as milligrams per gram dry basis (mg/g db) of the sample.

2.6. Total phenolic content (TPC) and total flavonoid content (TFC)

The extraction and analysis of total phenolic content (TPC) were performed using the Folin-Ciocalteu assay, as previously described (Chumroenphat et al., 2023). Absorbance was measured at 750 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). TPC was expressed as milligrams of gallic acid equivalent (GAE) per gram dry basis (mg GAE/g db). The total flavonoid content (TFC) of sample was also determined according to the previously reported method (Chumroenphat et al., 2023) and measured at 510 nm using a the same microplate reader. Results were expressed as milligrams of rutin equivalent (RE) per gram dry basis (mg RE/g db).

2.7. Phenolic acids and flavonoid compounds by HPLC

Phenolic acids and flavonoid compounds were analyzed according to previously established methods (Chumroenphat et al., 2023). One gram of sample powder was extracted with 20 mL of water/methanol (80,20, v/v) and shaken at 37 °C at 150 rpm for 12 h. The extract was filtered through a 0.22 μm nylon membrane filter and analyzed using HPLC with a C18 column (4.6 mm x 250 mm, 5 μm GL Sciences Inc., Tokyo, Japan). The mobile phase consisted of 1 % acetic (solvent A) and acetonitrile (solvent B). The gradient elution was performed as follows: from 0 to 5 min, linear gradient from 5 % to 9 % solvent B; from 5 to 15 min, 9 %solvent B; from15 to 22 min, linear gradient from 9% to11 % solvent B; from 22 to 38 min, linear gradient from 11 % to 18 % solvent B; from 38 to 43 min, from 18 % to 23 % solvent B; from 43 to 44 min, from 23 % to 90 % solvent B; from 44 to 45 min, linear gradient from 90 % to 80 % solvent B; from 45 to 55 min, isocratic at 80 %solvent B; from 55 to 60 min, linear gradient from 80 to 5 % solvent B and a re-equilibration period of 5 min with 5 % solvent B used between individual runs. The operated under gradient conditions at a column temperature of 38 °C, with an injection volume of 20 μL. Absorbance was measured using a photodiode array detector at wavelengths of 280 nm (gallic acid, protocatechuic acid, cinnamic acid, syringic acid, p-hydroxybenzoic acid and vanillic) and 320 nm (gentisic acid, chlorogenic acid, caffeic acid, ferulic acid, p-coumaric acid and sinapic acid) for phenolic compounds, and at 370 nm (apigenin, catechin, kaempferol, myricetin, quercetin and rutin) for flavonoid compounds. Results were expressed as micrograms per gram dry basis (μg/g db) for phenolic compounds and flavonoid compounds (Li et al., 2024). The HPLC parameters, including wavelength and linear equation of standards, were used in these studies of phenolic acids and flavonoid compounds, as demonstrated in Table S6 (supplementary material).

2.8. Fourier transform infrared spectroscopy (FTIR) measurement

The samples were examined using an FTIR instrument (Frontier) equipped with a UATR accessory (Perkin Elmer, USA) fitted with a Diamond/KRS-5 crystal composite (single bounce), in powder form without prior processing. Spectra data were collected over 32 scans with a force gauge of 110 units. The resolution was set at 4 cm⁻¹, covering the spectra range from 4000 to 400 cm⁻¹. The FTIR spectra in this study are presented in transmittance mode, which is the default output format of the FTIR instrument used. Background spectra were automatically subtracted by the software, PerkinElmer Spectrum IR version 10.6.1.942 (Perkin Elmer, USA).

2.9. Antioxidant capacity determination

The DPPH radical scavenging activity and ferric reducing antioxidant power (FRAP) of the samples were assessed following previously published methodologies (Chumroenphat et al., 2023). The DPPH assay involved mixing 20 μL of the extract or control (deionized water) with 180 μL of a 60 μM DPPH solution (dissolved in methanol), followed by incubation in the dark at room temperature for 30 min. Absorbance was then measured at 517 nm using a Varioskan Lux microplate reader (Thermo Fisher Scientific, Waltham, MA, USA), The standard trolox curve was used according to the equation of a linear equation, and results were calculated and expressed as milligrams of Trolox equivalent (TE) per gram dry basis (mg TE/g db). Concurrently, the FRAP assay was performed by combining 5 μL of each extract with 180 μL of FRAP reagent in a 96-well plate. The FRAP reagent was prepared by mixing 0.3 M acetate buffer (pH 3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl₃·6H₂O in a 10:1:1 (v/v/v) ratio, and incubated at 37 °C for 2 h. before use. After shaking the reaction mixture for 1 min and incubating at 37 °C for 15 min, absorbance was measured at 593 nm using the same microplate reader. The standard FeSO₄ curve was used according to the equation of a linear equation, and the FRAP values were expressed as milligrams of FeSO₄ per gram dry basis (mg FeSO₄/g db).

2.10. Determination of molecular weights of protein components

In this study, Western blot with anti-JAK2 and anti-STAT5 antibodies was used to analyze the molecular size of proteins in C. piriformis under different drying conditions. Western blot can provide more precise data result about the target proteins, including the detection of denaturation or loss of specific proteins, which is important to evaluate the effect of heat treatment on protein structure and size (Sule et al., 2023).

Protein extraction procedure.

Protein extraction was performed milligrams porously reported method (Li et al., 2024). Briefly, 10 mg of 0.02 % ascorbic acid was used to homogenize 1 g of the defatted sample powder, and the mixture was then filtered through medical gauze. The resulting filtrate was centrifuged at 10,000 ×g for 30 min. Western blotting analysis was subsequently performed using the recovered supernatant.

Evaluation of protein molecular weight using the Jess simple Western blotting analysis.

Protein molecular weight (MW) was evaluated by capillary-based Western blotting with the ProteinSimple Jess system (Protein Simple, CA, USA). Briefly, 1 × sample buffer, 1× fluorescence standard, and 40 mM dithiothreitol were mixed with cell lysates to prepare a master mix. After heating the mixture at 95 °C for 5 min, assay plate wells were filled with 3 μL of the denatured protein extract, 10 μL of each primary antibody (diluted1:20), HRP-conjugated anti-mouse secondary antibodies, chemiluminescent substrate, and protein normalization solution. The primary antibodies used were anti-JAK2 and anti-STAT5. MW standards (12–230 kDa) were supplied for each experiment via biotinylated ladder cartridges. Protein electrophoresis and blotting were fully within the Jess instrument using a plate-based and capillary system. Capillaries were modified to enable protein loading. Chemiluminescence detection and data analysis were performed using Compass software (Protein Simple, CA, USA).

2.11. Statistical analysis

All data were expressed as the mean of three replicates ± standard deviation (SD). One-way ANOVA followed the least significant difference (LSD) test was used to evaluate the data, with statistically significant differences considered at p < 0.05. Statistical analysis of the measured parameters was performed using MetaboAnalyst 6.0 (Chumroenphat et al., 2025). Partial least squares discriminant analysis (PLS-DA) was employed to identify key metabolites, with variable importance in projection (VIP) scores greater than 1.5 considered indicative of significant compounds. Additionally, hierarchical clustering analysis (HCA) was conducted to classify samples based on metabolic profiles. A dendrogram was generated to visualize clustering patterns and assess similarities among sample groups.

3. Results and discussion

3.1. Change in microstructure of C. piriformis under different drying conditions

The microstructure of fresh and dried C. piriformis samples was analyzed using scanning electron microscopy (SEM), as shown in Fig. 1. Free water was easily removed from starch during drying under different conditions, resulting in significant changes to the microstructure characteristics of C. piriformis. The starch granules of samples dried at 40 °C, 60 °C, and 80 °C exhibited morphology similar to fresh samples, whereas sun-dried starch granules were densely packed than those in other treatments. This difference may be attributed to the consistent temperature and rapid water evaporation during hot air oven drying, which induced mechanical stress on cellular components, leading to the disruption and fragmentation of starch granules and creating a loosened structure within the cellular matrix (Attanasio et al., 2004). In contrast, sun drying at ambient temperatures ranging from 20 to 35 °C caused gradual water evaporation from the parenchyma cells of C. piriformis. This slower drying process led to minor alteration in the microstructure alterations to the cellular matrix. Because sun drying depends on natural sunlight, it is susceptible to environment fluctuations, which may result in inconsistent drying rates (Attanasio et al., 2004). Hot air oven drying was found to preserve the structural integrity of C. piriformis starch granules compared to the fresh sample. These findings highlight the importance of selecting an appropriate drying method to achieve desired functional properties of starch (Zhang et al., 2022). Different drying conditions significantly influence surface structure and starch granules characteristics, thereby affecting their potential applications in food processing, including water absorption, gelatinization temperature, and swelling capacity.

Fig. 1.

Microstructure of C. piriformis; A: C. piriformis sample; B:fresh sample; C: sun dried; D: hot air oven dried at 40 °C; E: hot air oven dried at 60 °C; F: hot air oven dried 80 °C; image of fresh and dried sample using scanning electron micrographs: different drying (×200).

3.2. Change in free amino acids of C. piriformis under different drying conditions by LC/MS/MS

The amino acid concentrations in C. piriformis were exposed to different temperatures and drying conditions, including hot air oven drying at 40 °C (H40), 60 °C (H60), and 80 °C (H80), as well as sun drying (HS), were compared with those in the fresh sample (HF). The results revealed distinct differences in the preservation of essential and non-essential amino acids depending on the applied drying conditions. Lower temperatures (HS, H40 and H60) were more effective in preserving both essential and non-essential amino acids (Table S1). The highest total amino acid concentration was observed in fresh sample (941.3 μg/g db), while the highest essential amino acids content was recorded in the 40 °C treatment (619.6 μg/g). Conversely, the lowest concentrations of total, essential amino acids, and non-essential amino acids were found in the 80 °C treatment, with values of 484.20 μg/g and 313.02 μg/g, and 171.1 μg/g db, respectively. Non-essential amino acids were also most abundant in the fresh samples (322.77 μg/g), highlighting the degradation of amino acids at higher drying temperatures and the benefits of moderate drying conditions. The concentration of the essential amino acid Met was significantly higher in the H40 samples (162.4 μg/g db) compared to H80 sample (69.6 μg/g db). These results suggest that lower sun drying temperatures better preserved certain amino acids. During thermal drying, water release predominates throughout the decomposition process rather than melting, followed by the further breakdown of the condensation products depending on the characteristics of specific intermediates (Weiss et al., 2018). Arg followed a similar pattern, with the highest levels observed at 40 °C (104.5 μg/g), and a significant decrease at 80 °C (66.9 μg/g db). Ala and aspartic essential amino acids were also more effectively retained at 40 °C (50.4 μg/g db) but decreased substantially at 80 °C (34.9 μg/g db). Similarly, Asp concentrations were higher in the H40 treatment (23.0 μg/g db) compared to the H80 treatment (19.8 μg/g db). These findings confirmed that the drying method and temperature significantly influence the stability of amino acids in C. piriformis. Lower drying temperatures are more conducive to preserving amino acid integrity, whereas higher temperatures increased amino acid degradation. Understanding these effects is essential for optimizing drying processes to retain the nutritional and functional and properties of food products.

Furthermore, when comparing the amino acid content of C. piriformis after drying at 80 °C with fresh (HF) samples, the amino acids can be classified into three groups according to their stability. It was found that no amino acids could remain more than 80 % and were not classified as highly stable. Additionally, the moderate stability group (50–79.9 %) includes Arg, Ile, Leu, Thr, Val, Ala, Glu, Gly, Pro, and Tyr, which indicates some heat tolerance. The degraded group (less than 50 %) includes His, Lys, Met, Phe, Trp, Asn, Asp, Gln, and Ser. These amino acids showed significant decreases in their content after drying at high temperatures, reflecting their heat sensitivity and changes in their chemical structure. This classification facilitates the selection of optimal drying conditions to effectively preserve specific target amino acids, particularly for the development of functional food ingredients.

The analysis of amino acid profiles across the experimental groups (HF, HS, H40, H60, and H80) demonstrates the changes in amino acids resulting from the treatment, as shown in Fig.s2.

The heatmap (Fig. 2A) shows group-specific variations in amino acid concentrations influenced by temperature differences and drying conditions, with HF exhibiting elevated levels of Asp and Arg. Higher drying temperature significantly reduced amino acid content, with notable losses in heat-sensitive amino acids including Gln and Trp, particularly at 80 °C, likely due to thermal degradation and Maillard reactions. The PCA scores plot (Fig. 2B) shows overlapping clusters for HF and H40, indicating minimal impact of drying at 40 °C on the amino acid profile. In contrast, elevated temperatures (H60, H80) and sun drying (HS) resulted in distinct separation, indicating enhanced degradation of amino acids. The VIP score plot (Fig. 2C) identifies Arg, Met, and Phe as the most significant metabolites that differentiate the groups, highlighting their involvement in stress-related metabolic pathways. This observation aligns with the heatmap and hierarchical clustering results (Fig. 2D), indicating that HF and H40 display similar clustering patterns, whereas HS and H60 show distinct profiles. The findings highlight the sensitivity of amino acid concentrations to different drying conditions.

Fig. 2.

Relationship of free amino acids in C. piriformis under different drying conditions. (A) Heatmap illustrating amino acid intensity relationships across samples. (B) PCA scores plot showing group separation based on amino acid profiles. (C) VIP scores identifying key amino acids contributing to group differences. (D) HCA dendrogram clustering samples by amino acid relationship patterns. Drying conditions: HF (fresh), H40 (hot air 40 °C), H60 (hot air 60 °C), H80 (hot air 80 °C), HS (sun dried).

DAA, including Glu, Asp, Ser, Gly, Ala, and Tyr, play a crucial role in influencing the umami taste and overall flavor profile of food products. The total delicious amino acids content (∑DAA) of C. piriformis was significantly influenced by drying conditions, with the highest concentration observed in the fresh sample (HF: 199.3 μg/g db). Among the dried samples, hot air oven drying at 40 °C (H40: 193.4 μg/g db) retained the highest ∑DAA, comparable to the fresh sample, followed by drying at 80 °C (H80: 188.5 μg/g db). In contrast, lower retention was noted in samples dried at 60 °C (H60: 156.3 μg/g db) and under sun drying (HS: 145.5 μg/g db). These results indicate that moderate-temperature drying (40 °C) more effectively preserves delicious amino acids than drying at (60 °C) or uncontrolled sun drying, likely due to reduced oxidative or enzymatic degradation. Although the preservation of DAAs in C. piriformis under optimal conditions (H40 and H80) was slightly lower than that reported for Chinese chestnuts, it was comparable to levels observed in other nuts, such as walnuts subjected to similar drying treatments (Chen et al., 2018). These results highlight the potential of C. piriformis as a nutritionally valuable food source, particularly when processed using controlled high temperature drying. The findings provide guidance for optimizing drying conditions to improve the nutritional quality of C. piriformis, supporting its development as a functional foods and nutraceutical products.

3.3. Change in sugar content of C. piriformis under different drying conditions

This study examined the effect of drying on sugar profile of C. piriformis. The carbohydrate content of fresh C. piriformis was quantitatively evaluated and compared with samples subjected to various different drying. Conditions. The oligosaccharides analyzed included FOS, SCY, and RFN, while the soluble sugars measured were sucrose, glucose, and fructose, as shown in TableS2. Results showed that the H80 sample demonstrated the highest total sugar content (741.7 mg/g db), likely due to the effective preservation and increased concentration of sugars resulting from the degradation of complex carbohydrates into simpler, more soluble forms (Correia et al., 2009; Mensink et al., 2017). This supports previous findings that high temperatures facilitate polysaccharide hydrolysis, converting them into monosaccharides and disaccharides (Correia et al., 2009; Sakamoto et al., 2020). These effects were evident by the analysis of different sugar forms. FOS were most abundant in the fresh sample (HF: 12.8 mg/g db), suggesting that these sensitive compounds were partially retained during drying. In contrast, H80 had the highest levels of SCY and RFN, at 47.60 ± 0.88 and 40.97 ± 2.36 mg/g db, respectively, indicating that specific drying conditions could preserve or even enhance certain oligosaccharide concentrations (Mensink et al., 2017; Morgan et al., 2006). Soluble sugars such as sucrose, glucose, and fructose followed a similar trend, with H80 demonstrating significantly higher sucrose content (593.9 mg/g db) compared to the HF (494.6 mg/g db). This pattern suggests that the H80 sample, dried at a high temperature, showed increased concentration of these sugars as well as preserving them. This may be advantageous for products where more sweetness is desired (Schiffman et al., 2000). Moreover, higher drying temperatures gave higher reducing sugar contents (Correia et al., 2009). These findings indicate that the drying method impacts both the nutritional quality and sensory properties of dried foods. The ability of a drying process to preserve or concentrate sugars significantly promotes the functional and health-related attributes of the final product. Food processors must carefully select and optimize drying conditions based on the desired product specifications. This study highlighted the importance of modified drying approaches that accommodated the thermal sensitivity of sugars to maximize the preservation of beneficial compounds while enhancing the overall quality and appeal of dried food products (Zhou et al., 2023).

The sugar profiles of C. piriformis were significantly affected by different drying conditions, with fresh samples (HF) exhibiting the highest total sugar content, particularly for sucrose and glucose, as shown in Fig. 3. Among the drying conditions, hot air oven drying at 80 °C (H80) preserved sugar levels most similar to those of fresh samples, whereas sun drying (HS) and drying at 60 °C (H60) resulting in the most significant reductions, likely due to prolonged exposure to enzymatic and oxidative degradation. PCA analysis revealed clear group separation, with H80 clustering near HF, suggesting minimal sugar changes during high temperature drying. VIP scores indicated that sucrose and glucose are significant contributors to variability, as these sugars exhibited heightened sensitivity to degradation at lower drying temperatures (H40 and H60). The HCA analysis indicated that H80 samples exhibited greater compositional similarity to HF, whereas HS, H40, and H60 formed distinct clusters, indicating substantial sugar loss.

Fig. 3.

Relationship of sugar profiles in C. piriformis under different drying conditions. (A) Heatmap illustrating changes in sugar compounds (sucrose, glucose, fructose, stachyose, raffinose, and fructooligosaccharides) across samples. (B) PCA scores plot showing group separation based on sugar composition. (C) VIP scores identifying key sugars contributing to group differences. (D) HCA dendrogram clustering samples by similarity in sugar profiles. Drying conditions: HF (fresh), H40 (hot air 40 °C), H60 (hot air 60 °C), H80 (hot air 80 °C), HS (sun dried).

The percentage retention of individual sugars after drying at 80 °C, relative to fresh samples, revealed three distinct levels of thermal stability. The stable group, with retention ≥80 %, included FOS, which showed minimal change in concentration. The enhanced group, consisting of SCY, RFN, and sucrose, exhibited retention values exceeding 100 %, suggesting thermal processing facilitated the release of these sugars from bound forms through cell wall disruption or polysaccharide hydrolysis. No sugars were classified in the moderately stable group (50–79.9 %). In contrast, glucose and fructose belonged to the degraded group, with retention below 50 %, reflecting their susceptibility to heat-induced degradation. These findings highlight the importance of selecting appropriate drying temperatures to retain beneficial oligosaccharides and reduce monosaccharide degradation, ensuring their effective utilization as functional ingredients in functional food products. Previous studies have also demonstrated comparable sugar stability in thermally processed plant materials, indicating that elevated temperatures deactivate degrading enzymes, thereby reducing nutrient loss (Wang et al., 2017; Zhang et al., 2020). The results indicate that drying at 80 °C is optimal for enhancing sugar content in C. piriformis, thereby supporting its application in functional food and nutraceutical.

3.4. Change in organic acid content of C. piriformis under different drying conditions by HPLC

The analysis of C. piriformis exposed to different drying temperatures and conditions revealed that the preservation of organic acids is highly dependent on the drying method used. Organic acids are naturally present in fruits and vegetables (Kader, 2008). Organic acids naturally present in chestnuts, a species within the same genus as C. piriformis (Suárez et al., 2012). Among the most significant factors affecting taste and other sensory attributes of fruits and vegetables are their sugar and organic acid content (Kader, 2008). All the samples contained oxalic and citric acids, with the highest concentrations (64.2 mg/g db and 2.3 mg/g db) observed in samples dried at 60 °C (Table S3). Oxalic acid was well-preserved at moderate oven temperatures, while a reduction at 80 °C was attributed to volatilization and thermal degradation. Heat-induced interactions between nitrogen-free carboxylic acids and sugars likely contributed to an increased in inorganic acid such as citric acid (Delgado et al., 2018). Conversely, elevated drying temperatures led to the breakdown or transformation of other organic acids (Ilica et al., 2019). Malic acid content was similar to oxalic acid, with the highest levels at 60 °C (4.5 mg/g db). A decrease in the HS (2.1 mg/g db) further supports the observation that specific drying temperatures better preserve organic acids. Quinic acid displayed a higher concentration in the 80 °C samples (10.3 mg/g db), possibly due to a biotransformation process involving the conversion of chlorogenic acids into quinic acid and caffeic acids (Moon & Shibamoto, 2010). The synthesis and characterization of chlorogenic acid derivatives, including quinic acid, have previously identified various bioactive compounds with potential health benefits (Maas et al., 2008). Fumaric acid was the most abundant in the 80 °C sample, with a concentration of 9.2 mg/g db. This increase at higher temperatures may indicate either its thermal stability or transformation from other organic acids, such as through the dehydration of malic acid during thermal processing (Delgado et al., 2018; Ilica et al., 2019). In contrast, sun drying resulted in lower levels of fumaric acid and other organic acids, likely due to fluctuating temperatures and prolonged exposure during the drying period, which promoted degradation (Chen et al., 2019). The results showed that all organic acids in C. piriformis were highly stable after drying at 80 °C compared with HF. The amount of each organic acid remained close to or increased, reflecting their ability to be stable under high heat conditions. The order of organic acids from highest to lowest amount was oxalic acid > quinic acid > fumaric acid > malic acid > citric acid. These acids were all in the stable group, which may be due to the release of acids bound in the plant structure or chemical changes that resulted in the increase of these substances. The retention of organic acids during drying may significantly influence the sensory characteristics (e.g., taste and acidity) and the biological functionality of food ingredients and products. These findings underscore the importance of organic acids in contributing to the taste, nutritional value, and preservative properties of food products. Optimizing the drying process to maintain the organic acid profile is therefore essential for enhancing the quality, flavor, and shelf life of C. piriformis-derived foods.

The organic acid profiles of C. piriformis exhibited significant variation across different drying conditions, as shown in Fig. 4. The heatmap (Fig. 4A) shows variations in the composition of significant organic acids, including oxalic, fumaric, quinic, malic, and citric acids, among the samples. Fresh samples (HF) exhibited the highest concentrations of organic acids, particularly citric and malic acids, which were significantly reduced across all drying conditions. Hot air oven drying at 80 °C (H80) was more effective in retaining organic acids compared to other drying conditions. In contrast, drying at at 40 °C (H40) and sun drying (HS) led to the most significant reductions, likely due to prolonged exposure to oxidative and enzymatic degradation during the drying process. The PCA scores plot (Fig. 4B) demonstrated distinct differences among the sample groups according to their organic acid profiles. Fresh samples (HF) exhibited a distinct clustering, with H80 samples positioned nearer to HF, indicating minimal compositional alterations during high temperature drying. Samples dried at at 40 °C (H40) and 60 °C (H60), along with sun-dried samples (HS), showed significant deviations, indicating greater losses in organic acid content. VIP scores (Fig. 4C) indicated that oxalic and fumaric acids were the primary contributors to the observed differences, while malic and citric acids also had significant roles. These findings suggest that these organic acids exhibit increased susceptibility to degradation when subjected to prolonged or mild heat conditions. The hierarchical cluster analysis (HCA) dendrogram (Fig. 4D) validated the relationships among the drying conditions, with H80 closely clustering with HF, whereas H40, H60, and HS constituted separate clusters. This trend is consistent with previous studies showing that high-temperature drying is more effective in preserving organic acid content in plant materials due to rapid enzyme inactivation and reduced oxidation (Chan et al., 2009). These results suggest that hot air drying at 80 °C is optimal for maintaining organic acid composition in C. piriformis, providing valuable information for the food and nutraceutical industries in developing products.

Fig. 4.

Relationship of organic acid profiles in C. piriformis under different drying conditions. (A) Heatmap illustrating changes in organic acid composition across samples. (B) PCA scores plot showing group separation based on organic acid profiles. (C) VIP scores identifying key organic acids contributing to group differences. (D) HCA dendrogram clustering samples by similarity in organic acid profiles. Drying conditions: HF (fresh), H40 (hot air 40 °C), H60 (hot air 60 °C), H80 (hot air 80 °C), HS (sun dried).

that preserves health-promoting compounds.

3.5. Changes in total phenolic content and total flavonoid content of C. piriformis under different drying conditions

This study evaluated the effect of temperature and drying conditions on the total phenolic content (TPC) and total flavonoid content (TFC) of C. piriformis (Table S4). The highest TPC was observed in the H60 samples (68.0 mg GAE/g db), indicating enhancement or preservation of phenolic compounds at moderate temperatures, possibly due to increased extraction efficiency or a protective concentration effect. The TPC was significantly affected at higher drying temperatures, with a noticeable decrease in all components, especially at 80 °C. This reduction in TPC is attributed to heat-induced degradation (Chan et al., 2009). Moreover, the decline in TPC during dehydration may result from the binding of polyphenols to other substances (e.g. proteins) or alteration in their chemical structure (Martín-Cabrejas et al., 2009). In addition, thermal processing can induce non-covalent and covalent interactions between polyphenols (e.g., gallic acid, quercetin) and proteins (e.g., albumins or heat-denatured globulins), leading to reduced extractability and antioxidant activity. Additionally, high-temperature treatments can cause chemical transformations, including degradation, rearrangement, or conversion of phenolic glycosides to aglycones (Zhu et al., 2024). In contrast, hot air drying at 40 °C and sun drying at ambient temperatures (35–40 °C) yielded 55.7 mg GAE/g db and 62.4 mg GAE/g db, respectively. The polyphenols likely reacted oxygen during these treatment, promoting degradation of the sample (Prathapan et al., 2009). The highest TFC was also recorded in the H60 sample (1.3 mg RE/g db). Conversely, the HS sample exhibited the lowest TFC (1.1 mg RE/g db), suggesting significant flavonoid degradation under uncontrolled sun drying conditions, following a pattern similar to TPC. Furthermore, the increased value of TFC in the H60 treatment may reflect inconsistencies in flavonoid degradation or variation in extraction efficiency, suggesting that drying conditions significantly influence flavonoid retention (Chumroenphat et al., 2023; Chumroenphat & Saensouk, 2022). These findings demonstrate the complex relationship between heat and phytochemical stability, showing that both increases and decreases in phenolic and flavonoid contents can occur depending on the drying method. These findings are crucial for maximizing the health-promoting properties of phenolic and flavonoid compounds. The processing of C. piriformis and related products can be guided by knowledge of how various drying techniques influence these components to ensure the optimum retention of nutritional and medicinal properties.

3.6. Change in phenolic and flavonoid compounds of C. piriformis under different drying conditions by HPLC

The phenolic acid contents in five samples (HF, H40, H60, H80, and HS) exhibited significant variations under different drying temperatures, as shown in Table S5. Caffeic acid was the most abundant phenolic acids across all the samples, with the highest concentration found in the fresh sample (HF: 161.1 μg/g db) and the lowest in the sun-dried sample (HS: 81.2 μg/g db). Total phenolic acid content was also highest in HF (394.1 μg/g db) and lowest in HS (273.7 μg/g db). Gallic acid content ranged from 0.35 μg/g db in H40 to 0.51 μg/g db in H80, while protocatechuic acid varied between 24.0 μg/g db in HS and 27.0 μg/g db in HF. Remarkably, p-hydroxybenzoic acid content was highest in H80 (61.0 μg/g db), suggesting that higher drying temperatures enhanced certain phenolic compounds or degradation from high molecular complexes of phenolic acids. Vanillic acid levels highest in H60 (67.3 μg/g db) and decreased significantly in H80 (10.6 μg/g db), indicating sensitivity to higher drying temperatures. Chlorogenic acid was most abundant in HF (55.8 μg/g db) and lowest in H80 (32.0 μg/g db). These variations in phenolic acids content suggested that different drying temperatures and drying conditions influenced the stability and concentration of individual phenolic acids. High-temperature drying (H80) enhanced the levels of specific phenolic acids such as p-hydroxybenzoic acid but also indicated the degradation of others such as vanillic acid. This occurrence was attributed to the thermal sensitivity of phenolic compounds, where some compounds were stabilized while others were degraded under high temperatures. Similar trends have been observed in previous studies, where higher drying temperatures were associated with increased phenolic content in some cases and degradation in others. When comparing the phenolic acid contents in C. piriformis after drying at 80 °C with those in fresh samples, the compounds could be classified into three groups based on their thermal stability. The stable group, with retention above 80 %, included gallic acid, protocatechuic acid, and p-hydroxybenzoic acid, all of which tended to increase slightly after heat treatment. The moderately stable group, with 50–79.9 % retention, consisted of chlorogenic acid, caffeic acid, and p-coumaric acid, which remained at appreciable levels following drying. In contrast, the degraded group, with retention below 50 %, comprised vanillic acid, ferulic acid, and sinapic acid, reflecting their high sensitivity to elevated temperatures. These findings highlight the diverse thermal stability of phenolic acid compounds, which should be considered in selecting suitable drying conditions for preserving bioactive compounds in functional food development.

Total flavonoid compounds also showed significant differences among the samples. The highest total flavonoid compounds was observed in H60 (1124.7 μg/g db), while the lowest was in H40 (833.0 μg/g db). Rutin was the most predominant flavonoid in all samples, with the highest concentration in H60 (802.9 μg/g db) and the lowest in HS (641.0 μg/g db). Quercetin content ranged from 43.9 μg/g db in HS to 50.8 μg/g db in HF. Apigenin content was highest in HF (226.1 μg/g db) and lowest in HS (149.5 μg/g db), while myricetin content varied from 35.0 μg/g db in H80 to 40.4 μg/g db in HF. Kaempferol was not detected in any of the samples. These significant differences in flavonoid content among the samples suggested that drying temperature and method greatly influenced the maintenance of these compounds. The high total flavonoid compounds in H60 indicated that moderate drying temperatures were optimal for preserving flavonoids by creating a balance between sufficient heat to enhance extraction and moderate enough to prevent degradation. The high rutin content across all the samples supports its health benefits, given its well established vascular and anti-inflammatory properties, while elevated apigenin levels in HF underscored the importance of careful drying to preserve compounds with anti-inflammatory and anti-cancer potential. These results aligned with previous studies, which have shown that certain flavonoids were more stable at specific drying temperatures, thereby enhancing their antioxidant properties. The higher retention of quercetin, apigenin, and myricetin in HF compared to other samples suggests that specific drying conditions optimize the preservation of these bioactive compounds.

Additionally, the analysis of flavonoid compounds in C. piriformis after drying at 80 °C, compared with fresh samples, demonstrated that most constituents were relatively unaffected by thermal treatment. Remarkably, rutin, quercetin, and myricetin exhibited high thermal stability, maintaining concentrations comparable to those in fresh samples and were thus classified as stable. In contrast, apigenin showed a moderate decline in content nevertheless remained present at appreciable levels, indicating intermediate thermal resistance. These results underscore the capacity of certain flavonoids to preserve their structural integrity and bioactivity under elevated drying temperatures, which is essential for the formulation of functional foods and heat-processed phytochemical-based products. Therefore, optimizing drying parameters is essential for maintaining the nutritional and therapeutic values of the samples. Further research should investigate the specific mechanisms by which drying temperature affects flavonoid stability and retention, with the goal of maximizing the health benefits of dried products.

The drying conditions significantly influenced the phenolic and flavonoid compound profiles of C. piriformis, as shown in Fig. 5. The heatmap (Fig. 5A) demonstrated significant variations in key compounds, with fresh samples (HF) showing the highest concentrations of rutin, quercetin, and chlorogenic acid. In addition, drying at 80 °C (H80) was more effective in preserving these compounds compared to other drying conditions. Sun drying (HS) and drying at 40 °C (H40) led to significant degradation of phenolic compounds, probably due to prolonged exposure to oxidation and enzymatic activity. The PCA scores plot (Fig. 5C) revealed distinct samples clustering according to drying treatments, with H80 samples closely clustering with HF, suggesting minimal compositional alterations. VIP scores (Fig. 5B) indicated that rutin, apigenin, and vanillic acid significantly contributed to the variation among samples, with their increased retention in H80 suggesting that high-temperature drying effectively preserves phenolic content. The hierarchical cluster analysis (HCA) dendrogram (Fig. 5D) validated these findings, indicating that HF and H80 formed a closely related cluster, whereas H40, H60, and HS formed separate clusters as a result of increased phenolic degradation. The results are consistent with previous studies demonstrating that high-temperature drying effectively inactivates degradative enzymes, thereby preserving phenolic and flavonoid compounds in plant materials (Zhang et al., 2018). The study highlights the significance of selecting appropriate drying conditions, with hot air drying at 80 °C being optimal for preserving bioactive compounds in C. piriformis, thereby enhancing its applicability in functional food and nutraceutical industries.

Fig. 5.

Relationship of phenolic and flavonoid compound profiles in C. piriformis under different drying conditions. (A) Heatmap illustrating changes in phenolic and flavonoid compositions across samples. (B) PCA scores plot showing group separation based on phenolic and flavonoid profiles. (C) VIP scores identifying key phenolic and flavonoid compounds contributing to group differences. (D) HCA dendrogram clustering samples by similarity in phenolic and flavonoid profiles. Drying conditions: HF (fresh), H40 (hot air 40 °C), H60 (hot air 60 °C), H80 (hot air 80 °C), HS (sun dried).

3.7. FTIR analysis

The FTIR spectral analysis provided a detailed examination of the molecular composition of C. piriformis samples and the effect of different drying temperatures and conditions. The spectra explained the characteristic absorption peaks corresponding to specific functional groups, whose variations indicated chemical changes within the samples as presented in the above Fig. 6.

Fig. 6.

A: FTIR spectrum; B: Molecular weight distribution of proteins in C. piriformis.; HF: fresh sample; H40: hot air oven dried 40 °C; H60: hot air oven dried 60 °C; H80: hot air oven dried 80 °C; HS: sun dried.

The FTIR spectral analysis provided insights into the molecular structure of C. piriformis, revealing that different drying conditions led to prominent chemical alterations. Variations in peak intensities and shifts associated with hydroxyl, carbonyl, and amide groups indicated thermal degradation of phenolic compounds, flavonoids, amino acids, sugars, and organic acids, reflecting the breakdown of bioactive structures upon heat exposure. In the higher wavenumber region, a broad absorption band at ∼3280 cm⁻¹, attributed to O—H stretching, corresponded to hydroxyl groups in phenolics, carbohydrates, and water (Singh et al., 2014). The decreasing intensity of this band with rising temperature reflected disruption of hydrogen bonding interactions. Similarly, the absorption band at 1634 cm⁻¹, corresponding to C O stretching from amide and phenolic groups, exhibited shifts under high temperature drying (H80), indicating protein denaturation and phenolic degradation.

In the fingerprint region (1400–500 cm⁻¹), characteristic peaks such as 1077 cm⁻¹ (C—O stretching), 995–997 cm⁻¹ (C–O–C bending), 853–859 cm⁻¹ (CH₂ deformation), and 522–573 cm⁻¹ (pyranose ring skeletal vibrations) were observed, suggesting the presence of starch and oligosaccharides. These spectral patterns were consistent with FTIR profiles reported in chestnut species from Italy, China, and Thailand. Particularly, reductions in the peaks at 1001 cm⁻¹ and 925 cm⁻¹ in H80 samples indicated breakdown of glycosidic bonds and α-1,4-linkages, confirming thermal degradation of polysaccharides.

Collectively, the spectral changes confirm that oven drying at 80 °C causes significant structural degradation of bioactive components. These findings agree with the compound profiles determined via LC-MS/MS and HPLC, further supporting the conclusion that drying temperature profoundly influences the chemical integrity and functional food ingredients potential of C. piriformis.

3.8. Antioxidant activity

The antioxidant potential of the samples was investigated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity and the FRAP (ferric reducing antioxidant power) assay, with results expressed in milligrams of Trolox equivalent per gram dry basis (mg TE/g db) for DPPH and milligrams of ferrous sulfate per gram dry basis (mg FeSO4/g db) for FRAP, as shown in Table S4. The DPPH assay results were consistent across the samples, with the highest scavenging activity observed at 17.6 mg TE/g db, indicating a strong capacity to neutralize free radicals. The FRAP assay showed decreased antioxidant power, with the highest value recorded in the fresh sample (HF: 60.4 mg FeSO₄/g db). The declining trend in FRAP values suggested that the samples had varying levels of reductive potential, reflecting differences in the efficiency of reducing the ferric-tripyridyltriazine complex to its ferrous form. The decline observed in both assays indicated differences in sample preparation, storage, or inherent variability in sample antioxidant composition. In particular, the decrease in antioxidant activity at higher drying temperatures was closely associated with the degradation of heat-sensitive phenolic compounds (Miranda et al., 2010), as well as impacting the decrease of some phenolic acids (e.g., gallic acid, ferulic acid) and flavonoids (e.g., quercetin, rutin), which are known to contribute significantly to radical scavenging activity in chestnut (Chumroenphat et al., 2023; Kim et al., 2023). However, the results of both assays demonstrated strong antioxidant properties, emphasizing the importance of employing multiple analytical methods to capture the full spectrum of antioxidant capacity in food samples. Although this study observed on in vitro antioxidant assays (DPPH and FRAP) to evaluate the antioxidant activity of C. piriformis, these methods serve primarily as preliminary indicators. Several studies have established an associated between strong in vitro antioxidant activity and in vivo efficacy. For example, defatted walnut kernel extract that exhibited high DPPH and FRAP values significantly enhanced antioxidant enzyme activities (such as SOD, CAT, and GSH-Px) and reduced lipid peroxidation in mice with in vivo (Zhou et al., 2023), confirming biological proprieties relevance of in vitro measurements. The strong antioxidant activity detected in C. piriformis suggests its potential as a functional ingredient. However, further studies on in vivo and clinical studies are necessary to validate the potential health benefits”.

3.9. Protein molecular weight

The protein extracts from C. piriformis were separated based on molecular weight, as shown in Fig. 6. Utilizing a biotinylated protein ladder as a molecular weight standard, distinct protein bands were observed across the samples, each corresponding to a specific molecular weight. The fresh sample (HF) displayed a prominent protein band near the 66 kDa marker, indicative the presence of a protein species with a molecular mass in this range. The prominent protein band observed at approximately 66 kDa suggests a highly abundant protein, likely representing a structural, metabolic (vicilin-like) protein or albumin protein (Halima et al., 2022). The sun-dried sample (HS) exhibited more diffuse bands, suggesting protein degradation or structural modification due to the drying process. In the samples subjected to oven drying, H40 showed a band corresponding closely with the 40 kDa marker, while H60 exhibited a clear band just above the 66 kDa marker. The H60 sample, treated at the highest temperature, demonstrated a range of protein bands from below 40 kDa to just above the 66 kDa marker. This spread suggested thermal denaturation effects, where proteins unfold or aggregate, altering their mobility during electrophoresis. Additionally, the protein profile analysis shows that proteins disappear at H80, indicating significant denaturation and degradation at this high temperature. High temperatures cause proteins to unfold and break down into smaller, undetectable fragments. In contrast, lower temperatures (HF, HS, H40, H60) preserve protein integrity better. This emphasizes the importance of using moderate drying temperatures to maintain the structural and nutritional quality of C. piriformis nuts, as high-temperature drying compromises protein integrity and nutritional value. The intensity and sharpness of the bands provided additional insights into protein concentration and purity, respectively. Drying processes, especially at elevated temperatures, lead to changes in protein structure that affect their solubility and electrophoretic behavior (Malumba et al., 2008; Ye et al., 2024). The observed differences between the fresh and treated samples underscored the impact of drying conditions on the proteome of C. piriformis, with implications for its nutritional and functional properties.

In the food industry, selecting optimal processing conditions is essential to preserve bioactive compounds and ensure product quality. Different drying temperatures influence the retention or degradation of functional constituents such as amino acids, phenolic compounds, sugars, and organic acids. Therefore, understanding the thermal sensitivity of these compounds enables food manufacturers to design appropriate drying protocols that maintain the phytochemical compounds and functional properties of C. piriformis such as functional food ingredients in health drinks, instant soups, and bakery products. The data from this study can support the selection of appropriate processing methods for raw materials to enhance functional properties in terms of quality and quantity of bioactive compounds of C. piriformis in the food industries.

The application of suitable drying conditions not only helps to retain these health-promoting components but also improves product stability, shelf-life, and market value in the development of functional or value-added food products.

4. Conclusions

This study highlights the significant influence of drying methods and temperatures on the physicochemical, nutritional, and functional properties of C. piriformis. It must be noticed that different drying temperatures had variable effects on different groups of compounds. While drying at 80 °C showed better retention of some sugars and organic acids, drying at 40 °C was found to be more favorable for preserving heat-sensitive bioactive compounds, such as flavonoids, total phenolics, and amino acids, which contribute significantly to antioxidant activity. In addition, H40 was more effective in preserving cellular structure and minimizing structural degradation. Higher temperatures promoted the breakdown of proteins and carbohydrates, leading to reduced levels of bioactive compounds. FTIR and protein molecular weight analyses confirmed molecular alterations associated with elevated drying temperatures. Overall, hot air drying at 40 °C is recommended for optimal preservation of structural integrity, nutritional quality, and antioxidant properties, supporting its potential application as a functional food ingredient. Future studies on proximate composition and fatty acid profiles will provide a more complete nutritional assessment of C. piriformis, especially its lipid content related to oxidation and shelf-life stability.

CRediT authorship contribution statement

Theeraphan Chumroenphat: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Ananya Dechakhamphu: Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Data curation. Surapon Saensouk: Writing – review & editing, Writing – original draft, Resources, Methodology. Sirithon Siriamornpun: Writing – review & editing, Writing – original draft, Project administration, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material

Data availability

Data will be made available on request.