Hot Water Treatment Preserves Chinese Chestnut (Castanea mollissima Blume) Quality during Storage by Increasing Its Sugar Accumulation and ROS-Scavenging Ability

Abstract

Heat treatment is a widely used physical technology for postharvest fruit and crops. The Chinese chestnut cultivar “Kuili” has high sugar and amylose contents, and is popular among people. However, the chestnut quality decreases quickly after harvest. In order to maintain the chestnuts’ quality during storage, this study explores five hot water treatments for chestnuts: T1 (control, no treatment), T2 (50 °C), T3 (65 °C), T4 (75 °C), and T5 (90 °C) for 45 min. T1 was dried at ambient temperature, while the other heat treatments were dried at 30 °C for 30 min. After treatment, chestnuts were placed in plastic trays, covered with a 15 μm thick PVC film, and stored at 4 °C with 70% relative humidity; they remained in the same air for 120 days. Results indicated that T3 and T4 showed slight color changes while maintaining shell and kernel firmness, and their weight loss was reduced (+5–8%), as well as their decay rate (limited to within 20%). The T3, T4, and T5 treatments (from days 60 to 120) decreased their pest survival rates to <2%. Additionally, heat treatments facilitated the accumulation of total soluble sugar and increased the expression of sugar biosynthesis-related genes. Meanwhile, T3 and T4 delayed starch reduction (they maintained relatively higher contents, from 288 to ~320 mg g⁻¹ DW) and altered some starch biosynthesis genes. Furthermore, T2, T3, and T4 exhibited higher antioxidant activity and lower hydrogen peroxide (H₂O₂) and superoxide anions (O₂⁻) contents than T1. At the end of storage, the scores of T3 and T4 treatments were 55.1 and 52.3, and they ranked first and second among the five treatments, respectively. Therefore, these findings provide valuable insights for controlling postharvest losses in chestnuts.

1. Introduction

Chestnut (Castanea spp., Fagaceae family) is an economically and ecologically valuable species distributed mainly in Asia, Europe, Africa, and the Americas [1,2,3]. Chinese chestnut (Castanea mollissima Bl.) is an early-domesticated and -cultivated fruit tree widely planted in China, renowned for its high-quality nuts and wood [4,5], and its strong resistance to diseases and pests [6,7]. However, postharvest chestnuts are prone to decay and fungal infections, leading to short consumption periods and commercial losses [8,9]. Traditional chemical fungicides used for preservation do not align with public health needs; thus, the development of effective, cost-efficient, and eco-friendly chestnut preservation methods is urgently required [10,11,12].

Various handling methods have been investigated to maintain the quality and prolong the shelf or storage time of fruits, vegetables, and nuts. These methods include ultraviolet-B (UV-B) radiation, hyperbaric storage (high pressure), hydrothermal treatments (hot water or hot air), and radio frequency treatments [13,14,15,16,17]. To preserve fruit quality and reduce fungicide contamination and pest mortalities, hot water/air alone or these methods combined with other physical methods are widely used for treatment during fruit postharvest storage periods [18,19].

Fungi and pests are major threats that reduce shelf life and cause economic losses [16,20]. The combination of 500 W ultrasound and 55 °C hot water treatments inhibited Rhizopus stolonifer development in sweet potatoes during storage [21]. Hot water dips (40 °C for 20 min) significantly inhibited the growth of Colletotrichum musae, maintaining fruit quality with a better appearance and flavor during cold storage and its shelf life [18]. Similarly, hot water/air (heat) treatments were effective in reducing pest mortalities during fruit storage. For example, pest mortalities decreased ~20–80% when walnut temperatures at the cold spot reached 50 °C after 30, 40, and 55 min of radio frequency heating [20]. Thermal treatments had no impact on the moisture, firmness, and color of chestnuts when compared with the untreated controls [16]. Fresh-cut broccoli treated with hot air (48 °C for 3 h) exhibited higher chlorophyll content without significant differences in weight loss and respiratory rate compared to controls stored at 0 °C [22].

Combining heat (hot air/water) treatment with other technological methods significantly improved food or fruit quality and storage time. The application of specific radio frequency heating at a 5 °C/min rate and a 55 °C transition temperature reduced the crack ratio while maintaining the peroxide value and free fatty acid content [20]. Satsuma orange (Citrus unshiu) treated with 40 °C distilled hot water or hot electrolyzed functional water showed improved fruit quality by enhancing antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) during storage at 2 °C for 60 d [23]. Additionally, a hot air treatment at 34 °C for 24 h on “Rhapsody” tomatoes maintained higher ascorbic acid, isoascorbic acid, and carotenoid contents during storage at 10 °C for 60 d [24].

Utilizing a combined treatment of nitric oxide (NO) and controlled atmosphere (CA, 2–3% O₂ + 3% CO₂) preserved the postharvest quality of chestnuts by increasing the SOD, CAT, and POD activities [25]. When chestnuts were treated with the combination of cold water (15 °C for 5 days) and CA (15.20 kPa CO₂ and 3.04 kPa O₂), the decay rate was reduced and minimal color change was maintained, while an acceptable sensory quality was maintained [10]. The treatment of chestnuts with 90 °C hot water for 10 min effectively reduced their infection level by 95% [11]. Furthermore, chlorination with a sodium hypochlorite solution along with sorbic and propionic acids effectively inhibited the fungal infection on chestnuts postharvest [11]. However, the effects of the combination of hot water treatments and lower temperatures on chestnuts are seldom studied.

This research investigates the potential of hot water treatment for preserving Chinese chestnuts. Chestnuts were subjected to various temperatures (50 °C, 65 °C, 75 °C, and 90 °C) for 45 min, with untreated ones as controls, and stored at 4 °C for 120 d. Physical and chemical changes in the nut quality were assessed. Furthermore, antioxidant activities, such as SOD, CAT, POD, and ascorbate peroxidase (APX), were also measured to evaluate how hot water treatments affect chestnuts during storage periods. Additionally, carbohydrate biosynthesis-related gene expression was also analyzed. Finally, a sensory evaluation of nut quality during storage was conducted for each treatment, which indicated the enhanced effectiveness of hot water treatments in maintaining chestnut quality.

2. Material and Methods

2.1. Chestnuts Sampling

The Chinese chestnut cultivar of “Kuili” is famous for its high sucrose and amylopectin contents, and its relatively bigger nuts (~18 g/fruit). However, chestnuts are prone to decay, which decreases their quality during storage. The chestnut samples were harvested on 15 September, from ten-year-old trees in Lanxi City, Zhejiang Province, China (29°13′ N, 119°27′ E). The sunny weather was maintained for 3 days before and after the chestnut nut harvesting to prevent rot caused by rain, which can impact the humidity levels of the chestnuts. Healthy chestnut fruits were initially selected and transported to the laboratory of the Research Institute of Subtropical Forestry, Chinese Academy of Forestry. In this study, before treatment, the chestnuts were dipped in sterile distilled water for 3 min at a ratio of 1:2 (w/v) to remove the floating fruits, which are regarded as either rotten or dehydrated. After that, these chestnut samples were dried at room temperature at ~26 °C for further treatments.

2.2. Hydrothermal Treatments and Experiment Design

Five treatments were set up in this experiment, with each treatment including 2700 chestnuts, in three repetitions (in total, ~48.6 kg, ~16.2 kg/plastic trays), while untreated fruits served as the control (T1). The treatments involved immersing chestnut fruits in hot water using thermostatic equipment (SH050A, Chongqing, China, Chongqing Haosheng Technology Co., Ltd., http://www.hosontech.com/, accessed on 15 October 2022) at temperatures of 50 °C (T2), 65 °C (T3), 75 °C (T4), and 90 °C (T5) for 45 min (

Table 1. The parameter sets of hot water treatments.

Groups	Water Temperature (°C)	Treat Time (min)	Drying Temperature (°C)	Drying Time (min)
T1	Ambient temperature	No	Ambient temperature	30
T2	50	45	30	30
T3	65	45	30	30
T4	75	45	30	30
T5	90	45	30	30

). Before immersion, the fruits were placed in plastic trays under different temperatures for ~5 min to ensure that they reached the desired temperature. Following the hot water treatments, the chestnut fruits were dried with 30 °C hot air for 30 min and then air-dried under ambient temperature (~26 °C). Subsequently, 1000 fruits were placed in each of the three plastic trays and covered with a 15 μm thick PVC film to prevent water loss. These were subsequently stored at a constant 4 °C and 80–85% relative humidity under normal atmospheric conditions for up to 120 d. A similar number of untreated chestnut fruits were stored under the same conditions and used as the control. Quality parameters of the chestnut fruits were assessed before treatment and after treatment at 0, 30, 60, 90, 120 d of cold storage at 4 °C.

2.3. Color Determination

Chestnut fruits without physical injuries, visual blemishes, or infections were chosen and sorted based on their shape and size uniformity. Each group comprised 100 chestnut fruits (~1.8 kg). To determine the color of both the nut’s shell and peeled kernel, a colorimeter (Chroma meter CR-400, Konica Minolta Sensing, Inc., Tokyo, Japan) was used with a D65 light source, which provided the CIE L*, a*, and b* values [10,17]. Among them, L*, a*, and b* indicate lightness (varying from 0 = black to 100 = white), redness–greenness (positive = red, negative = green), and yellowness–blueness (positive = yellow, negative = blue). For color analysis, a total of 150 nuts were selected to determine the L*, a*, and b* values.

2.4. Texture Determination

Here, 300 chestnut fruits from three trays (~5.4 kg) of each group were selected for the texture determination. Both the fruit’s shell and peeled kernel texture were detected using a Texture Analyzer (GY-4-J, Zhejiang Top Cloud-Agri Technology Co., Ltd., Hangzhou, China, https://zjtop17.en.made-in-china.com/, accessed on 15 October 2022) at room temperature, and the results were shown as kg/cm².

2.5. Weight Loss Determination

Weight loss was reported as a percentage variation in the fruit weight at the beginning of testing [10]. Three hundred chestnut fruits (~5.4 kg) were marked and weighed before and after the treatments and during the storage at 0, 30, 60, 90, and 120 d. Each group had at least 100 nuts for three replicates, and the weight loss was expressed as the percentage relative to the initial weight during storage. The weight loss was calculated as follows [26]:

$$\mathrm{Weight} \mathrm{loss} \mathrm{rate} (\%)=\frac{W1-W0}{W1}\times 100\%$$

(1)

where W₁ is the initial weight of the chestnut fruit (day 0, g), and W₀ is the weight of the chestnuts at different storage times (day 30, 60, 90, and 120, g).

2.6. Decay Rate and Pest Survival Rate

Here, 300 chestnut fruits (~5.4 kg) were used for determining decay and pest survival rates, respectively. Even though we selected the good fruit preliminarily, bad chestnuts still existed. We first opened ~200 fruits to check and determine the decay rate and survival rate, before the hot water treatments. Then, at least 100 nuts were taken for three replicates in each group to calculate the decay and pest survival rates, with the results being expressed in percentages as follows [26]:

$$\mathrm{Decay} \mathrm{rate} (\%)=\frac{A}{B}\times 100\%$$

(2)

where A is the number of decayed nuts after storage at 30-day intervals and B is the initial number of nuts at the start.

$$\mathrm{Pest} \mathrm{survival} \mathrm{rate} (\%)=\frac{P1}{P0}\times 100\%$$

(3)

where P₁ is the number of surviving pests after storage at 30-day intervals and P₀ is the initial number of nuts at the start [27].

2.7. Determination of Total Soluble Sugar and Starch Contents

At the beginning of the treatments and on the 30-day intervals during storage, the total soluble sugars were quantified using the anthronesulfuric acid colorimetry method as described by He et al. (2020) [28]. Briefly, 1.0 g of finely ground chestnuts was mixed with 10 mL of distilled water extraction and placed in a boiling water bath for 20 min. After extraction, the solution was filtered through a 0.22 μm filter. Subsequently, 0.5 mL of the filtered sample was combined with 5 mL of concentrated sulfuric acid and 0.5 mL of anthrone ethyl acetate solution, and 1.5 mL of water was added. The mixture was thoroughly shaken and then boiled in a water bath for 12 min. After cooling to room temperature, the absorbance was measured at 620 nm using a spectrophotometer. Soluble sugar concentrations were determined using a standard curve ranging from 0 to 100 mg·L⁻¹ of sucrose. The results were calculated as follows:

Soluble sugar content (mg/g FW) = [(ΔA + 0.07)/8.55 ∗ V₁]/(W ∗ V₁/V₂) ∗ F = 1.17 ∗ (ΔA + 0.07)/W, where ΔA = detection value − control value, V₁ was the volume of added sample (0.2 mL), V₂ was the volume of extraction (1 mL), F was the dilution ratio (10×), and W was the fresh weight (0.1 g), and the results were shown as mg/g FW. Each treatment group underwent analysis in triplicate to ensure accuracy and reliability.

The starch content was measured according to Cao et al. (2007) [29], with minor modifications. Chestnut tissues weighing 1.0 g were immersed in 5 mL of 80% ethanol, followed by centrifugation at 8000× g at 4 °C for 5 min. This step was repeated thrice, with each wash using 5 mL of 80% ethanol. The pellet was then resuspended in 5 mL of distilled water, shaken for 2 min, and centrifuged at 8000× g for 5 min. The supernatant was collected and incubated in a boiling water bath. A 0.98 mL aliquot was combined with 0.02 mL of iodine solution, and the reaction was allowed to proceed for 10 min. The absorbance was measured at 660 nm, and starch content was determined by interpolation onto the standard curve. Each treatment group was analyzed in triplicate. The results were calculated as follows:

Starch content (mg/g FW) = [(A + 0.0295) ∗ V₁]/5.872/(W ∗ V₁/V₂) = 0.289 × (A + 0.0295)/W, where A was the extraction absorbance value at 620 nm, V₁ was the volume of sample added to the reaction system (0.2 mL), V₂ was the volume of extraction (1.7 mL), V₃ was the volume of sample, and W was the fresh weight (0.1 g), and the results were shown as mg/g FW. Each treatment group was analyzed in triplicate.

2.8. Antioxidant Enzyme and ROS Determination

The SOD, CAT, POD, and ascorbate peroxidase (APX) activities and the H₂O₂ and O₂⁻ contents were determined before the treatments and on days 0, 30, 60, 90, and 120 as per the manufacturer’s instructions and as previously reported using their corresponding detection kits (Suzhou Comin Biotechnology Co., Ltd., Suzhou, China) [30]. The determinations of SOD, CAT, POD, and APX were made using the ultraviolet spectrophotometer methods as follows:

Inhibition percentage (IP, %) = (A − B)/A ∗ 100%, where A was the control value, B was the detection value. SOD activity (U/g FW) = [IP/(1 − IP) ∗ V₁]/(W ∗ V₂/V₃) = 11.4 ∗ IP/(1 − IP)/W, where V₁ was the total volume of reaction liquid (1.026 mL), W was the fresh weight (FW, 0.1 g), V₂ was the volume of samples (0.09 mL), V₃ was the added extraction liquid volume (1 mL), and the results were shown as U/g FW.

CAT activity (μmol/min/g FW) = (ΔA − 0.0013)/0.2 ∗ V₁/(W ∗ V₂/V₃)/T = 4.458 (ΔA − 0.0013)/W, where ΔA = control value − detection value under the wavelength of 405 nm, V₁ was the total volume of reaction liquid (1.335 mL), W was the fresh weight (0.1 g), V₂ was the volume of samples (0.15 mL), V₃ was the added extraction liquid volume (1 mL), and T was the reaction time (10 min), and the results were shown as μ mol/min/g FW.

POD activity (U/g FW) = ΔA ∗ V₁/(500 ∗ V₂/V₃)/0.01/T = 2000 ∗ ΔA/W, where ΔA = the detection value at 1 min − detection value at 2 min under the wavelength of 470 nm, V₁ was the total volume of the reaction liquid (1.026 mL), V₂ was the volume of the samples (0.05 mL), V₃ was the added extraction liquid volume (1 mL), and T was the reaction time (1 min), and the results were shown as U/g FW.

APX activity (AsA, nmol/min/g FW) = ΔA/(ε ∗ d)/V₁ ∗ 109/(W ∗ V₂/V₃)/T = 1786 ∗ ΔA/W, where ΔA = detection value at 10 s − detection value at 130 s under the wavelength of 290 nm, ε: the molar absorption coefficient of AsA at 290 nm is 2.8 ∗ 10³ L/mol/cm, d was the optical path of the colorimetric dish, V₁ was the total volume of the reaction liquid (1 ∗ 10⁻³ L), V₂ was the volume of the samples (0.1 mL), V₃ was the added extraction liquid volume (1 mL), T was the reaction time (2 min), and W was the fresh weight (0.1 g), and the results were shown as nmol/min/g FW.

H₂O₂ content (μmol/10⁴ FW) = [(ΔA − 0.0006)/0.3744 ∗ V₁]/(W ∗ V₁/V₂) = 2.67 ∗ (ΔA − 0.0006)/W, where ΔA = detection value − control value, V₁ was the total volume of the reaction liquid (0.25 mL), V₂ was the added extraction liquid volume (1 mL), and W was the fresh weight (0.1 g), and the results were shown as μmol/10⁴ FW.

O₂⁻ content (nmol/g FW) = (ΔA + 0.0027)/0.0242 ∗ V₁/(V₂/V₃ ∗ W) ∗ 2 = 148.76 × (ΔA + 0.0027)/W, where ΔA = detection value − control value, V₁ was the total volume of the reaction liquid (1 mL), V₂ was the added extraction liquid volume (0.9 mL), V₃ was the added extraction liquid volume (0.5 mL), and W was the fresh weight (0.1 g), and the results were shown as μmol/10⁴ FW.

2.9. Visual Quality Evaluation and Sensory Evaluation during Storage

Chinese chestnut sensory properties include appearance, flavor, color, luster, and firmness. Ten healthy panelists (five men and five women aged 20–30) were trained to evaluate chestnut quality using the indicators in

Table 2. Evaluation criteria used for determination of sensory quality of chestnut fruit during storage.

Score (Points)	Grade (Descriptor)	Description (Detail)
9	Excellent	No mold is found in the shell and peeled kernel surfaces; shelled and peeled kernels have good smell; shell and peeled kernels have original color (not visual changes compared with day 0); shelled and peeled kernel are not decaying; peeled kernels have a sweet and juicy taste with a suitable firmness; the peels of the shell and kernel can be easily removed.
7	Good	No mold is found on the shell and peeled kernel surfaces; shell and peeled kernels have no bad smell; lighter or darker color changes of the shell and peeled kernels are slight but not noticeable; the shelled and peeled kernels are not decaying; the peeled kernel is sweet and juicy with no obvious water loss and is a little soft or hard; the peel of the shell and kernel can be easily removed.
5	Average	Mold is found on the shell or peeled kernel surfaces, with a <15% decay rate; shell and peeled kernels have a foul smell which is <15%; the color of the shell and peeled kernels is noticeably lighter or darker but <15%; the peeled kernel has shrunk because of water loss; the shell and peeled kernels have become abnormally softer or harder; the peels of the shell and kernel are a little hard to remove.
3	Poor	Mold is found on the shell or peeled kernel surfaces, with the decay rate reaching 40%; shelled and peeled kernels have a foul smell, but <40%; the color of shell and peeled kernel has become noticeably lighter or darker and reached 40%; the peeled kernel has noticeably shrunk; the shelled and peeled kernels have become abnormally softer or harder, with the ratio reaching 40%; the peel of the shell and kernel have become hard to remove.
1	Very poor	Mold is found on the shell or peeled kernel surfaces, with the decay rate reaching >80%; the shell and peeled kernel foul smell reaches >80%; the shell and peeled kernel colors have become noticeably lighter or darker and reach 80%; the peeled kernels have noticeably shrunk; the shell and peeled kernel have become abnormally softer or harder, with the ratio reaching 80%; the peel of the shell and kernel have become very hard to remove and have become inedible.
0	Bad	Extensive breakdown of the inner and outside portions of the fruit, and they have become inedible.

. All evaluations were conducted in booths under white illumination and at room temperature. The evaluation criteria used for evaluating the Chinese chestnut sensory qualities are shown in Table 2. A score of <5 is considered unsuitable for consumption.

2.10. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis

Total RNA was extracted from the peeled kernel of chestnut fruits using the Wolact^® Plant RNA Isolation Kit (Vicband, Hong Kong, China). qRT-PCR was performed as described previously [30]. Gene-specific primers were designed using Primer Premier 5 software (Biosoft International, Palo Alto, CA, USA) (Table S1). The elongation factor gene (EF1-α2, GenBank: FK868440) was used as the chestnut internal reference gene to normalize gene expression.

2.11. Statistical Analysis

Data were analyzed based on the one-way analysis of variance (ANOVA) using SPSS 19.0 software (IBM Corp., Armonk, NY, USA) and expressed as the mean ± standard error (SE). Tukey’s multiple-range test was used to compare the differences between the means at a significant level (p < 0.05).

3. Results and Discussion

3.1. Effects of Hot Water Treatments on Nut Quality and Visual Appearance

On day 0, no obvious shell damage was observed before and after the heat treatments. There were no visible molds, insect damage, sprouting, or dehydration in both the shell and kernel after the treatments (Figure 1). However, the shells of heat-treated chestnuts appeared slightly shinier compared to untreated ones.

As the storage time progressed, small black spots appeared on the of the T4 and T5 kernel surfaces on day 30. No visual changes were observed among T2, T3, and T1 during the same period. However, hot water-treated chestnuts, especially T5 kernels, showed some softening on day 30 of storage. Additionally, the T1, T4, and T5 kernels apparently shrunk during the storage period. T1 treatment showed large black spots after day 60, and small black spots continued to grow during the T5 treatment from day 30 to 120 during storage. Mold-infected kernels were observed among T1, T2, and T5 treatments after day 60. Despite T3 and T4 shells being infected by molds, the kernels maintained better optical quality by the end of storage.

Visible molds were detected on the chestnut shells on day 30 across different treatments (Figure 1). However, more infected molds of shells of chestnut were observed on day 60 among different treatments. The kernels had the highest mold contamination in the T1 treatment, followed by T2 and T5, which were severely infected by molds from days 90 to 120 during storage. T3 and T4 kernels showed slight mold infection by the end of storage. Among the shells during this period, T4 and T5 were badly affected by molds, while T1, T2, and T3 showed minimal shell infection.

3.2. Effects of Hot Water Treatments on Color Changes

Our current data indicate no differences in the L* values of the shell, both before and after heat treatments, on day 0 (Figure 2A). On day 60, the T5 treatment showed the highest L* values of the shell, but no distinct differences were observed among other heat treatments when compared to T1. Except for T1 treatments, heat treatments increased the L* values of the shell on day 120. Heat-treated kernels (T4 and T5) exhibited lower L* values than the untreated kernels on day 0, but their L* values increased by the end of storage (Figure 2B). No differences were found in the L* values before treatments or on day 60 during storage.

Regarding the a* values of the shell, there were no visible differences between different heat treatments before day 60 (Figure 2C). However, heat treatments resulted in higher a* values on day 120 compared to the T1 treatment. After heat treatments, the a* values of the kernels were lower than T1, and they remained significantly lower until day 60 (Figure 2D). There were no significant changes in kernel a* values between different treatments at the end of storage. For the b* values of the shell, there were no differences between different treatments after heat treatments (Figure 2E). T2 and T4 treatments maintained relatively higher b* values of the shell on day 60. However, heat treatments showed higher b* values of the shell than T1 on day 120. There were no differences in b* values between different treatments before day 60 during storage, but heat-treated kernels showed higher b* values at the end of storage (Figure 2F).

3.3. Effects of Hot Water Treatments on Weight Loss and Firmness

In the study, firmness differences in shell and kernel between various treatments compared with before treatment were not significant (

Table 3. Firmness of the shell and kernels of Chinese chestnuts before and after their heat treatments. Weight loss detection of the Chinese chestnuts before and after their heat treatments. Nd.—the date of firmness and weight loss were not detected before treatments; No sig.—no significant difference among treatments on day 0. Values represent means ± SD (all experiments were replicated thrice). Different letters indicate significant differences (p < 0.05) within the different lines.

			Before Treatments	After Treatments
				0 d	60 d	120 d
Firmness (kg/cm2)	Shell	T1	10.05 ± 1.2a	10.08 ± 1.18c	8.82 ± 1.84c	9.17 ± 1.44c
		T2	10.13 ± 1.3a	10.48 ± 1.06b	9.853 ± 1.64b	9.45 ± 1.07b
		T3	10.05 ± 1.12a	10.57 ± 1.95b	10.86 ± 1.63a	10.772 ± 1.53a
		T4	10.01 ± 1.9a	10.82 ± 1.08a	9.794 ± 2.08b	11.31 ± 1.14a
		T5	10.19 ± 1.1a	11.15 ± 1.31a	10.34 ± 0.97a	11.23 ± 1.5a
	Kernel	T1	4.95 ± 0.89a	5.14 ± 1.31a	4.3 ± 1.34b	4.1 ± 1.06c
		T2	4.9 ± 1.06a	4.97 ± 1.03b	4.95 ± 0.9ab	4.8 ± 1.13ab
		T3	5.03 ± 1.38a	4.91 ± 0.75b	5.03 ± 0.81a	5.06 ± 1a
		T4	4.99 ± 0.68a	4.86 ± 1.14b	4.97 ± 0.84ab	4.74 ± 1.15b
		T5	5.02 ± 1.77a	4.89 ± 0.89b	4.93 ± 1.09ab	4.86 ± 0.69ab
Weight loss (%)		T1	Nd.	No sig.	10.77 ± 1.75a	15.31 ± 2.21a
		T2	Nd.	No sig.	9.39 ± 2.01b	10.78 ± 1.84b
		T3	Nd.	No sig.	5.82 ± 155d	10.85 ± 2.11b
		T4	Nd.	No sig.	7.55 ± 1.63c	10.46 ± 2.09b
		T5	Nd.	No sig.	3.3 ± 0.94e	8.59 ± 1.31c

T1: no heat treatment, T2: 50 °C for 45 min, T3: 65 °C for 45 min, T4: 75 °C for 45 min, T5: 90 °C for 45 min.

). However, heat treatments resulted in slightly harder shells compared to untreated ones. The heat treatment groups showed higher shell firmness than the T1 and T2 treatments, with T5 having the highest firmness during storage. Conversely, kernel softening was observed in heat treatments compared to T1 on day 0. However, heat treatments increased kernel firmness during storage, with T3 showing the highest firmness on day 120.

Significant differences in weight loss were observed among the different treatments on day 0, but weight loss decreased slightly with rising temperatures on day 60 (Table 3). The weight loss reduction continued until day 120, with heat treatments delaying weight loss compared to the T1 treatment.

3.4. Effects of Hot Water Treatments on the Decay and Pest Survival Rates

Among the heat treatments, T5 exhibited the worst decay condition throughout the storage period, while T2, T3, and T4 showed significantly lower decay rates compared to T1 until day 90 (Figure 3). T3 and T4 demonstrated the best performance, with consistently lower decay rates during storage. The decay rates of T3 and T4 remained below 20% until the end of the storage period, whereas T5 had a decay rate exceeding 40%. The T1 and T2 treatments showed similar decay rates, ~30%, with no significant difference on day 120. Therefore, these results indicate that regardless of the temperature and treatment time, heat treatments extended the shelf life of fruits compared to untreated chestnuts, especially within a relatively short storage time, except for extreme temperatures like T5 (with a high temperature of 95 °C).

Our study demonstrated a decrease in pest survival rate with increasing temperature during hot water treatment (

Table 4. Pest survival rates (%) of Chinese chestnuts for different treatments during storage. Data are mean ± SD (n = 200 nuts for each of the treatments). Values not represented by the same letter are significantly different according to Tukey’s multiple-range test (p < 0.05).

		T1	T2	T3	T4	T5
Before treatments		5.6 ± 1.2a	5.4 ± 1.3a	5.2 ± 2.2a	5.3 ± 1.2a	5.5 ± 1.5a
After treatments	0 day	5.4 ± 1.4a	5.6 ± 1.4a	5.5 ± 1.2a	5.6 ± 1.5a	5.3 ± 1.6a
	30 days	6.6 ± 1.9a	6.3 ± 2.7b	4.3 ± 2.1c	3.5 ± 2.5d	2.4 ± 1.2e
	60 days	5.9 ± 1.4a	5.9 ± 2.5a	3.7 ± 1.3b	2 ± 1.9c	0
	90 days	6 ± 1.7a	6.1 ± 1.3b	3 ± 1.6c	2 ± 1.2d	0
	120 days	5.5 ± 2.1a	5.2 ± 1b	2.7 ± 1.1c	0	0

T1: no heat treatments, T2: 50 °C for 45 min, T3: 65 °C for 45 min, T4: 75 °C for 45 min, T5: 90 °C for 45 min.

). Before treatments, we detected the pest survival rate first, with an average value of 5.4%, and there were no significant differences among them. Among the treatments, T5 exhibited the lowest survival rate throughout storage, reaching 0% from day 60 to 120. T3 and T4 showed significantly lower survival rates than other heat treatments and reached 0% on day 120. T2, with temperatures below 50 °C, showed no significant difference in pest survival rate compared to T1 during storage.

3.5. Hot Water Treatments Increased the Sugar Content and Expression of the Carbohydrate Biosynthesis-Related Structural Genes

After 30 days of storage, the very hot water treatment of T5 resulted in abnormal values for both total soluble sugar (TSS) and total starch contents. The RNA extraction for gene expression analysis proved challenging due to the high temperature of 90 °C. Consequently, the T5 treatment was discontinued, owing to its presenting the highest decay rate during storage time, an undesirable appearance, and an unpleasant odor observed after day 60 of storage. While the other four treatments were continued and recorded during storage, the T5 treatment results are excluded from Figure 4, Figure 5 and Figure 6.

Our results indicated no significant difference in TSS between untreated and heat treatments (only including T2, T3, and T4) on day 0 after treatments (Figure 4A). Heat-treated chestnuts consistently showed higher TSS levels compared to T1 treatment throughout the storage of T2, T3, and T4 treatments.

Previous studies have verified that the total soluble sugar content in chestnuts increases during storage as a result of starch degradation. However, there is limited research on the related genes in chestnuts during the storage period. The gene expression of SS increased slightly, while T3 and T4 exhibited significantly higher gene expression than both T1 and T2 on day 0 (Figure 4B). Subsequently, all heat treatments showed higher gene expression compared to T1, with SS gene expression being consistently higher than T1 during storage time. Notably, there were no significant differences in HT gene expression between different treatments before treatment and on days 0 and 120 after treatments (Figure 4C). During days 30 to 90, heat treatments showed a slightly higher expression of HT than T1. Furthermore, on only days 30 and 60, heat treatments displayed relatively higher expression of UGP than T1, with no differences among T1 and heat treatments in the period starting before treatments and proceeding through day 90 and day 120 after treatments (Figure 4D).

Interestingly, T2, T3, and T4 exhibited higher expression of SPS than T1 during days 30 to 120 (Figure 5E). Similar trends were observed for T2, T3, and T4 for the expression of SUT and SWEET6 (Figure 4F,G). Additionally, for the expression of SWEET11, only T3 and T4 consistently showed higher expression than T1 (Figure 4H). Therefore, these findings indicate that hot water treatment accelerates sugar accumulation and upregulates related gene expression in Chinese chestnuts.

3.6. Hot Water Treatments Delayed the Total Starch Content Reduction and Increased the Expression of Some Starch Biosynthesis-Related Structural Genes

During storage, the total starch content reportedly decreased (Figure 5A). Towards the end of storage, T2 treatment had slightly higher total starch content than T1, with T3 and T4 treatments showing higher total starch content than T1. Heat treatments delayed the decrease in total starch content to some extent.

No significant differences in gene expression of DBE, SSS, SBE, and GBSS were observed among different treatments before treatment and on day 0 after heat treatments (Figure 5B–E). However, gene expression varied among heat treatments. For example, on day 30 after treatment, the DBE expression was higher in all heat treatments compared to T1, while the SSS expression was higher in all heat treatments compared to T1 on days 60 to 120 after treatments. The SBE expression showed no noticeable difference between the heat treatments and T1, except on day 90. Furthermore, only T4 had higher GBSS expression than T1 on days 30, 90, and 120, while other heat treatments showed minor changes compared to T1 during storage.

3.7. Hot Water Treatments Enhanced the Antioxidant Activity

Higher-temperature treatments significantly increased H₂O₂ and O₂⁻ contents compared to T1 treatment (Figure 6A,B). Among them, the T4 treatment had higher contents of H₂O₂ and O₂⁻ on day 120, while the other heat treatments showed slight differences compared to the T1 treatment. On day 0, the SOD, CAT, and POD activities in T3 and T4 were slightly higher than T1 treatment (Figure 6C–E). However, on days 60 and 120 after treatments, the SOD, CAT, and POD activities in T2, T3, and T4 were significantly higher than in T1 treatment. However, APX activity showed no significant differences among T1, T2, T3, and T4, consistent with the other three antioxidant activities (Figure 6F).

3.8. Effects of Hot Water Treatments on Sensorial Attributes

In this study, sensory scores for T2, T3, and T4 treatments gradually declined during storage, with T3 being the highest ranked at the end (

Table 5. Sensory quality scores for chestnut fruit during storage.

No. Treatments	Score during Storage (Day)
	0	30	60	90	120	Total Score	Rank
T1	9	7.7b	5.5b	5b	2.5b	44 ± 3.2b	4
T2	9	8.3ab	7.5a	5.1b	2.7b	45.6 ± 4.5b	3
T3	9	8.5a	7a	6.2a	5.4a	55.1 ± 3.1a	1
T4	9	8.4a	7.7a	5.9a	5.2a	52.3 ± 2.2a	2
T5	9	6.5c	5.7b	4.7c	0c	38.4 ± 3.0c	5

Values in the same column with different letters are significantly different (p < 0.05). T1: no heat treatments, T2: 50 °C for 45 min, T3: 65 °C for 45 min, T4: 75 °C for 45 min, T5: 90 °C for 45 min.

). The most significant decline occurred at day 120 in T5; thus, it was scored the lowest. Although T2 delayed sensory decline compared to T1 before day 60, there was no significant difference from days 90 to 120. Notably, T3 and T4 maintained relatively good color and luster in the shell and kernel throughout storage. T5 treatment at 95 °C had severe adverse effects, resulting in an unpleasant smell and the highest decay rate.

4. Discussion

Broccoli stems treated at 50 °C for 3 min showed delayed changes in their hue and L* values, thereby maintaining higher quality than the untreated ones [31]. Heat treatment also delayed the weight loss of broccoli during storage [31]. This study showed that heat treatment delayed the color changes of the nut’s shell and peeled kernel, as well as the weight loss (Figure 1 and Figure 2 and Table 3).

Hot water dips were found to improve disease resistance against fungi and maintain the fruit quality of organic bananas and yellow pitahaya [18,32]. Similarly, hot-air drying treatment reduced shell cracks in hazelnuts (Corylus avellana L.), with relative humidity identified as a key factor inducing shell cracks during progression [33]. Hydrothermal treatments can quickly inactivate the insect larvae and vegetative cells of spoiling microorganisms [10,11]. These findings suggest that heat treatments are efficient in reducing decay caused by various fungal infections. Our results showed that the decay rate and pest survival rate were decreased with the increased temperature; however, excessive temperature treatments caused a higher decay rate during the lower-temperature storage period, which might be due to the destruction of nut shells (Figure 3 and Table 4).

Heat treatments affected the sugar and starch synthesis and metabolism of crops during storage. In peach fruits, hot air treatment (40 °C and 90% humidity for 4 h) promoted anthocyanin accumulation and reduced the degradation of sucrose content [34]. Similarly, hot air convective drying at 50 °C for 10 h altered sugar contents in different chestnut varieties [19]. SUS and SUS-related genes were more highly correlated to the “calcification” process of chestnuts during storage [35]. In this study, heat treatments (such as T2, T3, and T4) maintained a higher sugar content and several carbohydrate biosynthesis-related structural genes (Figure 4). Superheated steam treatment with 10–30% moisture content prevented starch agglomeration during rapid pasting with hot water, preserving the starch microstructure [36]. Hot air convective drying (50 °C for 10 h) of chestnut slices increased glucose content, suggesting starch thermal hydrolysis [19]. During storage, the increased water loss resulted in the degradation of starch [37]. Our study showed that heat treatments delayed the water loss and starch degradation to some extent (Table 3 and Figure 5).

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂) and superoxide radical (O₂⁻), are typically present at low levels in postharvest horticultural crops under normal physiological conditions. However, these levels can rapidly escalate when fruits or vegetables encounter stressors such as mechanical damage, cold temperatures, or drought [38]. This excessive ROS production can trigger membrane lipid peroxidation, disrupting cell membrane integrity, and causing browning in fruits and vegetables [39]. Recent research indicates that boosting the activities of antioxidant enzymes, such as SOD, CAT, and APX, can inhibit ROS accumulation, thereby mitigating decay and browning in fruit [40]. In the case of chestnuts, CA and CA + NO treatments have been shown to enhance antioxidant activity, including that of SOD, CAT, and phenylalanine ammonia-lyase, thereby reducing ROS accumulation and malondialdehyde content, and preserving chestnut quality [25]. The antioxidant activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2′,2′-azinobis (3-ethyl-benzothiazline-6-sulfonic acid) (ABTS⁺) increased in ginger (Zingiber officinale) dried under 0.9 W/g microwave power and a hot air condition at 60–70 °C [41]. Turmeric (Curcuma longa L.) treated with hot-air microwave rolling blanching maintained higher total phenolic, DPPH, and ABTS contents compared to unblanched dried turmeric [42]. However, some studies showed that hot water treatment did not alter the total soluble phenolics and antioxidant capacity in mango (Mangifera indica L.) during storage [43]. This study demonstrated that an appropriate heat temperature for Chinese chestnuts maintained higher antioxidant enzymes and related genes, while reducing ROS accumulation (Figure 6).

Hot air treatment (40 °C for 4 h) of peach fruits increased aroma volatile emissions (esters and lactones) by upregulating different enzymes (alcohol acyltransferase, fatty acid desaturase acetyl coenzyme A transferase) [44]. Drying with hot air at 50 °C preserved the highest succinic acid content, contributing to the most taste activity value in Lentinula edodes products [45]. Combining CA with NO treatment delayed chestnut decline by enhancing antioxidant activity during storage and preserving flavor [25]. However, research on sensory evaluation in chestnut fruit is relatively scarce. Combined with the appearance and inner quality of nuts, we found hot water treatments could reduce the fungal infection and decay rate, maintaining an acceptable appearance and internal quality (Figure 1, Figure 2 and Figure 3 and Table 5).

Storage conditions, including cold temperature and humidity, significantly influenced the quality and shelf life of hydrothermal and high-pressure-processed chestnuts [17]. Although we developed an easy and effective method for chestnut treatment using hot water, further studies are needed to explore suitable storage conditions (e.g., storage temperature, humidity, optimum time, and packing materials) after hot water treatments for chestnut fruit.

5. Conclusions

Our findings demonstrate that hot water treatment at 50 °C for 45 min (T3) or 65 °C for 45 (T4) min effectively extends the postharvest shelf life and maintains the quality of chestnuts. During storage periods, higher total soluble sugar and starch contents were maintained. Hot water treatments combined with low temperatures, such as T3 and T4, effectively minimized color changes, weight loss, decay rate, and pest survival. Furthermore, these treatments did not negatively impact the physicochemical and sensorial quality of the chestnut fruit. Thus, hot water treatment offers a promising, economically viable, and practical approach for chestnut storage.