Matsutake (Tricholoma matsutake) S. Ito & S. Imai Singer, one of the rarest and most valuable natural medicinal mushrooms in the world, often hails as the “king of mushrooms”. It is rich in vitamins, proteins, polysaccharides, amino acid and other bioactive compounds (Wang, 2020). It exhibits various health benefits, including enhancing physical strength, boosting the immune system, possessing antioxidative properties, and exhibiting anti-tumor effects (Kim et al., 2009). Moreover, it is a rare and highly sought-after wild edible fungus that cannot be artificially cultivated.

Matsutake mushrooms possess a high in water content, and throughout the distribution process, they continue to exhibit significant physiological and respiratory activities. These processes lead to the degradation of their nutritional components, resulting in aging, browning, flavor changes, waterlogging of tissues, and microbial growth on their surface (Wang, 2013; Yang, 2015). Besides, the decay of matsutake fruitbody caused by microbiota invasion was the major postharvest the quality deterioration. Preservation methods for matsutake primarily focus on postharvest physiological preservation. Currently, widely utilized preservation techniques for edible fungi include chemical, physical, and biological preservation methods (He et al., 2024). For example, it found that ultraviolet light combined with a 3% hydrogen peroxide solution for cleaning edible fungi effectively inhibited mold growth (Guan et al., 2013). Low-temperature (4°C) treatments have been shown to delay aging, browning, and reduce metabolic activities while slowing microbial growth in black boletus (Kong et al., 2025). It reported that pulsed light treatment significantly reduced water loss during storage and delayed browning in Pleurotus ostreatus during the later stages of storage (Wang et al., 2025). Furthermore, a composite coating film consisting of slime peptide and sodium alginate helped preserve the shiitake mushrooms’ quality and prolong their shelf life (Qian et al., 2024). The treatment of Agaricus bisporus with equal amount 1:1 compounds of essential oil and sodium alginate to form a film significantly reduced the respiration rate, weight loss, and polyphenol oxidase (PPO) activity of the mushrooms compared to other treatments, this approach increased mushroom firmness, color, total phenolics content, and antioxidant capacity, thereby significantly improving their quality (Eliezer et al., 2021).

The general formula for isothiocyanates (ITCs) is R-N=C=S, are isomers of thiocyanates belonging to heteronuclear cumulative dienes which are a class of sulfur and nitrogen-containing compounds. Natural ITCs were primarily discovered from cruciferous plants which were produced as secondary metabolites when encountering biological stress, ITCs were generated through direct interaction with enzyme of sinigrinase within cells, which catalyzes the hydrolysis of glucosinolates in vacuoles (Wu et al., 2022). Sulforaphane (SFN), allyl isothiocyanate (AITC), phenyl isothiocyanate (BITC), and phenethyl isothiocyanate (PEITC) are examples of common ITC forms (Chen et al., 2020). ITCs are known to exhibit various biological activities, including tumor inhibition, alleviation of diabetes symptoms, protection of nerve tissue, promotion of bacterial growth, and potent antioxidant effects. These properties make isothiocyanates highly promising for practical applications in the food industry, human health care, and agricultural production (Kiat et al., 2019; Maeda and Murakami, 2024; Wu et al., 2022). For instance, treatment with 50 μLL^-1 IAITC significantly inhibits browning in Flammulina velutipe, preserves their structural integrity, reduces weight loss, and suppresses bacterial growth on the surface (Zhu et al., 2023). Treatment of mulberries with 5 μLL^-1 and 15 μLL^-1 of AITC at 5 °C effectively preserved higher levels of soluble solids and titratable acidity, inhibited respiration, maintained surface color and firmness, and extended the storage life of the fruit (Chen et al., 2015); treating blueberries with 5 μLL^-1 AITC at 10°C effectively suppresses microbial growth and reproduction, thus delaying fruit spoilage (Wang et al., 2009); treatment of blackberries with 2 μLL^-1 of AITC at 4°C and 80% relative humidity effectively preserved anthocyanin content, maintained surface color and physiological quality, and inhibited the development of gray mold (DoSu et al., 2023), and treatment with 5 μLL^-1 of AITC effectively reduces the respiration rate and weight loss of fresh-cut purple cabbage, while maintaining its quality and nutritional value, thereby improving postharvest shelf life (Jiang et al., 2018); AITC treatment extends their shelf life of tomatoes, inhibiting microbial growth, and prolonging the flavor of tomatoes (Diego et al., 2024). Furthermore, BITC effectively inhibits the development of gray mold in strawberries and delays their natural spoilage (Sun et al., 2021). But research on ITCs in postharvest contexts is limited, particularly in relation to matsutake, a high-value fresh food crop. This study expands the understanding of ITCs’ biological mechanisms in postharvest preservation and provides a foundation for further exploration and promotion of matsutake mushroom postharvest management. Matsutake (Tricholoma matsutake), a highly prized edible mushroom, is susceptible to rapid postharvest deterioration due to oxidative stress and microbial activity. Isoamyl isothiocyanate (IAITC), a natural volatile compound, has shown potential in preserving perishable produce by modulating oxidative and antioxidative homeostasis. This study explores the efficacy of IAITC in maintaining the postharvest quality of matsutake by regulating reactive oxygen species (ROS) levels, enhancing antioxidant enzyme activity, and suppressing lipid peroxidation. The findings highlight IAITC as a promising eco-friendly strategy to extend the shelf life and commercial value of matsutake.

Currently, the chemical preservation of mushrooms predominantly involves the use of edible coatings (Liu, 2020). However, these materials degrade slowly and typically require the addition of other compounds to enhance their preservative effects. In contrast, allyl isothiocyanate (IAITC), a type of isothiocyanate (ITC) derived from cruciferous plants, has attracted considerable interest in food science due to its natural origin, biodegradability, relative safety, and antimicrobial properties (Zhu et al., 2023). Matsutake (Tricholoma matsutake) is a highly valued edible mushroom with a short shelf-life due to rapid postharvest deterioration, primarily caused by oxidative stress (Christiana et al., 2025). Isoamyl isothiocyanate (IAITC), a natural volatile compound, has shown efficacy in extending the postharvest quality of matsutake by regulating oxidative and antioxidative homeostasis. There are some keys mechanisms of IAITC action (Afzal et al., 2023); reduction of oxidative stress through IAITC suppresses the accumulation of reactive oxygen species (ROS), such as superoxide anion (O₂-) and hydrogen peroxide (H₂O₂), which are major contributors to cellular damage in postharvest mushrooms. As well as, by inhibiting lipid peroxidation (measured via malondialdehyde, MDA), IAITC helps maintain membrane integrity, preventing cell leakage and senescence. The other was enhancement of antioxidant defense systems through IAITC upregulates the activity of key antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), which scavenge harmful ROS. And it also boosts non-enzymatic antioxidants (e.g., glutathione, GSH), further enhancing cellular resistance to oxidative damage. The last was delayed senescence and quality preservation by balancing oxidative and antioxidative processes, IAITC slows down protein degradation, reduces browning, and maintains firmness, color, and nutritional quality. This modulation helps extend the commercial shelf-life of matsutake while preserving its unique aroma and texture. IAITC treatment offers a promising natural strategy for postharvest preservation of matsutake by maintaining redox balance. Further research could explore its application in other perishable fungi and optimal treatment conditions for industrial use. Since buyers frequently use color as the main criterion to evaluate quality without direct contact, the color of mushrooms is a significant indicator of market value (Lei et al., 2018). The results of our investigation indicated that the matsutake treated with 10 μLL^-1 IAITC exhibited the best performance throughout the storage period, particularly in terms of weight loss, color, and firmness. The possible reason for this could be that IAITC reduced the consumption of organic substances (ASA, soluble protein, free amino acids, total phenolics etc.) in matsutake, thereby maintaining the chitin content and preserving the integrity of the cell membrane, which helped maintain the hardness of the matsutake. Additionally, IAITC inhibited the enzymatic browning (PPO) in matsutake, thereby preserving their color. On the other hand, 30 or 60 µLL^-1 concentrations may have caused damage to the matsutake, leading to the continuous degradation of organic substances to sustain life activities, damage to the cell membranes, and exacerbation of browning in the matsutake.

The soluble protein, a key nutrient and osmotic regulator, is also an essential component of metabolic enzymes and plays a critical role in regulating various physiological activities. Its content level reflects the state of the organism’s tissues (Wang et al., 2017). As we can be seen from the results, the early-stage treatment with IAITC likely inhibited the physiological metabolic activities of the fruiting bodies, leading to reduced nutrient loss and maintaining soluble protein content at a relatively low level. During the middle stage of storage, the quality of the fruiting bodies gradually declined, and the permeability of the cell membrane increased, causing the outflow of soluble proteins from within the cells (Li et al., 2019b). This resulted in a slight increase in soluble protein content during the later stages of storage. Free amino acids are the main source of umami and aroma in matsutake mushrooms, and they are closely related to the unique flavor of matsutake. The changes in free amino acid content during the growth, development, maturation, and aging of matsutake mushrooms are closely associated with physiological and biochemical metabolism. With the increase in storage time, the free amino acids and soluble protein content in all experimental groups increased, especially with a significant increase in soluble protein at 30 or 60 µLL^-1. This could be due to the increased permeability of the cell membrane, which causes the leakage of soluble proteins from within the cells. As the soluble protein content increased, the free amino acids content also increased. The soluble protein in matsutake degrade into free amino acids as storage time progresses, but free amino acids can also be oxidized into quinone-like substances, which leads to browning and changes in the umami and aroma of matsutake (Liu, 2020). The soluble protein and free amino acids content remained stable throughout the storage period at 10 µLL^-1, while free amino acids at 30 or 60 µLL^-1 began to boost from the sixth day, leading to an increase in the products of browning and further exacerbating the browning of matsutake.

Fungal cell walls are mostly composed of chitin, an unbranched polymer that is primarily composed of β-1,4-N-acetylglucosamine. Chitin is essential for preserving the structure of fungal cell walls. The higher the chitin content, the greater the rigidity of the hyphal cell wall (Munro, 2013). With the continued extension of storage time, matsutake mushrooms gradually become softer, and their chitin content shows a decreasing trend. The higher the chitin content, the more intact the cell membrane remains. As observed through scanning electron microscopy, matsutake mushrooms treated with 10 µLL^-1 isothiocyanate retained a relatively good morphology, indicating that this treatment effectively maintained chitin content and preserved the normal morphology of the matsutake. The possible reason for this is that it enhanced the antioxidant activity within the matsutake mushrooms, thereby slowing down the accumulation of MDA, maintaining cell membrane integrity, and preserving the hardness of the matsutake.

The experimental results showed that as the storage period increased, the activities of PPO and POD also increased, and the degree of browning intensified. This is because phenolic compounds are oxidized into quinones under the action of enzymes, and these quinones further polymerize to form brown, yellow, or black polymers, which lead to browning in matsutake tissues (Yang et al., 2009). Browning occurs when PPO and POD oxidize phenolic compounds, leading to the formation of brown substances. PPO and POD work synergistically in the formation of brown polymers (Gao et al., 2017, 2014). The main reason of matsutake browning is the oxidation of phenolic compounds by PPO (Kiat et al., 2019; Li et al., 2019a), and the activity of PPO directly correlates with the intensity of the browning reaction. Our research found matsutake treated with 10 μLL^-1 IAITC inhibited the activity of both PPO and POD, while maintaining the content of total phenolics, thereby reducing the oxidation of phenolics compounds and the production of secondary metabolites. This treatment also stabilized the free amino acids content, reducing the substrates for browning and maintaining the stability of matsutake color. On the other hand, at 30 or 60 µL L^-1, PPO activity increased rapidly during storage. This could be due to oxidative damage to the matsutake, which caused an increase in PPO activity, as well as an increase in free amino acids content and a reduction in total phenolics. These changes led to an increase in the substrates for enzymatic browning, which exacerbated the browning and aging of matsutake mushrooms.

Reactive oxygen species (ROS) are involved in various metabolic processes in plant cells, and they can be toxic, causing oxidative damage and altering cell structure and function (Gill and Tuteja, 2010). Superoxide, hydrogen peroxide, and hydroxyl radicals are examples of ROS that can build up and cause tissue damage in plants, which lowers the quality of fruits and vegetables and lowers their market value (Hodges et al., 2004). SOD and CAT help mitigate the harmful effects of ROS (Yan et al., 2020). A strong and water-soluble antioxidant, ASA can prevent or lessen ROS-induced deterioration in fruits and vegetable (Nasiri et al., 2018). A report by (Sharma et al., 2019). demonstrated that total phenolics compounds possess the potential to scavenge harmful ROS and enhance tissue antioxidant capacity. Damage to the integrity of the cell membrane and the structure of the cell wall is reflected in the accumulation of MDA, a hallmark of membrane lipid peroxidation (Yang et al., 2022). Our study indicated matsutake treated with 10 μLL^-1 IAITC maintained high levels of SOD and CAT activity, likely due to the upregulation of certain genes related to these enzymes by IAITC. By maintaining high SOD and CAT activity, IAITC reduced the ROS levels within the matsutake, thereby preserving the content of ASA and total phenolics. IAITC alleviated the ROS-induced damage to the cell membrane by maintaining antioxidant enzyme activity and antioxidant content, which in turn reduced MDA accumulation and preserved the normal structure of the matsutake cell membrane, the firmness of the matsutake was maintained, which helped delay aging. This finding is consistent with studies on mulberries (Chen et al., 2015), blueberries (Wang et al., 2009), purple cabbages (Jiang et al., 2018) and Flammulina velutipes (Zhu et al., 2023).

Through PCA visualization of the variable evaluation, we found that the weight loss rate, browning, and MDA, PPO, POD, and free amino acids showed positive correlations. Firmness was negatively correlated with MDA, weight loss rate, and browning, which is consistent with the previous discussion. The accumulation of MDA disrupted the integrity and structure of the cell membrane, leading to the softening of matsutake mushrooms, increased weight loss, and a water-soaked state in the tissue. The increase in PPO, POD, and free amino acids enhanced the activity of the enzymes responsible for enzymatic browning and increased the substrates, leading to the browning of matsutake. Nevertheless, firmness showed a positive correlation with antioxidant capacity (SOD, CAT, ASA, total phenolics), which also aligns with the previous discussion. This is because the treatment with 10 µLL^-1 IAITC improved the antioxidant capacity of matsutake, reduced oxidative damage, and maintained the levels of organic compounds such as ASA, total phenolics, and chitin content. This helped preserve cell integrity, reduce weight loss, and inhibit PPO activity, thus delaying the browning and aging process.

To summarize, the treatment with 10 μLL^-1 effectively preserves the original brightness of matsutake, reduces browning, mitigates quality loss, enhances antioxidant enzyme activity, and maintains antioxidant content. No significant differences were observed between the 30 μLL^-1 treatment and the control group. However, the 60 μLL^-1 treatment led to an increase in PPO activity and MDA content, resulting in a dull appearance and deterioration of the samples by the 4th day. These findings imply that excessive application of IAITC accelerates browning and aging processes. The 10 μLL^-1 treatment may exert a preservative effect by activating the self-defense mechanisms of matsutake. This treatment significantly enhanced the activities of resistance-related enzymes, including CAT and SOD, resulting in increased levels of antioxidant substances, such as ASA and total phenolics, thereby improving the antioxidant capacity. However, to further validate these findings, additional research is needed to examine a range of ROS metabolism indicators, including changes in free radicals. The above conclusion indicates that a 10 μLL^-1 treatment can effectively preserve matsutake, maintaining their nutritional components and enhancing antioxidant capacity. However, excessive application of IAITC disrupts the physiological metabolic processes of matsutake, leading to metabolic disorders, accelerating aging, and resulting in negative effects.

Isoamyl isothiocyanate (IAITC) effectively preserves the postharvest quality of matsutake (Tricholoma matsutake) by maintaining oxidative and antioxidative homeostasis. Through the suppression of reactive oxygen species (ROS) accumulation and lipid peroxidation, IAITC mitigates oxidative stress, thereby delaying cellular damage and senescence. Additionally, it enhances the activity of key antioxidant enzymes (SOD, CAT) and non-enzymatic antioxidants (e.g., glutathione), reinforcing the mushroom’s defense mechanisms. As a result, IAITC treatment helps retain the firmness, color, aroma, and nutritional value of matsutake, extending its shelf life. These findings highlight IAITC as a promising natural preservative for postharvest mushrooms, offering potential applications in food storage and commercial preservation technologies. Further studies could optimize treatment conditions and explore its efficacy in other perishable fungi.