Impact of airborne ultrasound on pore characteristics and cell wall components in shiitake mushrooms during microwave vacuum drying

Abstract

The pore structure of dried products dictates their rehydration, flavor retention, and processing adaptability, thus affecting market potential. This study investigated the impact of ultrasound amplitude on pore structure evolution in shiitake mushrooms during microwave vacuum drying (MVD). Results showed dried shiitake porosity first increased then decreased with rising amplitude: maximum porosity (65.13%) was achieved at 60% amplitude, and the largest average pore size (3041.78 nm) was observed at 70%. Ultrasound modified shiitake cell wall components via cavitation, mechanical vibration, and enzyme activity regulation, ultimately influencing pore structure. At 50%–70% amplitude, ultrasound accelerated cell wall degradation and induced pore expansion; above 70%, excessive energy reduced enzyme activity and caused pore collapse. This study offers insights for regulating the pore structure of dried products and clarifies the mechanism by which ultrasound affects pore structure.

Graphical abstract

Highlights

• •Ultrasound amplitude modulated pore formation in dried mushrooms.
• •Cellulose and chitin exhibited distinct crystallinity shifts during drying.
• •Cell wall-degrading enzymes were reactivated during drying.
• •Thermal stability of dried mushrooms was significantly enhanced.
• •Large-scale pores dominated the dried mushroom structure.

1. Introduction

Shiitake mushrooms are widely favored by consumers due to their rich nutritional content and unique flavor. However, the moisture content of fresh shiitake mushrooms is usually over 80%, which not only compromises the quality stability during storage but also imposes higher demands on storage and preservation technologies (Tian, Zhao, Huang, Zeng, & Zheng, 2016). As an efficient dehydration method, drying reduces the moisture content to inhibit spoilage, extend shelf life, and enhance flavor quality and commercial value.

Presently, hot air drying (HAD) is widely employed for the dehydration of shiitake mushrooms in industrial production. Nonetheless, the drying cycle associated with HAD typically exhibits a relatively prolonged duration. Prolonged heating not only leads to high energy consumption but also easily causes shiitake mushrooms to shrink, lose flavor compounds, and degrade nutrients. Compared with hot air drying (HAD), freeze drying (FD) can effectively retain the nutritional components and original flavor of shiitake mushrooms. However, FD has relatively high equipment costs, which limits its large-scale application in the processing of agricultural products such as shiitake mushrooms. Furthermore, FD barely drives the formation of characteristic flavor compounds in shiitake mushrooms, resulting in a final product with weak aroma (Chen, Xu, Zhang, Duan, Zhu et al., 2025; Yaman et al., 2023). Microwave vacuum drying (MVD) is a relatively novel drying technology that integrates the dual advantages of microwave heating and a vacuum environment (Zhou et al., 2026). It maintains product quality while ensuring drying efficiency and has been widely applied in agricultural product processing (Tu et al., 2025; Zhou et al., 2026).

To address the defects of uneven heating and severe volume shrinkage in MVD, our team previously combined airborne ultrasound (US) technology with MVD to develop airborne ultrasonic-coupled microwave vacuum drying (USMVD) technology, which was further applied to shiitake mushroom drying. Our previous studies showed that US can regulate the dielectric properties of shiitake mushrooms by altering their local water activity; meanwhile, it shortens the drying cycle by increasing the water migration rate, thereby alleviating local overheating and improving heating uniformity (Tu, Cheng, et al., 2025). In addition, another previous study found that USMVD technology can promote the release of characteristic flavor compounds (e.g., 1,2,4-trithiolane and γ-glutamyl peptides) from shiitake mushrooms during stewing, which is specifically reflected in enhanced product aroma and improved umami taste (Tu et al., 2025). This enhancement is related not only to US-promoted flavor formation but also to US-regulated internal pore structure. However, the mechanism by which US affects pore formation in shiitake mushrooms during MVD remains unclear.

Therefore, this study employed mercury intrusion porosimetry (MIP), Brunauer-Emmett-Teller (BET) specific surface area analysis, and scanning electron microscopic observation (SEM) to perform multi-dimensional characterization of the pore structure of shiitake mushrooms. By analyzing the dynamic changes in cell wall-degrading enzyme activity during drying and comparing differences in the content and structural characteristics of cellulose and chitin, this study systematically clarifies the action law and mechanism of US on the cell wall components of shiitake mushrooms treated with MVD.

2. Materials and methods

2.1. Materials

Fresh shiitake mushrooms were procured from a Yonghui Supermarket in Fujian Province, China. Damaged or immature mushrooms were excluded. The samples were refrigerated at 4 °C to equilibrate moisture distribution. The initial moisture content was 81.50 ± 0.25% (wet basis).

2.2. Drying treatment

Fresh shiitake mushrooms with uniform morphology (cap diameter: 6.0 ± 0.5 cm, thickness: 1.5 ± 0.2 cm) were selected, and their stipes were excised. For each drying experiment, 200 g of the preprocessed fresh shiitake mushrooms was evenly spread on the tray of a microwave dryer (Model KL-2D-2ZG; Kailing Microwave Equipment Co., Ltd., Guangdong, China). During ultrasonic treatment, the distance between the ultrasonic transducer probe and the sample surface was consistently maintained at less than 1 cm to minimize energy attenuation in the reduced-pressure environment. The technical specifications of this equipment were detailed in the study by Lei et al. (2021). Drying was terminated when the moisture content (wet basis, w.b.) of samples dropped below 13%. Specific operations for different drying methods are detailed as follows:

Microwave vacuum drying (MVD): Drying was initiated by activating the microwave generator once the vacuum gauge stabilized at −80 kPa (corresponding to an internal chamber pressure of 20 kPa). The microwave output power was set to 500 W, and the carrier table rotated at 1 rpm throughout the process.

When the vacuum gauge reading reached −80 kPa (chamber pressure 20 kPa), both the microwave generator and the airborne ultrasonic generator were simultaneously activated. Microwave parameters were consistent with MVD (output power: 500 W; carrier table rotation speed: 1 rpm). The ultrasonic frequency was fixed at 20 kHz with maximum power output of 340 W (actual power), while amplitudes were set to 50%, 60%, 70%, 80%, 90%, and 100% (corresponding sample labels: 50M, 60M, 70M, 80M, 90M, 100M).

Based on data obtained from prior research (Tu, Cheng, et al., 2025), MVD required a drying duration of 52 min. For USMVD, the drying times at different amplitudes were: 51 min (50%), 49 min (60%), 47 min (70%), 45 min (80%), 43.5 min (90%), and 42 min (100%).

2.3. Mercury intrusion porosimetry (MIP) experiments

The pore characterization of shiitake mushrooms was performed following the method described by Qiu et al. (2022). Measurements of USMVD samples at different ultrasonic amplitudes were carried out using a mercury injection instrument (Micromeritics AutoPore IV 9500, Micromeritics Instrument Co., Ltd., Shanghai, China), with the MVD sample serving as the control. The experiments were carried out on dried shiitake mushroom caps with dimensions of 50 mm × 50 mm × 50 mm. The pressure applied to the samples ranged 0–50MPa. The pressure ranged from 0 to 50MPa. Assuming ideal pores, the relationship between intrusion pressure (P) and pore diameter (d) was determined using the Laplace equation:

$$P=\frac{4\mathit{\gamma cos}\theta }{d}$$

where γ represents the surface tension of mercury (0.48 N/m) and θ denotes the contact angle (140°) for incomplete wetting between the pore surface and mercury. The pore size distribution was expressed as the incremental intrusion volume as a function of pore diameter.

2.4. Nitrogen adsorption (N₂A) experiments

N₂A experiments were performed using an AMI-TOP 200 instrument (Jingwei Gaobo Instrument Co., Ltd., Beijing, China) to obtain information on specific surface area, pore volume, and pore size distribution. The samples were first degassed at 70 °C for 12 h, and the specific surface area and pore volume were then determined at −196 °C. Pore volume was calculated using the BJH method based on the adsorption and desorption isotherms.

2.5. Scanning electron microscope (SEM)

The morphology of the truncated surfaces of shiitake mushrooms was observed using a field emission scanning electron microscope (Hitachi Regulus 8230, Tokyo, Japan). Dried shiitake mushrooms were brittle-fractured in liquid nitrogen, mounted on a sample stage, and coated with gold.

2.6. Cell wall-associated enzyme activities

Crushed shiitake mushrooms were suspended in 10 mL of 0.1 mol/L phosphate buffer (pH 7.0) and mixed in an ice bath. The mixture was then centrifuged at 12,000 rpm for 20 min at 4 °C, and the resulting supernatant was collected as the crude enzyme solution.

2.6.1. Cellulase activity

The CMC-Na solution (0.5 mL, 1%) was mixed with 0.5 mL of enzyme solution in an EP tube, and the reaction was incubated at 37 °C with oscillation for 1 h. Subsequently, the reaction was heated in a water bath at 90 °C for 15 min, then centrifuged at 8000g for 10 min. The supernatant was collected as the saccharification solution. Cellulase activity was determined by analyzing the supernatant using anthrone colorimetry at 620 nm, and cellulase activity was calculated by comparing the absorbance to a standard glucose curve (Kalita & Sit, 2024).

2.6.2. Chitinase activity

1 mL of colloidal chitin and 1 mL of crude enzyme solution were reacted at 55 °C for 1 h, and the supernatant was taken by centrifugation at 10,000 rpm for 10 min. The enzyme activity of chitin was calculated by the 3,5-dinitrosalicylic acid method using N-acetylglucosamine (NAG) as a standard (Su et al., 2025).

2.6.3. β-1,3 Glucanase activity

1 mL of enzyme solution was mixed with 1 mL of 1% kombucha polysaccharide solution in a colorimetric tube in a water bath at 37 °C for 60 min. A DNS reagent was added to produce a brownish-red compound, and the absorbance was measured at 540 nm. The enzyme activity was calculated by measuring the rate of reducing sugar production (Zhang et al., 2025).

2.7. Determination of cellulose content

The samples with different drying times were lyophilized, crushed, and passed through a 40-mesh sieve. A 0.5 g of the sample was combined with 100 mL of 80% ethanol, then subjected to a 90 °C water bath for 20 min. After cooling to room temperature, the mixture was centrifuged at 8000g for 10 min at 25 °C, and the supernatant was discarded. The residue was washed once with 150 mL of 80% ethanol and acetone, then dried to obtain the crude cell wall. Next, 100 mL of a chloroform:methanol (1:1) mixture was added to the crude cell wall, thoroughly mixed, and incubated at 90 °C for 30 min. The mixture was then cooled and centrifuged at 8000g for 10 min at 25 °C, and the supernatant was discarded. The residue was washed three times with 100 mL of distilled water. The precipitate was mixed with 100 mL of acetone, centrifuged at 8000g for 10 min, and the resulting precipitate was dried for using. A 0.1 g portion of the dried precipitate was combined with 5 mL water and 10 mL of H₂SO₄. The mixture was placed in the ice water bath for 30 min. Then centrifuged at 8000g for 10 min and collected the supernatant. The cellulose content was determined by anthrone colorimetry and calculated by glucose standard curve.

2.8. Determination of chitin content

1.0 g of crude cell wall powder was taken and mixed with 3 mL of 12 mol/L H₂SO₄. The mixture was then extracted in a water bath at 100 °C for 4 h. Subsequently, the solution was left overnight for extraction, and centrifuged at 10,000g for 10 min. 0.5 mL of the supernatant was adjusted to pH 7 with 0.1 mol/L NaOH solution, followed by centrifugation at 10,000g for 10 min. The absorbance value was measured at 530 nm by the color reaction between glucosamine and p-dimethylaminobenzaldehyde, and the content of chitin was calculated based on the standard curve.

2.9. Cellulose extraction

The dried shiitake mushrooms were ground to a powder. A 5.0 g sample of the powder was mixed with 100 mL distilled water, and the pH was adjusted to 7.0 with 0.6 mol/L NaOH. Subsequently, 0.02 g of neutral protease was added, and the mixture was stirred at 50 °C for 5 h to hydrolyze the substrate. The enzyme was inactivated by heating the sample to 90 °C. The sample was then placed in a water bath at 60 °C and extracted with 11 mL of 0.6 mol/L NaOH for 2 h. The resulting solution was centrifuged at 1000g for 15 min. The pellet was bleached with 5% H₂O₂, washed three times with ultrapure water, freeze-dried, and ground for use. (Bellesia, Carullo, Fachin, Caneva, & Farris, 2024).

2.10. Chitin extraction

A 10.0 g sample of shiitake mushroom powder was mixed with a 2 wt% NaOH solution at a material-to-liquid ratio of 1:40 and stirred at 80 °C for 1 day to remove proteins and glucans. Insoluble residue from filtration was re-dispersed in 100 mL of 5 wt% hydrogen peroxide, and heated at 80 °C for 5 h to remove pigments. After washing, 100 mL of 2 mol/L HCl was added to the residue and soaked at room temperature for 1 day. The sample was then treated with 100 mL of 5 wt% NaOH for 2 days to effectively remove residual glucans. The residue was filtered and washed between each treatment step until the pH was neutral. Finally, the sample was freeze-dried to obtain shiitake mushroom chitin, which was then ground for further use (Hamdan et al., 2023).

2.11. Fourier transform infrared (FT-IR)

For FT-IR analysis, 1–2 mg of the powder sample was mixed with 200 mg of KBr, ground uniformly, and pressed into a transparent pellet. The pellet was placed in an infrared spectrometer for analysis, with a wavenumber range set at 4000–400 cm⁻¹, the number of scans at 32, and the resolution at 4 cm⁻¹.

2.12. X-ray diffraction (XRD)

XRD spectra were obtained using a SmartLab X-ray diffractometer. Cu Kα1 radiation was used with a voltage of 40 kV and a current of 40 mA. The 2θ diffraction angle range was 5°–50°. The crystallinity index (CI) of shiitake mushroom cellulose was calculated using the following formula (1):

$$\mathit{CI}=\frac{I_{002}-I_{\mathit{am}}}{I_{002}}\times 100\%$$ (1)

where I₀₀₂ is the diffraction intensity of the (002) crystalline peak and I_am is the diffraction intensity of the peak in the amorphous region at 2θ = 18° (Zhang, Fang, Hu, & Qiu, 2024).

The crystallinity index (CI) of shiitake mushroom chitin was calculated using the following formula (2):

$$\mathit{CI}=\frac{I_{110}-I_{\mathit{am}}}{I_{110}}\times 100\%$$ (2)

where I₁₁₀ is the diffraction intensity of the (1 1 0) crystalline peak and I_am is the diffraction intensity of the peak in the amorphous region at 2θ = 16° (Totani, Tanihata, & Egi, 2025).

2.13. Thermogravimetric analysis

Thermogravimetric (TG) analysis was performed on shiitake mushroom cellulose and chitin using a TG analyzer (TG 8000, Tokyo, Japan) under a nitrogen atmosphere. Approximately 5.0 ± 0.1 mg of the sample was placed in an alumina crucible and heated from 40 to 600 °C at a rate of 20 °C/min under a nitrogen flow of 100 mL/min.

2.14. Statistical analyses

All experiments were performed in triplicate or more, and the results were expressed as the Mean ± SD. One-way ANOVA tests (with a significance level set at P < 0.05) and the subsequent follow-up analysis were carried out using SPSS 27 software. To create the visual illustrations, Origin 2023 software, which is developed by Origin Lab located in Northampton, Massachusetts, in the United States, was utilized.

3. Results and discussion

3.1. Pore structure analysis based on MIP experiments

Porosity, pore size, and pore size distribution are key parameters for characterizing food texture, and they have a significant impact on the mechanical, diffusional, and sensory properties of food. Firstly, mercury intrusion porosimetry was used to characterize the pore characteristics of shiitake mushrooms. As presented in Fig. 1 and Table 1, drying conditions have a significant impact on the pore structure of the samples, which is consistent with the findings reported by Qiu et al. (2022). It can be observed from Fig. 1, the pore size distribution range of shiitake mushrooms in the MVD group is 1000–40,000 nm, with a maximum pore size of approximately 11,000 nm. In contrast to the MVD group, the pore size distribution of those in the USMVD group exhibits a significant difference. Additionally, during the USMVD treatment process, ultrasonic amplitude also exerts a significant influence on the pore size distribution of shiitake mushrooms. Specifically, with the increase in ultrasonic amplitude, the pore size of shiitake mushrooms gradually expands and eventually stabilizes at 10,000–100,000 nm, while the total pore volume exhibits a trend of first increasing and then decreasing. The calculated results of the pore parameters of dried shiitake mushrooms are presented in Table 1. These results indicate that with the increase in ultrasonic amplitude, the porosity, average pore size, total pore volume, and total pore area of dried shiitake mushrooms all first increase and then decrease. This variation is associated with the ultrasonic cavitation effect, which can damage the microstructure to alter the pores, and the amplitude directly determines the intensity of this effect (Umana, Calahorro, Eim, Rossello, & Simal, 2022). Additionally, Ručigaj, Connell, Dular, and Genorio (2022) has also noted that either excessively high or low amplitude will affect the pore structure and material properties, and excessively high amplitude may even excessively damage the pore structure. In this experiment, when the ultrasonic amplitude ranges from 50% to 70%, the pore size and total pore volume of the internal pores in shiitake mushrooms increase simultaneously with the increase in amplitude. While when the amplitude exceeds 70%, excessive ultrasonic energy no longer optimizes the pore structure through the cavitation effect; instead, it triggers pore collapse, ultimately resulting in a decrease in porosity. Nevertheless, compared with the MVD group, ultrasonic treatment still significantly improves the interconnectivity and uniformity of the pore network in shiitake mushrooms.

Fig. 1.

MIP results of MVD shiitake mushrooms under different ultrasound effects.

Table 1.

Pore structure parameters of dried shiitake mushrooms based on mercury intrusion porosimetry.

	Porosity (%)	Average pore size (nm)	Total pore volume (mL/g)	Total hole area (m2/g)
MVD	41.72 ± 1.88e	177.24 ± 6.67f	0.71 ± 0.08f	21.72 ± 0.84e
50M	36.84 ± 1.15f	728.65 ± 25.61e	1.06 ± 0.12e	36.41 ± 0.78b
60M	65.13 ± 2.00a	2150.66 ± 50.29b	2.17 ± 0.09a	36.92 ± 1.05b
70M	51.38 ± 3.62d	3041.78 ± 94.67a	1.98 ± 0.06b	40.61 ± 0.74a
80M	56.63 ± 1.66c	1757.17 ± 51.23d	1.85 ± 0.07c	35.17 ± 1.10b
90M	59.00 ± 1.63b	1952.84 ± 58.78c	1.93 ± 0.07bc	32.46 ± 0.58c
100M	50.75 ± 2.16d	1759.92 ± 41.27d	1.72 ± 0.03d	29.59 ± 0.70d

Note: Each value is expressed as mean ± SD, and different letters indicate significant differences (p < 0.05) between different extractions; MVD represents the microwave vacuum dried shiitake mushroom group, and 50M–100M represent the microwave vacuum dried shiitake mushrooms prepared under the effect of different ultrasonic amplitudes, respectively.

3.2. Pore structure analysis based on N₂A experiments

To investigate the effect of ultrasonic treatment on the pore structure and adsorption capacity of shiitake mushrooms, N₂ adsorption/desorption tests were conducted by a surface and porosity analyzer. From Fig. 2(A), it can be observed that the nitrogen adsorption behavior of dried shiitake mushrooms exhibits three-stage characteristics: in the low relative pressure range (p/p₀ < 0.2), the adsorption capacity increases slowly; in the medium relative pressure range (0.2 < p/p₀ < 0.8), the adsorption capacity increases continuously with the increase of relative pressure; when the relative pressure reaches the high range (p/p₀ > 0.8), the adsorption capacity rises sharply, and all samples exhibit an H3 hysteresis loop during this process. This indicates that the adsorption-desorption isotherms of all samples conform to the typical characteristics of Type II isotherms. At the same p/p₀, the gas adsorption capacity of the MVD samples is significantly higher than that of the USMVD samples. Furthermore, as the ultrasonic amplitude increases from 50% to 100%, the gas adsorption capacity of the samples exhibits a gradual decreasing trend. BET and BJH equations were used to calculate the pore distribution and parameters of shiitake mushrooms, with the results presented in Fig. 2(B, C) and Table 2. Specifically, when the ultrasonic amplitude ranged from 50% to 70%, ultrasound exerted no significant effect on the specific surface area, total pore volume, or micropore volume of shiitake mushrooms; while when the ultrasonic amplitude exceeded 70%, both the specific surface area and total pore volume of the samples exhibited a decreasing trend. Fig. 2(C) presents the mesopore (2–50 nm) size distribution of shiitake mushrooms under different drying conditions. The results show that the mesopore distribution range of shiitake mushrooms is 2–20 nm; although ultrasonic amplitude has no significant effect on such distribution, their mesopore structure is observed to show a decreasing trend with the increase of ultrasonic amplitude. This phenomenon can be attributed to the fact that during the drying process, the mesopores of shiitake mushrooms are affected by both the cavitation effect and thermal effect of ultrasound, leading to further destruction or collapse of mesopores and the subsequent formation of larger pores (Ghamartale, Escrochi, Riazi, & Faghih, 2019). Notably, the mesopore distribution results obtained by the gas adsorption method and mercury intrusion porosimetry are inconsistent. The reason for such differences may lie in that mercury intrusion porosimetry is a destructive test, which requires the application of high pressure to force mercury intrusion into pores during the test process, and this operation ultimately alters the morphology of mesopores.

Fig. 2.

(A) N₂ adsorption-desorption isotherms of dried shiitake mushrooms at −196 °C and pore size distribution profiles for (B) micropores and (C) mesopores.

Table 2.

Pore structure parameters of dried shiitake mushrooms based on BET gas adsorption analysis.

	Specific surface area (m2/g)	Total pore volume (mm3/g)	Average pore size (nm)	Total volume of micropores (mm3/g)
MVD	1.36 ± 0.16abc	5.22 ± 0.29ab	20.18 ± 1.09a	0.49 ± 0.06b
50M	1.43 ± 0.14ab	5.38 ± 0.32a	18.44 ± 1.18b	0.56 ± 0.06a
60M	1.45 ± 0.09a	5.13 ± 0.24ab	17.94 ± 0.41b	0.53 ± 0.04ab
70M	1.39 ± 0.06ab	5.33 ± 0.36a	20.3 ± 0.82a	0.51 ± 0.04ab
80M	1.26 ± 0.11bc	4.54 ± 0.47c	18.9 ± 0.85b	0.46 ± 0.06b
90M	1.32 ± 0.09abc	5.11 ± 0.25ab	19.08 ± 0.74ab	0.51 ± 0.04ab
100M	1.19 ± 0.09c	4.77 ± 0.2bc	18.53 ± 0.89b	0.49 ± 0.03ab

Note: Each value is expressed as mean ± SD, and different letters indicate significant differences (p < 0.05) between different extractions; MVD represents the microwave vacuum dried shiitake mushroom group, and 50M–100M represent the microwave vacuum dried shiitake mushrooms prepared under the effect of different ultrasonic amplitudes, respectively.

3.3. Pore structure analysis based on SEM experiments

To intuitively compare the differences in the pore structure of dried shiitake mushrooms, scanning electron microscopy (SEM) was adopted to observe their microstructures at magnifications of 1000×, 5000×, and 30,000×, respectively. As shown in Fig. 3, at a magnification of 1000×, dried shiitake mushrooms exhibit an obvious pore structure, which is formed by hyphal stacking. The morphology of this pore structure is related to the inherent structural characteristics of shiitake mushrooms and the drying method used (Chen et al., 2025). Experimental results show that the USMVD group has more porous structures than the MVD group, indicating that ultrasound exerts a significant effect on the pore structure of shiitake mushrooms. Furthermore, when the microstructures of shiitake mushroom hyphae were observed at magnifications of 5000× and 30,000×, it was found that the mycelia of the USMVD group exhibited more significant twisting and deformation under the action of ultrasound, with obvious pores formed on the mycelium surface. Such phenomenon becomes more pronounced as the ultrasonic amplitude increases. In terms of drying mechanisms, during the MVD process, moisture evaporates gradually relying on the heat generated by microwaves, causing the shiitake mushroom tissue to shrink progressively and eventually form a relatively uniform and compact structure (Pirnazari, Esehaghbeygi, & Sadeghi, 2014). In contrast, during the USMVD process, the cavitation effect induced by ultrasound damages the cell wall structure, thereby creating additional moisture diffusion channels (Gong, Li, Li, Fan, & Wang, 2023). As the ultrasonic amplitude increases, this structural damage makes the mycelium cell walls looser, further promoting the formation of pores and surface pores.

Fig. 3.

Ultrasonic amplitude-driven microstructural evolution in USMVD-dried shiitake mushrooms: cross-sectional SEM morphology at 1000×, 5000×, and 30,000× magnifications.

3.4. Analysis of cell wall degrading enzyme activities

To elucidate the dynamic changes in cell wall-degrading enzyme activities during the drying of shiitake mushrooms, the activities of β-1,3-glucanase, chitinase, and cellulase were investigated under MVD and USMVD. As shown in Fig. 4(A–C), the enzymatic activities of β-1,3-glucanase, chitinase, and cellulase exhibited a consistent trend of significant reduction during MVD compared to fresh samples, with the lowest activity observed at the end of drying (50 min). This decline can be attributed to two main factors. First, most cell wall-degrading enzymes, including β-1,3-glucanase, chitinase, and cellulase, are heat-sensitive proteins. During drying, elevated temperatures induced structural denaturation of these enzymes, resulting in reduced activity. Second, as drying progressed, the moisture content of shiitake mushrooms decreased gradually. The lack of water caused cell wall-degrading enzymes to lose their catalytic capacity, which further lowered their activity (Perdana, Fox, Schutyser, & Boom, 2012). Unlike the continuous decrease in the activities of β-1,3-glucanase and cellulase throughout the drying process, chitinase exhibited secondary activation. At 20 min into the drying process, chitinase activity increased significantly, likely due to its high optimal temperature. This observation aligns with the findings of Waghmare and Ghosh (2010) who previously isolated a thermophilic chitinase from mushroom beds and reported an optimal temperature of 60 °C.

Fig. 4.

Dynamic changes in wall-degrading enzyme activities (A–C) and structural component contents (D–E) during USMVD drying of shiitake mushrooms with varied ultrasonic amplitudes. Note: Data are expressed as means ± standard deviation (n = 3).

A comparison of enzymatic activity between MVD and USMVD indicated that ultrasound exerted a notable impact on enzyme dynamics. During the early and middle stages of drying (0–30 min), enzymatic activity exhibited a trend of first increasing and then decreasing with rising ultrasonic amplitude at the same drying stage. Specifically, the highest enzymatic activity was observed when the ultrasonic amplitude reached 70%. On one hand, the disruption of cell wall structure induced by ultrasound exposed more substrate molecules and enzymes. This exposure enhanced the binding efficiency between enzymes and substrates and improved degradation capacity (Larsen, van der Weem, Caspers-Weiffenbach, Schieber, & Weber, 2021). On the other hand, ultrasonic waves may induce structural changes in enzymes, which in turn increase the catalytic reaction rates of enzymes. Furthermore, ultrasound energy can alter key physicochemical conditions of the reaction (e.g., temperature and pressure). These conditions are critical for optimizing enzymatic activity. However, excessive ultrasonic amplitude (>70%) can cause significant changes in both intracellular and extracellular environments. These changes lead to the rupture of cell walls and membranes. This phenomenon may be caused by conformational changes in enzyme proteins induced by ultrasound. These changes mask or expose active sites, thereby altering the ability of enzymes to bind substrates (Hou et al., 2020). In the later drying stages, as moisture content continued to decrease, the enzymatic activity of shiitake mushroom cell wall-degrading enzymes in the USMVD group was lower than that observed in the MVD group.

3.5. Analysis of changes in cellulose and chitin content

Cellulose and chitin, as fundamental components of the cell wall skeleton, play a crucial role in the pore evolution of dried shiitake mushrooms (Jiang, Wang, Xu, Jahangir, & Ying, 2010). The variations in cellulose and chitin content in microwave vacuum-dried shiitake mushrooms under different ultrasonic amplitudes are presented in Fig. 4(D) and (E). Results indicated that compared with fresh shiitake mushrooms (232.07 ± 4.15 mg/g), the contents of cellulose and chitin in both MVD and USMVD samples were significantly reduced. During the early and middle stages of drying (0–30 min), the contents of cellulose and chitin in shiitake mushrooms decreased rapidly. This decrease can be attributed to two factors: the degradation by enzymes and the absorption of microwave energy by water molecules. In the later stage of drying (30–50 min), the degradation rate of cellulose and chitin slowed down. This phenomenon is likely due to the reduced moisture content and the inhibitory effect of high temperature on enzyme activity. Notably, at the same drying time, the contents of cellulose and chitin in the USMVD group were lower than those in the MVD group. Additionally, with the increase in ultrasonic amplitude, the contents of cellulose and chitin decreased gradually. This may be because the cavitation and microjet effects induced by ultrasound could intensify the rupture of cell walls, resulting in a looser cell structure. This loose structure increases the contact area between enzymes and substrates, further accelerating the degradation of cellulose and chitin. Although the activity of cell wall-degrading enzymes gradually decreased in the late drying stage (30–50 min), the mechanical vibration generated by ultrasound still played a crucial role in influencing the contents of cellulose and chitin (Liao & Huang, 2022; Ni, Li, & Fan, 2021). It has been shown that while ultrasound- and microwave-induced mechanical vibrations do not directly degrade these polysaccharides, they can alter their morphological characteristics (Guo, Guo, Wang, & Yin, 2016). Such structural modifications may affect the efficiency of acid hydrolysis, leading to a reduction in cellulose and chitin content.

3.6. Fourier transform-infrared (FT-IR) analysis

As a basic index for assessing the structural properties of macromolecules, FT-IR can be applied to reflect the effects of different drying conditions on the structure of shiitake mushroom cell wall components. As shown in Fig. 5(A), the infrared spectrum of shiitake mushroom cellulose exhibited typical peaks of cellulose, including 3300 cm⁻¹ (O—H stretching vibration), 2911 cm⁻¹ (C—H stretching vibration), 1430 cm⁻¹ (CH₂ symmetric bending vibration), 1370 cm⁻¹ (C—H bending vibration), 1157 cm⁻¹ (C—O—C asymmetric stretching vibration of β-1,4 glycosidic bonds), and 1010 cm⁻¹ (C—O stretching vibration) (Li et al., 2022). Additionally, characteristic peaks corresponding to chitin amide I and II bands were observed at 1640 cm⁻¹ and 1550 cm⁻¹, respectively. The results showed that the infrared spectra of shiitake mushroom cellulose before and after drying exhibited peak shifts and intensity attenuation, but no new characteristic peaks emerged, and no existing peaks disappeared. This implied that MVD or USMVD treatment did not induce chemical bond cleavage or the formation of new functional groups. Compared with MVD, the USMVD-treated samples showed no significant changes in the absorption intensities of CH₂ (1430 cm⁻¹) and C—O—C (1157 cm⁻¹), indicating that the mechanical vibrations generated by ultrasound and microwaves did not disrupt cellulose polymerization, which preserved the integrity of cellulose chains. However, USMVD treatment may have altered the planar structure of cellulose, leading to variations in the intensity of certain absorption peaks. Notably, the infrared spectra of USMVD samples differed significantly from those of MVD-treated samples. With increasing ultrasound amplitude, absorption intensities at 3300 cm⁻¹, 2911 cm⁻¹, 1370 cm⁻¹, and 1010 cm⁻¹ increased, yet no new peaks or peak shifts were detected. This phenomenon demonstrated that ultrasonication did not alter the chemical composition of cellulose but disrupted its hydrogen bonding network through cavitation effects and mechanical vibrations (3300 cm⁻¹ and 2911 cm⁻¹ peaks were changed) (Rotaru, Fortuna, Ungureanu, & Olguta, 2024). Additionally, the localized high temperature and pressure generated by ultrasound can modify the structure of the crystalline and amorphous regions of cellulose. This might disrupt the amorphous (non-crystalline) regions of cellulose and facilitate the rearrangement of molecular chains in the crystalline regions, enhancing overall crystallinity (Wu et al., 2023).

Fig. 5.

Ultrasonic amplitude-driven structural response of cellulose (A–C: FTIR, XRD, crystallinity) and chitin (D–F: FTIR, XRD, crystallinity).

Fig. 5(D) shows the structural features of chitin in dried shiitake samples. 3260 cm⁻¹, 2878 cm⁻¹, 1624 cm⁻¹, 1554 cm⁻¹, 1376 cm⁻¹, 1315 cm⁻¹ absorption peaks indicate typical structures of chitin, corresponding to O—H stretching vibration, C—H stretching vibration of amide I band, and C—H stretching vibration of amide II band, respectively (Chen et al., 2021). It can be observed that the chitin of shiitake mushroom exhibited a single peak distribution at 1624.1 cm⁻¹ before and after drying. Consequently, the addition of ultrasound did not lead to significant changes in 1157 cm⁻¹ compared to MVD, which represented the glycosidic bonds in mushroom chitin. However, the cavitation effect of ultrasound may provide a special platform to disrupt the intramolecular and intermolecular hydrogen bonding network of chitin (Lu et al., 2013). Notably, the absorption peaks at 3260 cm⁻¹, 2878 cm⁻¹, and 1553.9 cm⁻¹ gradually increased with higher ultrasonic amplitudes. The disruption of hydrogen bonds by high-intensity ultrasound resulted in a more relaxed chitin structure, thereby exposing additional hydroxyl groups.

3.7. X-ray diffraction (XRD) analysis

Fig. 5(B) presents the XRD spectra of cellulose extracted from microwave vacuum dried shiitake mushrooms subjected to different ultrasonic amplitudes. The spectra reveal three major diffraction peaks at 12.2°, 19.8°, and 21.2°, corresponding to the (1 1 0), (1 1 0), and (0 2 0) crystal planes, respectively. These findings indicated that the cellulose was primarily in the II crystalline conformation (Liu, Fu, Zhang, & Liu, 2022). The experimental results showed significant changes in the XRD patterns of shiitake mushroom cellulose before and after drying. Specifically, the intensity of the diffraction peaks at 12.2° and 26.5° decreased, while the intensity at 21.2° increased. The 12.2° peak was typically associated with amorphous or low-ordered regions (such as the (1 1 0) crystal plane of cellulose II), and its reduction in intensity suggested a decrease in the amorphous content following drying. The enhanced intensity at 21.2° indicated a reduction in the spacing between crystal planes, signifying a tighter packing of molecular chains and increased ordering within the crystalline regions. In comparison to the MVD group, the diffraction peaks at 19.8° and 21.2° in the USMVD group continued to increase with higher ultrasonic amplitudes, while the peaks at 12.2° and 26.5° diminished. Finally, the crystallinity of mushroom cellulose was calculated by peak height method (Fig. 5(C)). The results showed that the crystallinity of the fresh sample was 46.47%, which decreased to 42.74% following MVD treatment, demonstrating that rapid drying reduced cellulose crystallinity. This reduction may be attributed to the rapid evaporation of water during the drying process, which caused displacement and rearrangement of molecular chains, disrupting the original crystalline structure (Montoya-Escobar et al., 2022). In the USMVD group, cellulose crystallinity initially increased and then slightly decreased as the ultrasonic amplitude raising, reaching a maximum value of 52.2% in the 70M group. This suggested that ultrasound may enhance cellulose crystallinity by promoting structural reorganization, which increased its crystalline (Rotaru et al., 2024). Additionally, ultrasound shortened the high-temperature treatment time of mushroom cellulose, preventing the reduction in cellulose crystallinity and crystalline size.

Fig. 5(E) presents the diffraction peak distribution of shiitake mushroom chitin under different drying conditions. The XRD patterns reveal four distinct diffraction peaks at 12.9° (0 2 0), 19.3° (1 1 0), 23.5° (0 1 3), and 26.4° (0 0 4), indicating that the chitin is in the β-type crystalline form (Hajji et al., 2014). Significant structural changes were observed in mushroom chitin following MVD treatment, as evidenced by a reduction in the intensity of all four diffraction peaks. Specifically, the decreased intensity of the 12.9° peak indicated that microwave treatment induced intense vibrations of the chitin's polar groups, disrupting hydrogen bonding and loosening the molecular structure. The reduction in the strong diffraction peaks at 19.3° and 26.4°, which correspond to the periodic arrangement of chitin molecules, implies a loss of structural order and a decrease in the crystalline region. The (0 1 3) crystal plane, associated with the periodic repetition of the molecular helical conformation, also exhibited a decrease in intensity, which may be attributed to the partial degradation of chitin molecules. Further analysis showed that the intensity of all four diffraction peaks in the USMVD samples gradually decreased with increasing ultrasonic amplitude compared to MVD. This suggested that ultrasonic treatment accelerated molecular motion, disrupting the originally parallel-aligned chitin structure and leading to increased structural disorder (Ablouh, Jalal, Rhazi, & Taourirte, 2020). Meanwhile, the thermal effects generated by USMVD likely caused a transient increase in chitinase activity, which hydrolyzed a portion of the glycosidic bonds, further compromising the integrity of the crystalline regions. It can be calculated that the crystallinity of chitin in Fresh group was 45.00% (Fig. 5(F)). MVD treatment destroyed the degree of crystallinity of chitin, with a more pronounced decrease observed as the ultrasonic amplitude increased.

3.8. Thermal gravimetric analysis (TGA)

The thermal degradation behavior of shiitake mushroom cellulose and chitin was analyzed through TG and DTG to evaluate the impact of USMVD on their thermal stability. As shown in Fig. 6(A), all cellulose samples exhibited a two-stage degradation process. The first stage (50–200 °C) was attributed to the evaporation of bound and adsorbed water within cellulose. At 250–360 °C, cellulose backbone underwent pyrolysis, which involved cleavage of glycosidic bonds, hydroxyl oxidation, and release of small-molecule gases such as water vapor, carbon dioxide, and volatile organic compounds (Zhang, Fang, Cheng, Li, & Liu, 2024). Compared to the Fresh group, cellulose from MVD and USMVD groups demonstrated superior thermal stability, with stability increasing incrementally at higher ultrasonic amplitudes. This enhancement could be attributed to the fact that drying induced cellulose to form more crystalline structures, and ultrasound can promote cellulose rearrangement during drying, which collectively increased crystallinity. Higher crystallinity generally correlates with greater thermal stability, as the densely packed crystalline regions restrict molecular motion and reduce thermal degradation susceptibility. However, in USMVD-treated samples, cellulose pyrolysis did not exhibit a strictly linear relationship with ultrasonic amplitude. This deviation may be attributed to the incomplete removal of hemicellulose, lignin, and protein residues during processing (Tang et al., 2024).

Fig. 6.

Impact of ultrasonic amplitude on thermal stability of cellulose and chitin in USMVD-dried shiitake mushrooms. (A, C) Thermogravimetric (TG) mass loss curves; (B, D) Derivative thermogravimetric (DTG) degradation profiles.

As shown in Fig. 6(C), the thermal degradation of chitin proceeded through three main stages. The thermal degradation of chitin proceeded through three main stages. The experimental results indicated that mass loss during this stage decreased following drying treatment, suggesting effective removal of bound water within chitin molecules. The second stage of thermal degradation (180–280 °C) was associated with the deacetylation of chitin. During this process, C—N bond was broken within chitin molecules resulted in the release of volatile compounds such as acetic acid and CO₂, leading to mass loss (Qiao et al., 2015). As shown in Fig. 6(D), weight loss was more rapid and pronounced in the MVD and USMVD groups compared to the Fresh group at this stage, and it first increased and then decreased with the rising of ultrasound amplitude. This trend suggested that ultrasonic treatment disrupted the crystalline regions of chitin, exposing additional acetyl groups and making the material more reactive and susceptible to thermal degradation. At 310–370 °C, the main chitin polymer chain underwent cleavage, began to carbonize and experienced a rapid decline in mass (Machado et al., 2024). The results showed that, compared with fresh samples, chitin dried by MVD and USMVD exhibited higher thermal stability, and the stability was further improved with the increase in ultrasound amplitude. This deviation may be attributed to increased hydrogen bonding within chitin molecules. Ultrasonic treatment disrupted the ordered structure of crystalline regions, leading to molecular rearrangement within amorphous regions. This increase in molecular degrees of freedom facilitated intermolecular hydrogen bonding, thereby delaying structural collapse during thermal degradation. Additionally, the mechanical effects of ultrasound may promote cross-linking among chitin molecular chains, strengthening intermolecular interactions and further enhancing heat resistance, ultimately improving chitin's thermal stability in high-temperature environments.

4. Conclusion

This study characterized the multiscale pore characteristics of shiitake mushrooms treated by USMVD and analyzed the effects of USMVD on the content and structural properties of shiitake cell-wall components. During the USMVD process, the surface of shiitake mushroom mycelia cracked and fragmented. With the increase in ultrasound amplitude, the pore walls of micropores and mesopores in shiitake mushrooms collapsed and fused, forming macropores. Eventually, a large number of megaporous structures in the range of 10,000–100,000 nm were formed. These differences in porous structures are associated with changes in the properties of cell wall components. In the USMVD process, ultrasound significantly affects the activity of cell wall-degrading enzymes, promoting the degradation of cellulose and chitin, thereby reducing their content. Although USMVD did not alter the functional-group structure of cellulose or chitin, however appropriate ultrasound treatment facilitated the local rearrangement of cellulose molecular chains and the formation of more regular microcrystals. This led to an increase in their crystallinity, which in turn affected their thermal stability. This study provides a reference for the directional modulation of the multiscale pore structure in dried food products and offers data support for the study of pore and flavor delivery efficacy.

CRediT authorship contribution statement

Dongkun Tu: Methodology, Investigation, Conceptualization. Hanbing Xiao: Methodology, Investigation. Lifen Li: Methodology, Investigation. Ye Xu: Methodology, Investigation. Lujie Cheng: Methodology, Investigation. Yingting Zhao: Writing – review & editing, Supervision, Funding acquisition. Yong Lin: Writing – review & editing. Qisen Xiang: Writing – review & editing, Supervision. Yuting Tian: Writing – review & editing, Supervision, Funding acquisition.

Ethical guidelines

Ethics approval was not required for this research.

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.

Data availability

Data will be made available on request.