Incorporation of mushroom powder: enhancing structure and flavor in pea-mung bean composite protein

Abstract

This study investigated pea-mung bean composites protein (PMX) produced via High-moisture extrusion (HME) with shiitake mushroom powder (XM, 0–30%). XM addition significantly altered the PMX ‘s structure and properties: hardness ranged between 1526 and 1642 g, texturization degree between 1.28 and 1.54, expansion ratio decreased to 0.814, and bulk density increased to 1.58 g/cm³. Water and oil holding capacities peaked (3.27 g/g and 1.88 g/g, respectively) at 20% XM. Molecularly, XM increased disulfide bonds (up to 8.42 μmol/g), modified ionic, and enhanced rheological properties (G′/G″) and thermal stability at 20% XM. FTIR indicated higher ordered secondary structures (56.6%) at this level, while SEM revealed a dense, anisotropic fibrous structure. Flavor improved as XM masked undesirable pea protein off-notes (e.g., 1-octen-3-ol) and increased beneficial aldehydes/alcohols (e.g., benzaldehyde) and flavor-active amino acids (glutamate: 3.21 mg/g). PCA identified 20% XM as optimal for sensory quality, beyond which natural aromas were masked. Overall, 20% XM synergistically optimized fiber structure, cross-linking, hydration, and flavor for good texture.

Introduction

The enhancement of living standards has heightened public health awareness, leading to a steady increase in the demand for alternatives to animal meat for various reasons. In such circumstances, particular emphasis is placed on plant protein foods¹. Simultaneously, the “clean label” movement compels food producers to substitute synthetic additives with natural counterparts², propelling new opportunities within the food industry. Numerous enterprises have consequently increased their investments in the research, development, and production of plant-derived alternatives, unveiling a variety of plant-based products. By utilizing ingredients such as soy and pea protein, processed through high-moisture extrusion technology, they produce products that exhibit fibrous, meat-like textures³. This innovation presents an optimal choice for vegetarians while simultaneously appealing to health-conscious and environmentally aware consumers seeking reduced meat consumption⁴. Combining protein blends with extrusion can enhance the nutritional and functional quality of plant-based products^5–7. Consequently, this investigation centers upon a methodological approach integrating plant proteins with mushrooms to fabricate structured, protein-rich sustenance.

Pea protein, recognized for its high essential amino acid content, has extensive applications in the food industry due to its unique processing properties. Its excellent emulsifying properties⁸, foaming performance, and fluid stability are beneficial for forming an organized fiber structure in high-moisture extrusion. Pea protein also has health benefits such as anti-oxidant, anti-hypertensive⁹, anti-inflammatory, and cholesterol-lowering effects¹⁰. The research results of Chen et al. ¹¹ show that organized protein products prepared with pea protein as the raw material have similar quality to meat products. Mung bean contains over 20% protein, and is rich in vitamins, minerals, and essential amino acids¹¹. It has a wide range of uses, including as a snack, sprout, and bean flour, and health benefits such as detoxification, cholesterol reduction^6,12,13, anti-tumor and anti-inflammatory effects. Its remarkable thermal stability, along with superior water- and oil-binding capacities, makes it highly suitable for thermally processed food applications¹⁴. Studies have demonstrated that pure pea protein exhibits limited extrudability and texturization potential, but these properties can be significantly improved by blending it with other proteins¹⁵. Traditional methods often rely on chemical reagents to extract legume proteins, whereas starch-rich legumes can be directly dispersed in water, allowing proteins to co-solve with starch granules, which can then be separated through fractionation^16,17, achieving a greener and environmentally friendly extraction¹⁸. Most plant-based organized proteins are prepared using soybeans, peas, and other legumes as the main raw materials. During the extrusion process, the high temperature and high pressure inevitably cause the oxidation of unsaturated fatty acids, resulting in the production of a beany flavor that is difficult for consumers to accept. To improve the market acceptance of plant-based organized proteins, it is necessary to modify and mask their flavor¹⁹.

Shiitake mushroom (Lentinula edodes) is the second largest edible mushroom in global production, favored for their unique flavor and rich nutritional components²⁰. Although shiitake mushrooms are rich in dietary fiber, vitamins, and other nutrients and are widely used as seasonings and auxiliaries in food production—particularly in extrusion applications^21,22—their incorporation into extruded protein products to enhance nutritional quality and functionality has been explored, yet the effects on texture and functional properties remain to be elucidated. They are rich in dietary fiber, vitamins, and other nutrients, often used as seasonings and auxiliaries in food production, especially in extruded food applications. In this study, mushroom powder was used as a filler to improve the texture and appearance of pea and mung bean composite textured protein while providing essential nutrients, including protein and dietary fiber.

This study aims to incorporate mushroom powder into composite organized protein products prepared by high-moisture extrusion technology to examine the effects of varying mushroom powder levels on the quality, structure, physical and chemical properties, and flavor of these organized protein products. The findings obtained will reveal their potential as functional ingredients in plant-based food formulations.

Results and discussions

Quality characteristics analysis

Texture represents a critical quality indicator in the pea-mung bean composite (PMX), where fibrous structural development dictates meat-analog functionality. The effect of shiitake mushroom powder (XM) addition on the texture characteristics of textured composite protein (PMX) was demonstrated in Table 1. A significant influence was observed on the hardness, gumminess, and chewiness of PMX by the XM content (p < 0.05). Specifically, an initial increase followed by a decrease was observed in these parameters as the XM addition was elevated. The phenomenon was attributed to the structure stabilization of the squeezed protein matrix by dietary fiber and other components in the XM within the optimal concentration range, which is found to enhance textural properties²³. Dietary fiber forms a denser network structure by regulating the interaction between protein molecules, improving water distribution, and enhancing thermal stability. For example, the β-glucan and other dietary fiber components rich in mushroom powder can interact with protein through hydrogen bonding and hydrophobic action, thus improving the gel strength and water holding capacity²⁴. However, at excessive XM additions, interference with protein molecular interactions was detected, resulting in structural loosening and reduced hardness/gumminess. Excessive XM may introduce excessive starch, disrupting the cross-linking network between protein molecules²⁵. Research has shown that when the addition amount of mushroom powder exceeds 10%, its high water absorption may cause uneven local water distribution, weakening the continuity of the protein matrix²⁶. In the XM group, the texturization degree exhibited a similar trend, attaining its peak value (1.42) with the 20% addition. This optimal performance was linked to effective gap-filling between protein fibers by XM dietary fiber components, which were observed to strengthen the protein network. Concurrently, the expansion ratio was minimized (0.814) and bulk density was maximized (1.58 g/cm³) at this concentration. These outcomes were explained by the compaction of the protein structure through XM-mediated expansion inhibition.

Table 1.

Effects of different addition amounts of XM on the protein structure, texturization, expansion ratio and bulk density of PMX

Sample	Hardness/g	Gumminess/g∙sec	Chewiness	Texturization degree	Expansion ratio	Bulk density/g∙cm3
PMX0	1611.33 ± 9.71b	1449.67 ± 18.23c	1375.33 ± 14.50c	1.54 ± 0.02a	1.063 ± 0.011d	1.07 ± 0.01a
PMX5	1525.67 ± 9.87d	1454.67 ± 13.58c	1385.33 ± 14.84c	1.38 ± 0.01b	1.043 ± 0.025d	1.05 ± 0.04a
PMX10	1578.33 ± 9.71c	1459.33 ± 10.11c	1396.67 ± 14.19bc	1.35 ± 0.02bc	1.013 ± 0.010d	1.08 ± 0.05a
PMX15	1587.67 ± 14.98c	1462.33 ± 12.50c	1415.67 ± 11.93ab	1.28 ± 0.02bc	0.955 ± 0.006c	1.27 ± 0.08c
PMX20	1642.33 ± 17.62a	1565.33 ± 11.93a	1432.33 ± 11.50a	1.42 ± 0.06ab	0.814 ± 0.004a	1.58 ± 0.03a
PMX25	1635.33 ± 13.80a	1547.67 ± 9.50ab	1415.67 ± 12.01ab	1.27 ± 0.03bc	0.831 ± 0.006b	1.42 ± 0.03b
PMX30	1622.33 ± 9.71ab	1543.67 ± 14.57b	1417.33 ± 8.50b	1.23 ± 0.01 d	0.841 ± 0.008bc	1.35 ± 0.09bc

Different letters in the same column indicate a significant difference at p < 0.05.

PMX₀, namely the organized protein with 0% XM added, the digit in the subscript of PMX indicates the amount of XM added, the same as below.

Color analysis

As a vital quality indicator for consumer acceptability, color properties fundamentally influence the marketability of the pea-mung bean composite (PMX). The influence of shiitake mushroom powder (XM) addition on the color properties of flavor composite protein (PMX) was investigated in Table 2. Significant alterations were observed in PMX coloration due to XM incorporation (p < 0.05). Specifically, the lightness (L*) value was significantly reduced following XM addition, which was primarily attributed to the inherent darker pigmentation of XM. Furthermore, the extruded PMX samples were found to exhibit substantially darker coloration compared to the XM-free control (PMX₀), with this darkening effect being linked to non-enzymatic browning reactions occurring during thermal processing.

Table 2.

Effect of different addition amounts of XM on the color of PMX

Sample	L*	a*	b*	ΔE	Simulated color
PMX0	79.12 ± 0.84a	4.52 ± 0.29a	20.16 ± 0.36a	27.55 ± 0.81d
PMX5	77.12 ± 0.41b	4.00 ± 0.31b	18.44 ± 0.40b	27.77 ± 0.56d
PMX10	75.80 ± 0.40b	4.10 ± 0.23b	18.26 ± 0.32bc	28.69 ± 0.54c
PMX15	75.08 ± 0.53 cd	3.76 ± 0.30b	17.92 ± 0.33bc	29.00 ± 0.49bc
PMX20	74.36 ± 0.46 d	3.88 ± 0.23b	17.78 ± 0.41c	29.52 ± 0.57b
PMX25	71.58 ± 1.10e	4.48 ± 0.16a	18.14 ± 0.64bc	32.11 ± 0.67a
PMX30	71.18 ± 0.40e	4.72 ± 0.24a	17.84 ± 0.13c	32.32 ± 0.32a

Different letters in the same column indicate a significant difference at p < 0.05.

PMX₀, namely the organized protein with 0% XM added, the digit in the subscript of PMX indicates the amount of XM added, the same as below.

In extrusion processing, high-temperature treatment can trigger non-enzymatic browning reactions, resulting in deepened colors. Research has shown that increased extrusion temperatures can significantly promote the Maillard reaction and caramelization, especially when the raw materials contain protein and reducing sugar (such as a mixture of protein and starch). For example, Srinivas Jr et al. ²⁷ found that when the extrusion temperature was increased from 190 °C to 232.5 °C, the product color became significantly darker, and the sensory score decreased, which was directly related to the accumulation of Maillard reaction intermediates. Furthermore, Obradović et al. ²⁸ confirmed through mathematical modeling that fiber-rich ingredients (such as carrot powder) accelerate the Maillard reaction during high-temperature extrusion, resulting in a decrease in L* value and an increase in total color difference (ΔE). Yu et al. ²⁹ further pointed out that the addition of whey protein concentrate (WPC) would enhance the darkening effect of the extruded product, which was attributed to the interaction between amino groups in protein and reducing sugars. In these reactions, amino acids present in the protein were reacted with starch-derived reducing sugars through Maillard chemistry, leading to melanoidin formation. The Maillard mechanism was confirmed as a complex amino-carbonyl interaction, resulting in characteristic brown polymers that define non-enzymatic browning. A progressive increase in total color difference (ΔE) was quantified with escalating XM concentrations (p < 0.05), demonstrating that XM addition not only modulated lightness parameters but also exerted global chromatic effects.

Water and oil holding capacity (WHC and OHC)

The water-holding and oil-holding capacities of PMX represent critical functional properties determining product quality and processing performance. The effect of shiitake mushroom powder (XM) addition on the water‑holding capacity (WHC) and oil‑holding capacity (OHC) of PMX is shown in Fig. 1. As can be seen from the figure, with the continuous increase in the addition ratio of XM, the water-holding capacity (WHC) and oil-holding capacity (OHC) of the samples both showed a trend of first increasing and then decreasing. When the addition amount of XM was 20%, the water-holding capacity of the organized protein reached its maximum value of 3.27 g/g, and the oil-holding capacity reached its maximum value of 1.88 g/g. This phenomenon was mainly attributed to the fiber and porous structure of the XM itself, whose pores can absorb and retain water or oil³⁰. However, when the addition amount of XM continued to increase, the starch contained in the XM would destroy the aggregation of protein, affecting the structure of the pressed samples, resulting in structural destruction, and thus affecting their water-holding and oil-holding capacities. Such structural disruption reduces the porosity of the squeezed samples, increases micro-fracture, and thus weakens their water-holding and oil-holding capacities³¹. Additionally, mechanical shear forces during processing may exacerbate the disintegration of fiber structures, further reducing functional properties³². This structural change leads to a decrease in WHC and OHC.

Fig. 1. Effect of different addition amounts of XM on the water and oil holding capacity of PMX.

Values are mean ± SD. Means with different letters are significantly different (p < 0.05).

Determination of sulfhydryl groups (SH) and disulfide bond (DB) content

Sulfhydryl-disulfide bond conversions play a fundamental role in governing the structure and functional stability of PMX. The effect of shiitake mushroom powder (XM) addition on the sulfhydryl and disulfide bonds of flavor compound protein (PMX) is shown in Table 3. As shown in the table, with the increase of XM addition, the content of free sulfhydryl is lower than that of PMX₀, while the total sulfhydryl content increases. In addition, the content of disulfide bonds shows a trend of first increasing and then decreasing with the increase of XM ratio. This may be due to the fact that the cysteine content in the raw materials will affect the interaction between protein molecules during the reaction process, especially the conversion reaction of sulfhydryl-disulfide bonds. Shiitake powder contains a large amount of cysteine, and adding a suitable amount of shiitake powder can make up for the lack of cysteine content, promote the formation of disulfide bonds, and facilitate the production of extruded protein fiber structure. When the addition amount reaches 20%, the disulfide bond content of the composite protein can reach up to 8.42 μmol/g. However, the addition of excessive shiitake powder makes PMX unstable, resulting in a decrease in TSH and DB content, which may be due to the loose structure of PMX. Low-density crosslinked networks are prone to molecular rearrangement during storage, leading to the breakage of disulfide bonds and the release of free thiols³³.

Table 3.

Effect of different addition amounts of XM on the SH, DB and intermolecular forces of PMX

Sample	FSH (μmol/g)	TSH (μmol/g)	DB (μmol/g)	Ionic bond	Hydrogen bond	Hydrophobic interaction
PMX0	27.36 ± 0.67a	36.74 ± 0.65e	4.69 ± 0.67 d	0.032 ± 0.005cd	0.147 ± 0.009b	1.597 ± 0.045a
PMX5	24.33 ± 0.70bc	38.55 ± 0.67cd	7.11 ± 0.58c	0.047 ± 0.013bc	0.142 ± 0.012b	1.308 ± 0.006b
PMX10	23.35 ± 0.92 cd	37.88 ± 0.61de	7.27 ± 0.11bc	0.022 ± 0.003 d	0.136 ± 0.014b	1.256 ± 0.050c
PMX15	24.52 ± 0.46bc	40.98 ± 0.86ab	8.23 ± 0.09ab	0.108 ± 0.012a	0.098 ± 0.003c	1.083 ± 0.003e
PMX20	22.79 ± 0.74 d	39.63 ± 0.95bc	8.42 ± 0.88a	0.064 ± 0.018b	0.181 ± 0.002a	1.201 ± 0.029d
PMX25	25.36 ± 0.32b	41.80 ± 0.22a	8.22 ± 0.08ab	0.017 ± 0.018 d	0.098 ± 0.005c	1.157 ± 0.010d
PMX30	25.20 ± 0.68b	40.67 ± 0.55ab	7.73 ± 0.40abc	0.060 ± 0.009b	0.076 ± 0.008d	1.025 ± 0.012f

Different letters in the same column indicate a significant difference at p < 0.05.

PMX₀, namely the organized protein with 0% XM added, the digit in the subscript of PMX indicates the amount of XM added, the same as below.

FSH free sulfhydryl groups, TSH total sulfhydryl groups, DB disulfide bond.

Intermolecular Forces

Table 3 shows the effect of the amount of XM added on the intermolecular forces within PMX. Quantitative analysis further supports these trends: relative ionic bond (IB) content increased from 0.032 in PMX₀ to a peak of 0.108 at PMX₁₅, then declined to 0.060 at PMX₃₀; hydrogen bond (HB) content decreased from 0.147 (PMX₀) to 0.098 (PMX₁₅) and then rose to 0.181 at PMX20 before dropping to 0.076 at PMX₃₀; hydrophobic interactions (HI) decreased progressively from 1.597 (PMX₀) to 1.083 (PMX₁₅), with a partial rebound to 1.201 at PMX₂₀ and a further decline to 1.025 at PMX₃₀. These patterns are consistent with hydrophilic polysaccharides in XM (e.g., β-glucans) competing with free water for binding sites, which weakens the hydration layer’s shielding of intermolecular interactions in PMX and can enhance protein thermodynamic stability³⁴.

At ≥20% XM (PMX₂₀-PMX₃₀), the interaction profile shifts—IB drops from its peak and HB either increases or declines, while HI partially recovers then decreases—suggesting a more compact protein network, consistent with macroscopic hardness trends and with rheology showing increased (or sustained) storage modulus (G′) and loss modulus (G″).

Rheological measurements

The effect of different amounts of shiitake mushroom powder (XM) on the rheological properties of flavor compound organized protein is shown in Fig. 2. The relationship between the amount of mushroom powder added and the change in rheological properties of organized protein may be closely related to the interaction mechanism between its polysaccharides, fiber, and protein^35,36. The experimental results show that the apparent viscosity increases with the increase of mushroom powder addition, and the storage modulus (G’) and loss modulus (G”) increase significantly when the addition is within 20%. This may be attributed to the following mechanism: the polysaccharides and dietary fiber in the mushroom powder form a stable complex network structure with the organized protein through hydrogen bonds and hydrophobic interactions, increasing the cohesion and molecular cross-linking density of the material³⁷. Meanwhile, adding XM may enhance the exposure of hydrophobic groups, promoting the disulfide cross-linking between protein aggregates, thus improving the viscoelastic response of the system. For example, glycosylation can alter the β-fold and α-helix conformation of mushroom protein, increasing the density of intermolecular cross-linking³⁶. However, when the amount of mushroom powder added exceeds 20%, the modulus decreases, and excessive polysaccharides and fiber form independent discontinuous phases, interfering with the originally stable hydrogen bond or disulfide bond network between protein molecules, resulting in a decrease in the homogeneity of the system. Under high addition amounts, the flowability of the material is limited, and separation occurs during shearing, and some polysaccharide chains form local dense regions due to excessive molecular entanglement, weakening the overall elasticity and structural stability³⁷. The loss factor of the extruded mixture is less than 1, indicating that the elasticity of the extruded mixture is greater than its viscosity. The addition of 20% mushroom powder makes the viscoelasticity of the product system better.

Fig. 2. Effect of different addition amounts of XM on the rheological properties of PMX.

A Viscosity, B storage modulus, C loss modulus, D loss factor.

Differential scanning calorimeter (DSC)

The effect of shiitake mushroom powder (XM) addition on the thermal properties of flavor compound protein (PMX) is shown in Table 4. As can be seen from the table, at a lower addition level, i.e., 5% mushroom powder addition, the peak temperature Tp of PMX decreases, the termination temperature Tc decreases, and the enthalpy value ΔH decreases, indicating that the starch granules in the extruded mushrooms become denatured, and the starch molecules transform from an ordered state to a disordered state, accompanied by energy changes, and the ΔH value decreases, releasing less heat, indicating that the starch granules expand, and the dense structure of the crystallinity decreases, and the heat resistance of PMX is improved. As the addition amount of mushroom powder increases, ΔH shows a trend of first increasing and then decreasing, which may be due to the occurrence of Maillard cross-linking reactions, ultimately resulting in a decrease in enthalpy; Tp shows a similar trend, indicating that the thermal stability of PMX is improved. Similar studies have shown that the addition of 15% mushroom powder to bread increased ΔH from 0.42 J/g to 0.86 J/g, and the gelatinization temperature range expanded to 17.89 °C³⁸, which is consistent with the initial effect of XM on PMX. Additionally, the addition of whey protein also had a similar non-linear effect on the thermal properties of corn starch, indicating that this is a universal phenomenon in multi-component extrusion systems.

Table 4.

Effect of different addition amounts of XM on the thermal characteristics of PMX

Sample	T0/°C	TP/°C	TC/°C	ΔH/J·g−1
PMX0	97.75 ± 0.65b	111.81 ± 1.97ab	129.99 ± 1.20ab	6.15 ± 0.51a
PMX5	97.70 ± 0.59b	108.73 ± 2.94c	126.56 ± 2.64c	3.10 ± 0.63bcd
PMX10	99.23 ± 0.83ab	112.46 ± 3.39ab	129.60 ± 3.71ab	4.00 ± 0.38bcd
PMX15	99.66 ± 0.23ab	114.62 ± 0.34a	131.07 ± 1.63ab	4.41 ± 0.64b
PMX20	100.60 ± 0.22a	116.33 ± 3.38a	136.00 ± 0.78a	4.17 ± 0.82bc
PMX25	94.29 ± 2.51c	107.52 ± 2.19c	124.40 ± 2.24c	2.69 ± 0.23 d
PMX30	99.71 ± 1.29ab	112.70 ± 2.34ab	128.44 ± 5.80c	2.98 ± 0.79 cd

Different letters in the same column indicate a significant difference at p < 0.05.

PMX₀, namely the organized protein with 0% XM added, the digit in the subscript of PMX indicates the amount of XM added, the same as below.

Scanning electron microscopy (SEM) observation

The effect of shiitake mushroom powder (XM) addition on the microstructure of PMX is shown in Fig. 3. Scanning electron microscope (SEM) images clearly reveal the significant change in the surface structure of PMX with the addition of XM. When the content of XM is 0%, the surface of the organized protein is relatively rough, and there are uneven holes. This rough surface structure may be due to the fact that the fiber-like structure formed by the interaction between protein molecules is not dense enough. As the addition of XM increases, these holes are gradually filled with mushroom powder, and the surface of the sample changes from a fish-scale-like layered structure to a flatter state. When the addition of XM reaches 20%, the surface of the sample is the flattest, and the distribution of pores is uniform, indicating that mushroom powder and organized protein have formed a good interaction, enhancing the density and uniformity of the structure. However, when the addition of XM is further increased, the surface of the sample becomes rough again. This may be because too much mushroom powder causes the space between proteins to be over-filled, destroying the original structure, making the surface irregular again. Similar findings have been reported in the study by Webb et al.³⁹. The SEM images further explain the effect of mushroom powder addition on organized protein, which is consistent with the quality characteristics mentioned earlier.

Fig. 3. Effect of different addition amounts of XM on the scanning electron microscopy (SEM) of PMX.

The letters in the figure respectively represent the samples A PMX₀, B PMX₅, C PMX₁₀, D PMX₁₅, E PMX₂₀, F PMX₂₅, G PMX₃₀.

Fourier transform infrared spectroscopy (FTIR)

Figure 4A shows the infrared spectra for different amounts of XM added. The wavenumbers in the range of 3428 cm⁻¹ fall within the broad region of overlapping O–H and N–H stretching vibrations, which are characteristic of altered water binding states and strengthened hydrogen-bonding networks of the protein amide A band during extrusion. The wavenumber 2918 cm⁻¹ corresponds to C–H stretching vibrations in aliphatic amino acid side chains, indicating the exposure of hydrophobic groups due to thermal-mechanical effects. The band at 1658 cm⁻¹ belongs to the amide I band, associated with C=O stretching vibrations in the protein backbone; while still positioned in the characteristic α-helix region, the decreased intensity reveals that extrusion shear forces partially unwound α-helices. The wavenumber 1531 cm⁻¹ corresponds to the amide II band, primarily related to the coupling of N–H bending and C–N stretching vibrations, corroborating the reorganization of the peptide backbone’s hydrogen-bonding environment. The band at 1450 cm⁻¹ is assigned to C–H bending vibrations involving scissoring modes of CH₂/CH₃ groups, with its elevated relative intensity reflecting enhanced hydrophobic interactions. The wavenumber 1400 cm⁻¹ corresponds to C–O stretching vibrations in the side chains of acidic amino acids, suggesting that aspartic/glutamic acid residues may have participated in salt-bridge formation or the Maillard reaction under extrusion temperatures.

Fig. 4. Effect of different addition amounts of XM on the infrared spectra and molecular weight of PMX.

A FTIR spectra, B relative content of protein secondary structure, C molecular weight of PMX. Lower arrows indicate peak wavenumbers. Values are mean ± SD. Means with different letters are significantly different (p < 0.05).

The impact of shiitake mushroom powder (XM) supplementation on the secondary structure of PMX was analyzed (Fig. 4B). Sequential alterations in protein conformation were observed as XM concentration increased. The α-helix. content of PMX was initially elevated, followed by subsequent reduction. For β-turn structures, a gradual decrease was recorded at 5–10% XM incorporation, which was further reduced to 20% addition before demonstrating renewed growth. β-sheet content exhibited an initial increment before declining, while irregular coil structures were first diminished then subsequently enhanced. Correspondingly, the protein’s ordered structure displayed progressive augmentation followed by diminishment with increasing XM levels, whereas disordered structures manifested an inverse trend. At 20% XM loading, the ordered structure content was quantified at 56.58%, representing a 5.62% enhancement compared to PMX₀. These findings suggested that moderate XM incorporation could effectively promote protein structural organization, while excessive addition was found to compromise protein.

Protein electrophoresis analysis of PMX

The effect of different amounts of shiitake mushroom powder (XM) on the electrophoresis of flavor compound protein is shown in Fig. 4C. As shown in the figure, the molecular weight of the protein in the compound protein without mushroom powder is concentrated at 52, 42, 33, 22, 19, and 12 kDa. With a small amount of mushroom powder added (5–10%), the protein electrophoresis shows a band around 80 kDa, which may be due to during the extrusion process, the high temperature and high pressure conditions trigger the Maillard reaction between proteins and reducing polysaccharides in the mushroom powder, resulting in cross-linking between molecules and forming larger molecular weight complexes^40–42.

As the amount of mushroom powder added increases, the color of the low molecular weight bands below 25 kDa becomes lighter, and some small molecular subunits of PMX are destroyed, and eluted. When the amount of XM added exceed 10%, the concentration of polysaccharides and reducing sugars in the extrusion system increased, causing the Maillard reaction rate to accelerate, and some low-molecular-weight subunits (such as small peptide segments of PMX) cross-link or are encapsulated with sugars, resulting in a lighter color of low-molecular-weight bands in electrophoresis⁴³. This phenomenon is related to the decrease in solubility and increase in aggregation tendency of proteins⁴⁴. Meanwhile, high temperatures may disrupt the secondary structure of proteins (such as α-helix and β-fold), leading to subunit dissociation and elution. For example, soy protein forms insoluble aggregates during extrusion due to enhanced hydrophobic interactions⁴⁵.

However, it can be seen that with 15–20% mushroom powder added, the color of the low molecular weight bands is still darker than that without mushroom powder, which indicates that the appropriate addition of mushroom powder has a protective effect on the protein of PMX. This may be because the high water-holding capacity of mushroom powder can reduce the actual temperature of the extrusion system, slowing down the denaturation rate of protein⁴⁵.

Sensory evaluation

The experimental results of the sensory evaluation of PMX with different addition amounts of shiitake mushroom powder are shown in Fig. 5A. The sample with PMX0 addition obtained the lowest total sensory score, especially in terms of flavor, with an unpalatable beany taste that was difficult to mask. With the addition of shiitake mushroom powder, the sensory evaluation of the textured protein was significantly improved. In particular, when the addition amount of shiitake mushroom powder was 20%, the highest scores were achieved in flavor, color, taste, texturization degree, and total score. When the addition amount of shiitake mushroom powder was 25–30%, although the beany odor could be masked more effectively, the aroma of the food itself was also masked.

Fig. 5. Effect of different addition amounts of XM on the sensory evaluation and PCA analysis of PMX.

A sensory evaluation, B PCA analysis diagram of electronic nose.

Electronic nose measurement

The electronic nose analysis results of the effects of shiitake mushroom powder (XM) addition on textured composite protein flavor are shown in Fig. 5B. After the electronic nose measurement of the volatile flavor substances of the samples, principal component analysis (PCA) was performed. The PCA method can effectively highlight the differences in volatile substances between samples. The size of the variance contribution rate directly reflects the representation ability of the principal component to the original multidimensional information. In general, when the cumulative variance contribution rate exceeds 85%, it can prove the reliability of the analysis method. In this study, the first principal component obtained a variance contribution rate of 75.9%, and the second principal component was 16.0%. The sum of the two reached 91.9%, far exceeding the benchmark value of 90%, indicating that the core information of all samples was fully represented. The visualization analysis results clearly show that there are significant differences in the volatile components between the PMX₀ and PMX₂₀ samples, which verifies the improvement effect of mushroom powder addition on the flavor characteristics of organized protein. Notably, the two groups of data points occupy different regions in the principal component analysis map without overlap, which further confirms the applicability of the PCA method in this study.

Identification of volatile flavor compounds measurement

Based on the electronic nose experiment and sensory evaluation results, the identification of volatile flavor substances of PMX₀ and PMX₂₀ showed that the addition of XM had a significant impact on the main volatile compounds of organized protein in Table 5. Among the alcohol compounds, 1-octen-3-ol, is one of the main sources of beany flavor⁴⁶. After adding 20% mushroom powder, the content decreased significantly. This may be because in PMX₀, its concentration has exceeded the threshold for the bean-like odor, making it the main source of the unpleasant odor; whereas 1-octen-3-ol, which is present in the shiitake mushroom powder itself, shows a decrease in concentration after being combined with other ingredients due to the interaction with the matrix. The content of 2-pentylfuran, which has a grassy and leafy flavor⁴⁷, also decreased, and it is also considered one of the main sources of beany flavor. This confirms that mushroom powder has a significant inhibitory effect on beany flavor. In fact, at certain concentrations, different types of compounds together contribute to the overall pea-like odor of the pea protein⁴⁸. Benzaldehyde has a sweet almond flavor, and this structured compound has been shown to contribute to the flavor of mushrooms. Regarding mushroom flavor masking compounds, benzaldehyde, which possesses a sweet almond flavor and has been proven to contribute to mushroom aroma, increased from 257.25 ng/g to 745.04 ng/g. Nonanal, an important flavor component that provides fatty and floral characteristics in plant protein systems, increased by 120% from 482.49 ng/g to 1073.87 ng/g. Furthermore, 1-hexadecanol showed an increase (61.36 to 978.49 ng/g), providing a mellow mushroom aroma and strengthening the flavor foundation⁴⁹. Newly emerged compounds in PMX₂₀ including decanal (439.40 ng/g) and 8-octadecenal (378.97 ng/g), both lipid degradation products of mushrooms, enriched the fatty and citrus notes⁵⁰. Experimental research demonstrates that when the mushroom powder addition reaches 20%, PMX can be significantly improved through a dual mechanism: chemical inhibition of beany odorants and synergistic aroma masking by released mushroom-derived aldehydes and alcohols, directly enhancing the product’s fatty flavor characteristics and overall acceptability.

Table 5.

Content of main volatile flavor compounds and free amino acid of PMX₀ and PMX₂₀

Name	Chemical formula	CAS	Content (ng/g)
			PMX0	PMX20
1-Octen-3-ol	C8H16O	3391-86-4	390.90	138.18
1-Tetradecanol	C14H30O	112-72-1	331.96	577.00
Cedrol	C15H26O	77-53-2	119.45	211.63
1-Hexadecanol	C16H34O	36653-82-4	61.36	978.49
Benzaldehyde	C7H6O	100-52-7	257.25	745.04
Nonanal	C9H18O	124-19-6	482.49	1073.87
Decanal	C10H20O	112-31-2	N.D.	439.40
Undecanal	C11H22O	112-44-7	N.D.	242.02
Tridecanal	C13H26O	10486-19-8	131.66	809.15
8-Octadecenal	C18H34O	56554-94-0	N.D.	378.97
Isovanillin	C8H8O3	621-59-0	3046.69	N.D.
Caryophyllene	C15H24	87-44-5	1612.35	N.D.
3,5-Octadien-2-one	C8H12O	38284-27-4	544.73	572.17
2-Amylfuran	C9H14O	3777-69-3	1227.17	31.15
Amino acid types	PMX0	PMX20	Taste threshold (μg/100 mg)	TAV (PMX20)
Essential amino acid content (μg/100 mg)
Threonine	7.7	20.1	260	<0.1
Valine	10.9	66.3	40	1.66
Tryptophan	7.2	9.9	30	0.33
Isoleucine	4.5	35.1	90	0.39
Leucine	6.4	116.7	190	0.61
Phenylalanine	24.9	164	90	1.82
Histidine	3.8	20.9	20	1.05
Lysine	14.4	126.8	50	2.54
Non-essential amino acid content (μg/100 mg)
Aspartic acid	10.6	31.5	100	0.32
Serine	6.6	92	150	0.61
Glutamic acid	26.2	96.4	30	3.21
Glycine	3.5	18	130	0.14
Alanine	8.4	60.1	60	1.00
Cysteine	61.3	71.7	–	–
Tyrosine	21.1	135.1	90	1.50
Arginine	24	126.4	50	2.53
Proline	7.8	17.3	300	<0.1
Total essential amino acids	79.8	559.8
Total non-essential amino acids	169.5	648.5
Total amino acids	249.3	1208.3

PMX₀, namely the organized protein with 0% XM added, the digit in the subscript of PMX indicates the amount of XM added, the same as below.

N.D Not Detected.

Free amino acid analysis

The free amino acid content of two kinds of composite organized proteins, PMX₀ and PMX₂₀, is shown in Table 5. Taste activity value (TAV) method is used to evaluate the contribution of various nonvolatile flavor substances to the taste of shiitake mushroom⁵¹. With the addition of 20% mushroom powder, the total amino acid content in the organized protein increased significantly, with both essential and non-essential amino acids exhibiting an upward trend. Research has shown that by analyzing the TAV, the contribution degree of individual compounds to the overall flavor of food can be determined. Among the free amino acids, glutamic acid (TAV = 3.21), lysine (TAV = 2.54), and arginine (TAV = 2.53) have the most significant contribution to flavor. Glutamic acid is famous for its umami characteristics and can enhance the perception of saltiness, which is the key reason why the flavor of plant-based organized protein is improved after adding mushroom powder. Lysine, although bitter, is covered by umami and sweet taste and does not have taste activity⁵¹, can increase the rich flavor of food, which is also one of the reasons why mushroom powder can give food a thick and heavy taste. Additionally, arginine, as a basic amino acid, also plays an important role in the formation of food flavor.

In summary, this study shows that adding shiitake mushroom powder (XM) to pea–mung bean protein composites modulates structure, hydration, and flavor after high-moisture extrusion, with 20% XM emerging as the optimal level. At this level, products showed high hardness (up to 1642 g), a favorable texturization degree (1.28–1.54), reduced expansion (0.814), increased bulk density (1.58 g/cm³), and peak water- and oil-holding capacities (3.27 and 1.88 g/g, respectively). Molecular analyses showed increased disulfide bonds (to 8.42 μmol/g) and altered ionic, hydrogen-bonding, and hydrophobic interactions, consistent with enhanced viscoelasticity, a higher proportion of ordered secondary structures (56.6%), and more compact, anisotropic microstructures. Thermal analysis revealed non-linear Tp and ΔH responses, with Tp peaking at 116.33 °C at 20% XM. Flavor improved through suppression of beany markers (e.g., 2-pentylfuran, 1-octen-3-ol) and enrichment of aldehydes/alcohols (e.g., benzaldehyde, nonanal, decanal), as corroborated by electronic-nose PCA (91.9% cumulative variance explained) and higher sensory scores at 20% XM. Collectively, 20% XM offers a practical formulation window to co-engineer fibrous structure, cross-linking density, hydration, and flavor in pea–mung bean high-moisture extrusion systems.

Methods

Preparation of mung bean protein extraction solution

Pea protein (PP) was obtained from Yuwang Ecological Food Co., Ltd (Shandong, China). Peeled mung beans and shiitake mushroom powder (XM) were purchased at a local supermarket (Zhengzhou Dennis Department Store Co., Ltd., Henan, China). Protein, lipid, starch and dietary fiber contents of the XM (dry basis) employed in this study were 24.4 ± 0.1 g/100 g, 0.68 ± 0.12 g/100 g, 27.4 ± 0.2 g/100 g and 36.4 ± 0.6 g/100 g, respectively.

The peeled mung beans were homogenized (A25, Shanghai Ouhe Machinery Equipment Co., Ltd., Shanghai, China) with deionized water at a ratio of 1:15 (w/v), i.e., 1 g mung beans per 15 mL water, and then ultrasonically treated for 20 min using an ultrasonic cleaner with a power of 300 W (SB-1200DT, Ningbo Xinzhi Biotechnology Co., Ltd., Zhejiang, China). After that, the sample was homogenized for 1 min for 5 cycles at a speed of 10,000 rpm. Finally, the sample was centrifuged at 3500 rpm for 20 min to remove the precipitate (starch and other macromolecules) and collect the supernatant, which was then centrifuged again under the same conditions. The supernatant obtained was the mung bean protein extraction solution (MP). The soluble protein content of MP can reach 680 mg/100 g, determined by the bicinchoninic acid (BCA) method.

Extrusion heat treatment

Samples were prepared by incorporating shiitake mushroom powder (XM) into pea protein powder in gradients (0%, 5%, 10%, 15%, 20%, 25%, 30% concentration increments, m/m). The 100 g composite mixture was combined with mung bean protein MP while adjusting the moisture content to target moisture content of 60%. Subsequent extrusion processing was conducted using a twin-screw extruder (Process 11, Thermo Fisher Scientific Inc., Wisconsin, USA), equipped with a screw of 11 mm diameter and a 40:1 L/D ratio, operating at 160 rpm and the barrel was equipped with seven heating zones set to 40, 50, 60, 70, 90, 150, and 150 °C. As the torque sensor was uncalibrated, specific mechanical energy (SME) could not be accurately measured. Future efforts will incorporate online torque measurement for precise SME quantification. To facilitate replication, the complete screw configuration is provided, allowing inference of relative energy inputs. The texturized protein composite (PMX) was collected upon achieving thermal-mechanical equilibrium in the extruder. Fresh samples were subjected to textural quality assessment, while lyophilized counterparts were pulverized through 100-mesh sieve for subsequent structural characterization and protein property analysis.

Textural properties

Following the methods of R. Zhang et al.⁵² with slight modification, the sample was compressed with a P/36R probe at a speed of 1 mm/s, strain of 30%, for 5 s, including hardness, gumminess, chewiness properties, were obtained by analyzing the texture curve, using a texture analyzer (TA-XT Plus, Stable Micro System, UK).

The sample was cut into a shape of ~2 × 2 × 2 cm, and an A/MORS cutter was used for cutting. The triggering force was 5 g, the shearing degree was 60%, the pre-measurement speed was 2 mm/s, the test speed was 1 mm/s, and the post-measurement speed was 2 mm/s. The transverse shear force (FT) measures the resistance of the sample when a force is applied perpendicular to the fiber orientation, and the longitudinal shear force (FL) measures the resistance of the sample when a force is applied parallel to the fiber orientation, and the texturization degree was calculated as the ratio of FL to FT to indicate the alignment and strength of the protein fibers, and the measurement was repeated 10 times.

Expansion ratio and bulk density

Expansion ratio (ER) is expressed as the ratio of extruded product diameter to the die diameter⁵³. The extrudate diameter was measured through digital vernier callipers and an average of 10 randomly measurements was considered as diameter of PMX.

$$\mathit{Expansion}\mathit{Ratio}=\frac{\mathit{Extruded}\mathit{Product}\mathit{Diameter}}{\mathit{Die}\mathit{Diameter}\left(\mathrm{5mm}\right)}$$ 1

Bulk density (BD) was determined by measuring the PMX dimension using a digital vernier caliper and calculated as the ratio of mass to volume of the product, represented in g/cm³⁵⁴.

$$\mathrm{BD}=\frac{4\times \mathrm{m}}{\mathrm{\pi }\times {\mathrm{d}}^2\times \mathrm{L}}$$ 2

Where, L is extrudate length (cm), d is diameter (cm) and m is mass of PMX (g).

Color analysis

The L*, a*, and b* values of the sample surface were measured using a colorimeter (CR-400, Konica Minolta, Tokyo, Japan). Five different locations on the sample surface were randomly selected for measurement and the average value was taken. The color difference value (ΔE) of the extrudate was calculated using Equation (a standard white plate: L* = 98.2, a* = −1.0, and b* = 1.1).

$$\mathrm{\Delta }\mathrm{E}=\sqrt{{\left(\mathrm{\Delta }\mathrm{L}\right)}^2+{\left(\mathrm{\Delta }\mathrm{a}\right)}^2+{\left(\mathrm{\Delta }\mathrm{b}\right)}^2}$$ 3

Water and oil holding capacity (WHC and OHC)

The method of Mazaheri et al.⁵⁵ was used for reference and improved. The freeze-dried sample was crushed and screened with 100-mesh sieves to obtain sample powder. 1.5 g of the sample powder was weighed and placed in a 50 mL centrifuge tube, and then 30 mL of deionized water (soybean oil) was added. After vortexing for 10 min at room temperature, the mixture was centrifuged at 4000 rpm for 20 min, and the supernatant was discarded. The water and oil-holding capacity of plant protein meat was calculated according to the following formula:

$$\mathrm{WHC}\left(\mathrm{OHC}\right)(\mathrm{g}/\mathrm{g})=\frac{m_2-m_1}{m}$$ 4

Where: m was the mass of the sample (g); m₁ was the sum of the mass of the sample and the mass of the centrifugal tube (g); m₂ was the mass of the centrifugal tube after removing the supernatant (g).

Determination of sulfhydryl groups (SH) and disulfide bond (DB) content

The sulfhydryl groups and disulfide bond content were determined as described by Sun et al.⁵⁶, with some modifications. 15 mg of PMX was dispersed in 5 mL of Tris-Glycine buffer and Tris-Glycine-urea buffer to test the free SH and total SH, respectively. The protein sample solution was prepared by stirring (RH basic 1, IKA group, Staufen, Germany) at room temperature for 1 h. It was then centrifuged at 10,000 rpm for 10 min at 4 °C. Following this, 20 μL of Ellman’s reagent was added. After a 5-min incubation, the absorbance was measured at 412 nm using a Multimode microplate reader (Spark, Tecan Trading AG, Switzerland). The total SH (TSH) and free SH (FSH) content, and DB content were calculated according to the following formula:

$$\mathrm{TSH}\left(\mathrm{FSH}\right)\left(\mathrm{\mu }\mathrm{mol}/\mathrm{g}\right)=\frac{73.53\times A_{412}\times \mathrm{D}}{\mathrm{C}}$$ 5

$$DB\left(\mathrm{\mu }\mathrm{mol}/\mathrm{g}\right)=\frac{\mathrm{TSH}-\mathrm{FSH}}{2}$$ 6

Where: A₄₁₂ was the absorbance of the supernatant at 412 nm; D was dilution multiple, the dilution multiple of FSH was 5.02, and the dilution multiple of TSH was 10; C was the concentration of protein dispersed in different buffers (mg/mL).

Determination of intermolecular forces

The method of Zhang et al.⁵⁷ was referred to with minor modifications. A centrifuge tube was taken with 0.1 g of PMX, and 10 mL of solutions for disrupting intermolecular forces (using 0.05 mol/L phosphate buffer as the solvent) were added to dissolve the protein. These solutions were: Solution A (0.05 mol/L NaCl solution); Solution B (0.6 mol/L NaCl solution); Solution C (0.6 mol/L NaCl solution + 1.5 mol/L urea); Solution D (0.6 mol/L NaCl solution + 8 mol/L urea). PMX was mixed with the above solutions, homogenized for 1 min, and stirred magnetically for 1 h to ensure sufficient reaction. The mixture was then centrifuged at 6000 × g for 20 min, the supernatant was taken, and the protein content was determined by the BCA method. The effect of ionic bonds was represented by the difference in protein content dissolved in buffers A and B; the effect of hydrogen bonds by the difference in protein content dissolved in buffers B and C; the hydrophobic interaction by the difference in protein content dissolved in buffers C and D.

Rheological measurements

Apparent viscosity: Extrudate powder was dispersed in deionized water to obtain a 20% (m/m) suspension, which was equilibrated overnight at 4 °C. Prior to testing, samples were equilibrated at room temperature for 30 min. Rheological measurements were performed on a rheometer (HAAKE MARS 40, Thermo Fisher Scientific, USA) using a 50-mm parallel-plate geometry with a 1.0-mm gap. Excess sample was trimmed, and the sample was allowed to rest for 2 min. Measurements were conducted at 25 °C over a shear-rate range of 0.1–100 s⁻¹. Apparent viscosity was recorded as a function of shear rate.

Frequency sweep: Extrudate powder was prepared as described above. A 50-mm parallel-plate geometry was used at 25 °C. Frequency sweeps were performed over an angular-frequency range of 0.1–100 rad s⁻¹ within the linear viscoelastic region, and G′, G″, and tan δ (G″/G′) were recorded using HAAKE RheoWin software.

Differential scanning calorimeter (DSC)

According to the method of Ma et al.⁵⁸, with some modifications, the differential scanning calorimeter (DSC) (Q100 Series, TA Instruments, New Castle, USA) was used to analyze thermal properties. Samples (3 mg), to which 9 μL of deionized water was subsequently added, was sealed and equilibrated at room temperature for 24 h. Subsequently, the crucible was heated from 20 °C to 160 °C at a rate of 10 °C/min, with an empty crucible serving as the reference.

Scanning electron microscopy (SEM) observation

The microstructure of freeze-dried protein extrudate was observed using a scanning electron microscope (Sigma300, Carl Zeiss AG, Oberkochen, Germany). The sample was glued with conductive adhesive tape and sprayed with gold for 1 min. The microstructure of the PMX was observed after scanning by electron microscope, and at an accelerating voltage of 3 kV with a magnification of 5 K.

Fourier transform infrared spectroscopy (FTIR)

According to the method of Liu and Hsieh⁵⁹, the freeze-dried sample powder and potassium bromide were mixed and ground evenly according to the ratio of 1:100, and the transparent sheet was obtained by a press (HY-12, Tianguang New Optical Instrument Technology Co., Ltd., Tianjin, China), which was placed in the spectrometer (TENSOR 27, Bruker, Germany). The scanning conditions were as follows: the resolution was 4 cm⁻¹, the scans were 64, and the measuring range was 4000–400 cm⁻¹. The 1700–1600 cm⁻¹ (amide I) region was fitted and analyzed by PeakFit 4.12 software (version 4.12, SPSS Inc., Chicago, USA).

Protein electrophoresis analysis of PMX

According to the method of Cagnin et al.⁶⁰ with some modifications, SDS-PAGE experiments used a discontinuous gel system, where the separation gel concentration was set to 12% and the stacking gel was 5%. Sample pre-treatment included: the powder after freeze-drying was dissolved in phosphate buffer, centrifuged at 10,000 r/min for 20 min, and the protein concentration of the resulting supernatant was adjusted to 1 mg/mL. The protein sample was mixed with the buffer at a ratio of 4:1, then heated at 95 °C in a metal bath for 5 min, followed by cooling to room temperature in ice water. The sample volume was 10 µL, the initial electrophoresis voltage was set to 120 V, and when the sample entered the separation gel, the voltage was increased to 180 V. Electrophoresis was terminated when the dye front was 1 cm from the bottom of the gel. The staining process used Coomassie Bright Blue R-250 with shaking for 60 min, followed by destaining, with the destaining solution changed every 60 min until the bands were clear. The gel imaging device (iBright, Thermo Fisher Scientific Inc., Wisconsin, USA) was used to capture the image of the protein electrophoresis spectrum.

Sensory evaluation

Drawing from the method of Usman et al.⁶¹, with certain modifications, a panel of 10 professionally trained sensory evaluators (5 men and 5 women) conducted the experiment in a serene, neutral environment. Toothpicks were provided, during the evaluation, and each evaluator was required to rinse their mouth with purified water between samples to eliminate any residual flavor interference. The evaluation criteria encompassed flavor (20 points), color (20 points), taste (30 points), and texturization degree (30 points), culminating in a total score of 100 points. Scoring standards and evaluation bases were established for each criterion as “excellent (high score range), good (medium score range), and fair (low score range).” Ultimately, the scores from all evaluators were collected and averaged to ensure the objectivity and reliability of the evaluation results.

Electronic nose measurement

The sample (2.5 g) was weighed and transferred into a 50 mL centrifuge tube. The tube was sealed and equilibrated for 30 min prior to analysis via headspace injection. The following analytical conditions were applied: the sampling time was set to 1 s per group, the sensor self-cleaning time was maintained at 100 s, the sample injection time was fixed at 5 s, the injection flow rate was adjusted to 400 mL/min, and the data acquisition time was configured to 100 s. Data points between 69 and 71 s were extracted for summarization and subsequent analysis. Principal component analysis (PCA) was conducted on the acquired dataset.

Identification of volatile flavor compounds measurement

The sample was pretreated using headspace solid-phase microextraction technology. 1.0 g of the crushed sample was accurately weighed and placed in a 20 mL headspace bottle, after which 50 μL of cyclohexanone (66 ppm concentration) was added as an internal standard. A DVB/CAR/PDMS composite extraction head was selected, and static extraction was performed at a constant temperature of 60 °C for 30 min. Following extraction completion, the extraction head was rapidly inserted into the gas chromatograph inlet, where thermal desorption was carried out at 250 °C for 3 min, after which the extraction head was removed. For instrument parameter settings, optimized gas chromatography separation conditions and mass spectrometry detection parameters were utilized for component analysis. The column oven temperature program was controlled as follows: the initial temperature was maintained at 50 °C for 5 min, followed by a temperature ramp to 260 °C at 10 °C/min, which was then held for 8 min. High-purity helium was employed as the carrier gas in constant flow mode (1.0 mL/min), with a non-split injection method being adopted. The mass spectrometry detector parameters were configured as follows: the ionization mode was set to electron impact (EI) mode, the electron energy was adjusted to 70 eV, the ion source temperature was controlled at 210 °C, and the transmission line temperature was set to 300 °C. The volatile flavor component content was calculated using the specified formula.

$$\begin{array}{c}\text{Volatile}\text{flavors}\text{ubstance}\text{content}\left(\mathrm{ng}/\mathrm{g}\right)=\frac{\text{Volatile}\text{peak}\text{area}}{\text{Internal}\text{standard}\text{peak}\text{area}}\times \frac{\text{Internal}\text{standard}\text{substance}}{\text{PMX}\text{Quality}}\hfill \end{array}$$ 7

Free amino acid analysis

The determination of free amino acids in the sample was conducted in accordance with GB5009.124-2016 “Determination of Amino Acids in Food”, with an amino acid analyzer being utilized for analytical measurements. Post-column derivatization with benzene-1,2-dicarbaldehyde was adopted to facilitate the detection of the compound.

The taste activity value (TAV) was calculated as the ratio of the concentration of taste compounds to their respective threshold values. Compounds with TAV scores >1 were classified as taste-active. The taste thresholds were obtained from established literature sources⁶².

Statistical analysis

At least three independent replicates were performed for each test, with values expressed as mean ± standard deviation. The data were visualized using Origin 2022 (Origin Lab Corporation, Massachusetts, USA). All statistical analyses were performed by SPSS Statistics 22 (IBM, Armonk, New York, USA), using ANOVA and Duncan’s tests to analyze significant differences between mean values (p < 0.05).

Author contributions

Yunlong Li: Conceptualization, Writing-review and editing, Project administration, Resources. Shunzhang Ma: Writing-original draft, Formal analysis. Jilin Dong: Software, Data curation, Methodology and Investigation. Zhe Cheng: Conceptualization, Supervision. Ruiling Shen: Funding acquisition, Project administration, Validation, Resources.

Data availability

Data will be made available on request. No custom code or mathematical algorithm was developed for this study. Details regarding the specific codes used can be found in the references cited.

Code availability

No custom code or mathematical algorithm was developed for this study. Details regarding the specific codes used can be found in the references cited.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41538-025-00666-7.