Optimizing gonadal development and muscle flavor quality in the male Chinese mitten crabs (Eriocheir sinensis): Utilization of soy isoflavones in crustaceans

Abstract

Nutritional regulation is the key strategy for improving aquatic products quality. This research evaluated the impacts of soy isoflavones on gonadal development and muscle quality in male Chinese mitten crabs (Eriocheir sinensis). Crabs were fed diets containing soy isoflavones (0, 33.14, 71.36, or 373.26 mg/kg) for 12 weeks. Results revealed that soy isoflavones may regulate gonadal development through “eyestalk-accessory gonad-testis” endocrine axis. Moreover, they promoted protein deposition in muscles and enriched highly unsaturated fatty acids (HUFAs) and total polyunsaturated fatty acids (PUFAs). In addition, soy isoflavones increased the muscle fibers density while reducing diameter, and significantly increased the umami amino acids proportion and equivalent umami concentration (EUC). This research shows that soy isoflavones can effectively enhance the gonadal development, nutritional value, flavor characteristics, and health benefits of aquatic foods, with a recommended supplementation amount of 71.36 mg/kg in crab feed.

Highlights

• •Soy isoflavones promote gonadal development in male Chinese mitten crabs (E. sinensis).
• •Optimal soy isoflavone supplementation is 71.36 mg/kg.
• •Soy isoflavones regulated the “eyestalk- accessory gonad -testis” endocrine axis in E. sinensis.
• •Soy isoflavones increased PUFAs and HUFA levels in muscle, enhancing nutritional value.
• •Soy isoflavones enhanced the umami intensity of the muscles in E. sinensis.

1. Introduction

In global food production, aquaculture is now among the fastest-growing sectors (Ahmad et al., 2021). Although aquatic foods provide important support for global dietary health, their nutritional diversity is often oversimplified through conceptual categorization as generic “seafood or fish”. Current evaluation systems emphasize only their protein and energy value, neglecting their unique nutritional matrix, which is rich in ω-3 fatty acids (DHA, EPA), trace elements (such as calcium and iron), and vitamins (including B12 and D). Data on nutrition reveal that aquatic foods are superior to terrestrial animal sources within the top seven nutrient-rich food categories (Golden et al., 2021). Moreover, aquatic animal-sourced foods accounted for 15 % of the global animal protein supply and 6 % of the total protein supply as of 2021 (FAO, 2024). Therefore, among animal protein production systems, aquaculture has emerged as one of the most sustainable methods to for protein supply due to its outstanding resource utilization efficiency and environmental friendliness. Among aquaculture species, crustaceans, as important sources of aquatic food protein, have a higher protein content per unit than mollusks and algae do (Boyd et al., 2022).

The Chinese mitten crabs (Eriocheir sinensis), with its umami-rich texture, tender flesh, and unique flavor, is highly popular among consumers in Asian countries such as China, Japan, and South Korea (Qiu et al., 2023). In China, E. sinensis serves as a typical representative of the synergistic development of traditional dietary culture and modern sustainable agriculture, with annual production reaching nearly 890,000 tons in 2023, accounting for the majority of the global market share (China Fishery Statistical Yearbook, 2024). Before the commercial release of E. sinensis, high-quality diets are typically fed for fattening for approximately one month to significantly enhance gonadal development, nutritional quality, and flavor (Shao et al., 2013). The degree of gonadal development and edible yield are key indicators for evaluating the fattening effect, which directly affects the market release time and price of crabs (Shao et al., 2014). Besides the gonads, the hepatopancreas and muscles are also the major edible tissues. Reports suggest that consumers' emotions may be more positive when they consume crab meat (Ding et al., 2022); therefore, relevant research has focused on improving the quality and optimizing the flavor of crab meat. Genetic breeding, optimization of feed formulations and improvement of the breeding mode and environment are common measures used to enhance aquatic product quality (Wang, He, et al., 2021). Additionally, growing evidence have shown that diet ingredients strongly influence the flavor profiles and nutritional value of aquaculture products (Divya et al., 2020; Yue et al., 2022; Zhu et al., 2024). The well-grown crabs have plump and succulent muscles with a fresh and slightly sweet taste. This flavor is attributed primarily to nonvolatile taste-active compounds, such as free amino acids, soluble sugars, flavor 5′-nucleotides and succinic acid (Chen & Zhang, 2007), among which free amino acids make a particularly significant contribution to the taste (Wang et al., 2016). The composition of fatty acids is another critical factor for assessing the nutritional quality of aquatic products and closely associated with gonad maturation (Wu et al., 2010). Although male E. sinensis are larger in size and have a greater meat yield than females, researchers seem to be more interested in the development of ovaries and nutritional quality in female crabs.

Soy isoflavones are plant flavonoids, the main bioactive components of which include genistein, daidzein and glycitein. Their unique chemical structure endows them with multiple physiological functions. Despite having a nonsteroidal structure, the phenol ring of soy isoflavones enables them to bind to the estrogen receptor (ER) and exert estrogenic and/or antiestrogenic effects (Cederroth & Nef, 2009). Additionally, the functional hydroxyl group in its structure can stabilize free radicals by resonantly donating electrons and hydrogen atoms, thereby forming relatively stable flavonoid radicals (Dias et al., 2020). Thus, the multiple functional properties of soy isoflavones as phytoestrogens are not concurrently achievable by any other additives, which makes them have unique advantages in the development of functional feeds. Current research has primarily focused on their effects on reproductive development (Nuzaiba et al., 2020), sex differentiation (Inaba et al., 2022), and antioxidant functions (Cao et al., 2020) in aquatic animals. In contrast, studies investigating their impacts on muscle texture and nutritional value are extremely limited. (D'Souza et al., 2005; Yang et al., 2019; Yang et al., 2022). Current studies in crustaceans have covered exclusively on growth, lipid metabolism, and immune function of E. sinensis and white shrimp (Litopenaeus vannamei) (Chen et al., 2011; Shi et al., 2024). Notably, the effects of soy isoflavones on reproductive development and muscle nutritional quality of E. sinensis remain unexplored.

Therefore, this research selected male E. sinensis as the research subject to systematically evaluate the potential utility of soy isoflavones as functional feed additives for regulating gonadal development and muscle quality. These results not only provide new theoretical evidence for the nutritional regulation system of crustaceans but also offer innovative strategies for enhancing the flavor profiles and nutritional quality of aquatic food.

2. Materials and methods

2.1. Animal ethical guidelines

All animal experimental procedures in this study were adhered strictly to the Guidelines for the Care and Use of Laboratory Animals from East China Normal University. The experiments were approved by the University's Animal Ethics Committee (Approval No. F20201002).

2.2. Diet formulation

The basal diet was supplemented with 0, 40, 80, or 400 mg/kg soy isoflavones (CAS NO. 574-12-9, purity ≥98 %, from Xi'an Tiankang Biotechnology Co., Ltd.) to formulate four isonitrogenous and isolipidic diets. The composition and relative abundance of soy isoflavones were daidzein (about 70 %), genistein (about 20 %) and others (about 10 %). The appropriate supplemental levels of 40 and 80 mg/kg were based on our previous studies (He et al., 2024). When soybean meal is selected as the protein source for fattening feeds of E. sinensis, the proportion is generally 10 %–20 % (Chen et al., 2025; Shao et al., 2013; Zhang et al., 2024), with the soy isoflavones containing approximately 2.0–2.5 mg/g of soybean meal (Xiao et al., 2011; Zhang et al., 2015). Based on this, it can be estimated that the feed contains 200–500 mg/kg of soy isoflavones. Accordingly, the basal diet was added 0, 40, 80, and 400 mg/kg soy isoflavones. In each diet, the analyzed concentrations of soy isoflavones by high-performance liquid chromatography (HPLC, LC-20CE, Shimadzu Corporation, Japan) were 0, 33.14, 71.36 and 373.26 mg/kg. The highest level of soy isoflavones was 373.26 mg/g, which was within the middle range (200–500 mg/kg). Thus, this dose enables scientific investigation into the biological effects of high soy isoflavones levels in E. sinensis. The 0 mg/kg group was set as the control (C), with the remaining groups labeled SL, SM, and SH. First, all ingredients were ground, then sifted to 60 mesh, weighed, finely milled, and thoroughly mixed. The soy isoflavones and choline chloride were dissolved in oil and water, respectively, and thoroughly mixed with other feed ingredients. An F-26 II pelletizer was used to extrude and pellet the mixture into 2 mm particles. After air-drying in a cool, airy indoor environment until reaching around 10 % moisture, the particles were stored in airtight bags at −20 °C. Supplementary Table S1 details the basal diet formulation and proximate composition.

2.3. Crabs and feeding trial

The same batch of male E. sinensis that had just undergone reproductive molting was obtained from Nantong Haida Biotechnology Co., Ltd. (Jiangsu, China), and the growth trial was conducted at the Experimental Base of the Shanghai Fisheries Research Institute (Shanghai, China). For acclimation to experimental conditions, the crabs were temporarily cultured in a 9 m × 2 m concrete pond for 7 days, during which they were fed a basal diet. Before the end of the temporary culture, 30 similarly sized crabs (137.97 ± 3.13 g) were randomly selected for dissection, and the initial gonadosomatic index (GSI) was determined to be 1.08 ± 0.07. A total of 192 healthy crabs (140.85 ± 2.14 g) were randomly distributed into 24 tanks (300L), each containing 8 crabs and 6 treatment replicates. Each tank was provided with six arched plastic tubes that act as shelters to reduce fighting between the crabs. The daily feed weight was set at 3 % of the crab body weight for 12 weeks, and fed three times daily (07:00, 14:30, and 20:30). The residual feed and feces were removed when the water in the tank was renewed (approximately 1/3) every day. The water was maintained at 23–28 °C for temperature, 7.3–8.1 for pH, >7.0 mg/L for dissolved oxygen, and <0.05 mg/L for ammonia-N content.

2.4. Sample collection and performance evaluation

After the crabs fasted for one day, their weight and carapace length were measured. Crabs were anesthetized on slurry ice, six crabs were randomly sampled from each group for dissection, and the hepatopancreas, gonads, and muscles were accurately weighed to calculate the relevant indices. The small pieces of muscle tissue from the third foot and corresponding abdominal area were then carefully excised and fixed in Bouin's reagent for histological analysis; another piece of abdominal muscle tissue was collected for evaluation of collagen content and centrifugal loss. Finally, the remaining hepatopancreas, gonads, muscles and eyestalks were stored at −80 °C for subsequent use. The calculation formulas of the relevant parameters are as follows:

$$\text{Testis index}\left(\mathrm{TI},\%\right)=100\times \text{testis}\mathrm{wet}\text{weight}\left(\mathrm{g}\right)/\text{body}\mathrm{wet}\text{weight}\left(\mathrm{g}\right);$$

$$\text{Accessory gonad index}\left(\mathrm{AGI},\%\right)=100\times \text{accessory gonad}\mathrm{wet}\text{weight}\left(\mathrm{g}\right)/\text{body}\mathrm{wet}\text{weight}\left(\mathrm{g}\right);$$

$$\text{Gonadosomatic index}\left(\mathrm{GSI},\%\right)=100\times \text{gonad}\mathrm{wet}\text{weight}\left(\mathrm{g}\right)/\text{body}\mathrm{wet}\text{weight}\left(\mathrm{g}\right);$$

$$\text{Hepatopancreas index}\left(\mathrm{HSI},\%\right)=100\times \text{hepatopancreas}\mathrm{wet}\text{weight}\left(\mathrm{g}\right)/\text{body}\mathrm{wet}\text{weight}\left(\mathrm{g}\right);$$

$$\text{Meat yield}\left(\mathrm{MY},\%\right)=100\times \text{meat}\mathrm{wet}\text{weight}\left(\mathrm{g}\right)/\text{body}\mathrm{wet}\text{weight}\left(\mathrm{g}\right);$$

$$\text{Total edible yield}\left(\mathrm{TEY},\%\right)=\mathrm{GSI}+\mathrm{HSI}+\mathrm{MY};$$

$$\text{Condition factor}\left(\mathrm{CF},\mathrm{g}/{\mathrm{cm}}^3\right)=\text{body}\mathrm{wet}\text{weight}\left(\mathrm{g}\right)/\text{carapace}{\text{length}}^3\left(\mathrm{cm}\right)$$

$$\text{Survival}\left(\%\right)=100\times \left(\text{final crab number}/\text{initial crab number}\right).$$

2.5. Proximate composition analysis

The Association of Official Analytical Chemists were used to analyze the proximate compositions of the muscles and diets (AOAC, 1995). The oven drying method at 105 °C was used to measure moisture content via drying samples to constant weight. The Kjeldahl method (Kjeltec™ 8200) was employed for crude protein measurement, while crude lipids were determined via the chloroform/methanol method (Folch et al., 1951). The ash content was measured by incinerating samples in a muffle furnace at 550 °C for 6 h.

2.6. Biochemical analysis

Commercial kits (Jiancheng Bioengineering, Nanjing, China) were used to measure the malondialdehyde content (MDA; Cat. No. A003-1), superoxide dismutase (SOD; Cat. No. A001-3), glutathione peroxidase (GSH-Px; Cat. No. A005-1) and total antioxidant capacity (T-AOC; Cat. No. A015-2) activities in the muscle. The collagen content was determined by measuring the hydroxyproline (Cat. No. A030-2; which accounts for 13.4 % in collagen) content in the muscle. In addition, other commercial kits were used to measure total sulfhydryl groups (S0141S and S0138S, Beyotime Biotechnology, Shanghai, China) in the muscle, as well as the content of gonadal inhibiting hormone (GIH, YJ290712, Shanghai Enzyme Biotechnology Company, Shanghai, China) in the eyestalks. All steps complied strictly with kit instructions.

The method of Du et al. (2022) was used to measure centrifugal loss. At room temperature, fresh muscles were weighed (W₁, approximately 10 g) and then centrifuged (2000 rpm, 30 min). After the surface moisture was removed with filter paper, the samples were reweighed (W₂). Centrifugal loss (%) = 100 × (W₁ − W₂)/W₁.

2.7. Histological analysis

The abdominal and foot muscles of the crabs were fixed with Bouin's solution, followed by dehydration and transparency, paraffin embedding, sectioned at 5 μm thickness and H&E staining. A light microscope (Nikon Ds Ri2, Tokyo Metropolis, Japan) was used to select six visual fields at 10 × 40 magnification randomly. For each fiber, the major axis and the perpendicular minor axis of the cross-section were measured, with the mean value recorded as the fiber diameter. The number of myofibers in each visual field was counted, and the diameters and lengths of six individual myofibers were measured using ImageJ software.

2.8. RT-qPCR analysis

Total RNA was extracted from the testis, accessory gonad and muscle using RNAiso Plus (9109, Takara, China), and RNA quantity and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo, Wilmington, USA). Subsequently, the PrimeScript™ RT Reagent Kit (Takara, Dalian, China) was subsequently used for reverse transcription. The CFX96 Real-Time PCR system (Bio-Rad, USA) was employed for PCR amplification, with program parameters and reaction conditions as previously described (He et al., 2024). Reference genes β-actin and S27 were selected for relative gene expression quantification via the 2^−ΔΔCt method. (Livak & Schmittgen, 2001). The primer information used was shown in Supplementary Table S2.

2.9. Fatty acid analysis

The extracted crude lipid (2 mg) was methylated with 1 mL of 0.5 mol/L methanolic KOH (65 °C, 30 min) followed by 14 % BF3 in methanol (75 °C, 30 min). After sequential addition of 1 mL HPLC-grade n-hexane (>95 % purity) and 1 mL ddH₂O, the mixture was vortexed and centrifuged (3000 rpm, 5 min), concentrated under nitrogen gas flow to obtain fatty acid methyl esters. Finally, gas chromatography–mass spectrometry (GCMS-QP2010 SE, Shimadzu Co., Kyoto, Japan) was used for analysis. The specifications of gas chromatograph column were 0.25 mm internal diameter, 0.20 μm film thickness and 100 mm length. The carrier gas helium flow rate was 1.7 mL/min. The injector and flame ionization detector temperatures were set to 250 °C and 200 °C, respectively. The program was 120 °C (held for 1 min), 240 °C (held for 30 min), with a total run time of 60 min per sample. Fatty acid data are expressed as relative content (percentage of total fatty acids).

2.10. Free amino acids (FAAs) and nucleotides analysis

Muscle FAAs were extracted using trichloroacetic acid (TCA), following the method of Konosu (1982) with minor modifications. Specifically, the muscle sample (1.0 g) was accurately weighed and placed in a 50 mL centrifuge tube, added TCA solution (15 mL, 5 %) and homogenized (10,000 rpm, 1 min). The homogenate was sonicated (5 min) in an ice-water bath, incubated (2 h), centrifuged (12,000 g, 10 min, 4 °C) and collected supernatant. The supernatant pH was adjusted to 2.0 using 1 M and 6 M NaOH solutions, made up to 25 mL with ultrapure water, and filtered through a 0.22 μm syringe filter. Quantitative analysis was performed using an automatic amino acid analyzer (L-8500, Hitachi, Japan), and the specific measurement procedures were carried out by Sichuan Willtest Technology Co., Ltd.

Muscle nucleotides (2.0 g) were extracted using high-performance liquid chromatography (HPLC-1260, Agilent, USA) according to the method reported by Qiu et al. (2023). The muscle tissue (2.0 g) was crushed in liquid nitrogen, mixed with TCA solution (25 mL, 0.1 %, v/v) for 30 min and centrifuged (12,000 g, 5 min, 4 °C). The supernatant was collected, made up to 50 mL with sterile water, and filtered through a 0.45 μm membrane filter. Analysis was performed at 40 °C using an LC20AD HPLC system equipped with a column (4.6 mm × 250 mm, 5 μm film thickness). Mobile phase A comprised 0.1 M dipotassium hydrogen phosphate (40 mL) and 0.01 M potassium dihydrogen phosphate (960 mL). Mobile phase B was HPLC-grade methanol. Operated at 254 nm detection and 0.6 mL/min flow rate.

2.11. Taste activity value (TAV) and equivalent umami concentration (EUC) analysis

As described by Scharbert and Hofmann (2005), TAV represents the ratio of amino acids or nucleotides concentration in a sample to their respective taste thresholds; when TAV exceeds 1, it is considered a significant contribution to the taste. EUC represents the comparison of the umami intensity generated by the synergistic effect of umami amino acids and nucleotides with that of monosodium glutamate (MSG), expressed as the equivalent MSG concentration (Yamaguchi et al., 1971). The calculation formula is as follows:

$$\mathrm{EUC}=\sum {\mathrm{a}}_{\mathrm{i}}{\mathrm{b}}_{\mathrm{i}}+1218\left(\sum {\mathrm{a}}_{\mathrm{i}}{\mathrm{b}}_{\mathrm{i}}\right)\left(\sum {\mathrm{a}}_{\mathrm{j}}{\mathrm{b}}_{\mathrm{j}}\right),$$

EUC: equivalent umami concentration, g MSG/100 g;

a_i: umami amino acids concentration (Asp or Glu), g/100 g;

b_i: the relative umami potency coefficient of umami amino acids compared with MSG (Asp, 0.077; Glu, 1);

a_j: umami 5′- nucleotides concentration (inosine monophosphate, IMP; guanosine monophosphate, GMP; adenosine monophosphate, AMP), g/100 g;

b_j: the relative umami potency coefficient of umami nucleotides compared with IMP (IMP, 1; GMP, 2.3; AMP, 0.18);

1218: is determined by the synergistic coefficient of the g/100 g concentration employed.

2.12. Statistical analysis

Data were analyzed in SPSS version 20.0, with all results presented as the means ± SEMs (standard errors of the means). Normality and homogeneity tests were first conducted, followed by one-way ANOVA and Duncan's multiple-range tests to assess intergroup differences. Statistical significance was indicated by a P-value <0.05. To determine if the results followed a linear or quadratic pattern, orthogonal polynomial contrasts were performed. Finally, Origin 2021 was used to perform principal component analysis (PCA) on the muscle property data of the abdominal and foot regions.

3. Results

3.1. Gonadal development and muscle yield

Gonadal development degree determines the nutritional value and market price of male crabs (He et al., 2014). The effects of soy isoflavones on gonadal development and muscle yield in male E. sinensis was presented in Table 1. In this study, dietary supplementation with soy isoflavones did not significantly affect the TI (P > 0.05), but 71.36 mg/kg soy isoflavones significantly increased the AGI and GSI (P < 0.05), indicating that soy isoflavones substantially promoted gonadal development in male crabs. Further analysis revealed that the increase in the GSI appeared to depend mainly on the increase in accessory gonads, as TI did not differ significantly across groups. This phenomenon might be associated with the use of male E. sinensis which after reproductive molting in this study. This occurs because the testis and vas deferens develop rapidly before reproductive molting, whereas the maturation of the accessory gonads mainly occurs after reproductive molting. (Jiang et al., 2024). Endocrine hormones play a core regulatory role in the reproductive development of crustaceans, with GIH serving as a key negative regulator (Kleijn et al., 1994). Feeding 33.14 and 71.36 mg/kg soy isoflavones significantly reduced the content of GIH (P < 0.05), suggesting that they may promote gonadal development in crabs by modulating the synthesis and secretion pathways of endocrine hormones.

Table 1.

Effects of soy isoflavones on the gonadal development and muscle yield in male E. sinensis.

Parameters	Groups				ANOVA	Regression analysis
						Linear		Quadratic
	C	SL	SM	SH	P Value	Adj. R2	P Value	Adj. R2	P Value
TI (%)	0.18 ± 0.01	0.19 ± 0.01	0.20 ± 0.01	0.18 ± 0.01	0.434	−0.047	0.939	0.023	0.307
AGI (%)	1.37 ± 0.05a	1.72 ± 0.14ab	1.95 ± 0.16b	1.74 ± 0.12ab	0.029	0.147	0.036	0.283	0.012
GSI (%)	2.24 ± 0.06a	2.52 ± 0.14ab	2.88 ± 0.20b	2.55 ± 0.10ab	0.029	0.106	0.067	0.238	0.022
HSI (%)	5.76 ± 0.33a	6.72 ± 0.16ab	6.65 ± 0.53ab	7.37 ± 0.31b	0.036	0.263	0.006	0.231	0.024
MY (%)	20.58 ± 0.54a	25.46 ± 1.28b	24.13 ± 1.28b	27.24 ± 0.72b	0.004	0.445	0.003	0.427	0.011
TEY (%)	29.51 ± 0.34a	34.67 ± 0.19b	33.66 ± 0.58b	36.22 ± 0.70c	< 0.001	0.559	< 0.001	0.643	< 0.001
CF (g/cm3)	0.66 ± 0.02a	0.69 ± 0.01ab	0.70 ± 0.01b	0.73 ± 0.02b	0.013	0.397	0.001	0.367	0.005
Survival (%)	87.50 ± 4.17	91.67 ± 5.45	93.75 ± 3.05	85.42 ± 4.92	0.550	−0.032	0.837	0.001	0.373
GIH (μg/mL)	302.87 ± 5.13b	285.29 ± 3.44a	281.79 ± 7.55a	289.42 ± 4.26ab	0.047	0.061	0.093	0.191	0.018

Note: Values were presented as mean ± SEMs (standard error of the mean, n = 6), and in the same line marked with different superscripts are significantly different (P < 0.05). Adj. R² = adjusted R square. TI: testis index; AGI: accessory gonad index; GSI: gonadosomatic index; HSI: hepatopancreas index; MY: meat yield; TEY: total edible yield; CF: condition factor; GIH: gonadal inhibiting hormone. C: control diet; SL: 33.14 mg/kg soy isoflavone diet; SM: 71.36 mg/kg soy isoflavone diet; SH: 373.26 mg/kg soy isoflavone diet.

Beyond GSI, the HSI, CF, MY and TEY are also critical quality indices for E. sinensis. This study found that HSI and CF significantly increased as soy isoflavones supplementation levels rose. Specifically, the SH group showed a significant increase in HSI compared to the control group (P < 0.05), and the CF of both SM and SH groups had significantly higher (P < 0.05). Moreover, compared with the control group, soy isoflavone supplementation significantly increased MY and TEY (P < 0.05), with SH group exhibiting the highest TEY among all groups. During gonadal development, the hepatopancreas provides a material basis and physiological guarantee for gonadal development by storing, transferring, and distributing nutrients. A suitable dose of soy isoflavones can significantly enhance lipid oxidation for energy supply in the hepatopancreas (Shi et al., 2024), thereby reducing protein consumption and promoting protein deposition. This metabolic shift effectively improved the MY and TEY, ultimately increasing the CF. In conclusion, soy isoflavones may promote gonadal development in male crabs by regulating hormone levels, while also enhancing their condition factor, thereby increasing their commercial value.

3.2. Proximate composition of the muscle

The nutritional quality attributes of edible tissues are reflected by their proximate composition (Yuan et al., 2020). In mature E. sinensis, the muscle comprises the largest proportion of the edible parts. As shown in Table 2, the moisture and ash contents in the muscle did not differ significantly (P > 0.05). In the SM and SH groups, the crude protein content was significantly increased (P < 0.05), and the crude lipid content was significantly increased in the SL and SM groups (P < 0.05). Our previous study demonstrated that soy isoflavones improve lipid transport in the hepatopancreas (He et al., 2024). The significant increase in muscle crude lipid content in this study, further supporting this conclusion. However, a significant increase in the HSI was observed after supplementation with 373.26 mg/kg soy isoflavone, while crude lipid content in muscle showed no alteration. This phenomenon suggested that high doses of soy isoflavones may lead to abnormal lipid accumulation in the hepatopancreas rather than facilitating lipid redistribution to peripheral tissues. On the other hand, consistent with findings in grass carp (Ctenopharyngodon idella), soy isoflavones have been shown to significantly enhance protein deposition in the muscle (Yang et al., 2019). Nevertheless, the underlying mechanisms have not been fully elucidated. The possible mechanisms may involve the suppression of protein catabolism (Rehfeldt et al., 2009) and the activation of protein synthesis via the target of rapamycin (mTOR) signaling pathway (Yang et al., 2019). The results revealed that soy isoflavones significantly enriched the conventional nutritional composition of crab meat.

Table 2.

Effects of soy isoflavones on the proximate composition of the muscle in male E. sinensis.

Parameters	Groups				ANOVA	Regression analysis
						Linear		Quadratic
	C	SL	SM	SH	P Value	Adj. R2	P Value	Adj. R2	P Value
Proximate composition
Moisture (%)	82.52 ± 1.17	82.01 ± 0.29	81.74 ± 1.33	81.42 ± 0.53	0.860	−0.010	0.370	−0.086	0.676
Crude protein (%)	12.53 ± 0.32a	13.84 ± 0.69ab	14.17 ± 0.50b	15.00 ± 0.46b	0.020	0.331	0.002	0.307	0.008
Crude lipid (%)	1.19 ± 0.18a	1.77 ± 0.10b	1.89 ± 0.23b	1.12 ± 0.16a	0.012	−0.056	0.983	0.415	0.004
Ash (%)	1.58 ± 0.05	1.52 ± 0.03	1.47 ± 0.02	1.46 ± 0.03	0.111	0.417	0.014	0.397	0.041

Note: Values were presented as mean ± SEMs (standard error of the mean, n = 6), and in the same line marked with different superscripts are significantly different (P < 0.05). Adj. R² = adjusted R square. C: control diet; SL: 33.14 mg/kg soy isoflavone diet; SM: 71.36 mg/kg soy isoflavone diet; SH: 373.26 mg/kg soy isoflavone diet.

3.3. Muscle fiber characteristics and PCA of muscle

H&E staining of the abdominal and foot muscles was shown in Fig. 1A, and the impacts of soy isoflavones on muscle fiber characteristics in male E. sinensis were shown in Table 3. The diameter of abdominal and foot muscle fibers was significantly reduced by 33.14 mg/kg soy isoflavones supplementation, while muscle fiber density was increased (P < 0.05). For foot muscle fibers, supplementation with 71.36 mg/kg significantly decreased the diameter (P < 0.05), while 373.26 mg/kg significantly increased the length (P < 0.05). As shown in Fig. 1 B, the PCA analysis integrated variables of muscle fiber characteristics from different crab muscle parts under varying levels of soy isoflavone supplementation. The first two principal components collectively accounted for 78.9 % of the total variance (PC1 = 49.3 %, PC2 = 29.6 %). The results indicated that soy isoflavones had similar effects on abdominal and foot muscle fibers, but their impact on foot muscle fibers was more pronounced. Specifically, Low dosages of soy isoflavones (33.14 and 71.36 mg/kg) showed a positive association with muscle fiber density but an inverse relationship with fiber diameter and length. In contrast, high supplementation levels (373.26 mg/kg) had the opposite effect.

Fig. 1.

The H&E staining of the abdominal and foot muscles (40×), with the scale representing 100 μm (A), and PCA analysis (B) in the muscle. In Fig. A, “C” denotes cross sectional, and “L” denotes longitudinal sectional. MFDi: muscle fiber diameter; MFL: muscle fiber length; MFD: muscle fiber density. C C: control diet; SL: 33.14 mg/kg soy isoflavone diet; SM: 71.36 mg/kg soy isoflavone diet; SH: 373.26 mg/kg soy isoflavone diet.

Table 3.

Effects of soy isoflavones on the muscle fiber characteristics in male E. sinensis.

Parameters	Groups				ANOVA	Regression analysis
						Linear		Quadratic
	C	SL	SM	SH	P Value	Adj. R2	P Value	Adj. R2	P Value
Abdominal muscle
MFDi	395.58 ± 18.10b	362.54 ± 21.05ab	317.25 ± 7.72a	366.42 ± 13.57ab	0.020	0.067	0.118	0.248	0.019
MFL	1625.12 ± 108.46	1995.39 ± 92.36	1946.00 ± 126.15	1841.02 ± 153.39	0.178	0.004	0.308	0.119	0.101
MFD	26.37 ± 2.78a	28.82 ± 2.66a	36.08 ± 1.58b	28.04 ± 1.84a	0.033	0.004	0.307	0.147	0.073
Foot muscle
MFDi	429.57 ± 34.66bc	369.31 ± 21.02ab	350.30 ± 18.53a	494.44 ± 13.07c	0.001	0.023	0.227	0.463	0.001
MFL	1779.60 ± 94.62a	2014.36 ± 108.89a	1982.42 ± 161.94a	2702.04 ± 65.72b	<0.001	0.482	<0.001	0.544	<0.001
MFD	6.00 ± 1.04a	8.22 ± 1.03ab	9.77 ± 1.85b	5.42 ± 1.67a	0.010	−0.045	0.969	0.316	0.007

Note: Values were presented as mean ± SEMs (standard error of the mean, n = 6), and in the same line marked with different superscripts are significantly different (P < 0.05). Adj. R² = adjusted R square. MFDi: muscle fiber diameter; MFL: muscle fiber length; MFD: muscle fiber density. C: control diet; SL: 33.14 mg/kg soy isoflavone diet; SM: 71.36 mg/kg soy isoflavone diet; SH: 373.26 mg/kg soy isoflavone diet.

The properties of muscle fibers determine muscle texture. As the primary component of muscle, proteins crucially regulate muscle fiber development (Yun et al., 2022). In this study, soy isoflavones were found to promote the accumulation of muscle nutrients. Additionally, the textural firmness of muscle is associated with decreased myofiber diameter and increased density (Wang et al., 2024). At low dosages (33.14 and 71.36 mg/kg), soy isoflavones significantly reduced muscle fiber diameter in both abdominal and foot muscles while increasing density, thereby improving muscle textural firmness and elasticity. Furthermore, PCA analysis revealed a more pronounced response of foot muscles to soy isoflavones. This may be attributed to the fact that foot muscles, which are primarily used for support and movement, require greater mechanical strength and endurance, increasing their sensitivity to nutrients.

3.4. Antioxidant capacity and muscle quality biochemical analysis

In muscles, soy isoflavone supplementation significantly increased collagen content and the activities of T-AOC and GSH-Px while simultaneously reducing MDA content (P < 0.05, Fig. 2A, C, and F). Furthermore, MDA content showed an initial decrease followed by an increase as soy isoflavone supplementation levels rose, with the lowest value observed in the SM group (P < 0.05, Fig. 2D). Additionally, the SM and SH groups showed significantly higher total sulfhydryl group content in muscles compared to the control group (P < 0.05, Fig. 2 E). Although centrifugal loss of muscle gradually decreased, soy isoflavones significantly impacted neither the centrifugal loss nor SOD activity (P > 0.05, Fig. 2B and E).

Fig. 2.

Effects of soy isoflavones on the antioxidant capacity and muscle quality in male E. sinensis. Means (n = 6) and standard errors (vertical bars) are shown for the values, and groups marked with different lowercase letters show significant differences (P < 0.05). Adj. R² = adjusted R square. (A) T-AOC: total antioxidant capacity; (B) SOD: superoxide dismutase; (C) GSH-Px: glutathione peroxidase; (D) MDA: malondialdehyde; (E) centrifugal loss and total sulfhydryls group content of muscle; (F) collagen protein. C: control diet; SL: 33.14 mg/kg soy isoflavone diet; SM: 71.36 mg/kg soy isoflavone diet; SH: 373.26 mg/kg soy isoflavone diet.

During storage, the most common chemical deterioration of meat is the oxidation of proteins and lipids, which leads to off-flavors, discoloration, the formation of harmful substances, and a decline in nutritional quality (Shah et al., 2014). Since people became aware of the toxicological effects of synthetic antioxidants and other safety concerns, attention has gradually shifted toward natural antioxidants (Kumar et al., 2015). Soy isoflavones constitute secondary metabolites naturally generated in soybean growth, and the hydroxyl groups in the molecular structure of soy isoflavones can act as electron and hydrogen donors, thereby neutralizing free radicals. After supplementation, the content of total sulfhydryl groups in the muscle increased significantly, whereas the content of MDA decreased significantly. Total sulfhydryl groups are easily oxidized, resulting in a decrease in their content, which is commonly used to reflect the degree of protein oxidation. In contrast, MDA is used to reflect the degree of lipid oxidation (Xiong et al., 2009). Both the degree of protein and lipid oxidation are correlated with meat tenderness (Zhang et al., 2016). This study found that soy isoflavones significantly increased antioxidant enzyme activities like T-AOC and GSH-Px, increased the ability to reduce H₂O₂ to H₂O, and protected total sulfhydryl groups from oxidation. Interestingly, although muscle MDA content was significantly lower in all soy isoflavones supplemented groups compared to the control, it was significantly higher in the SH group than in the SL and SM groups. This may be attributed to HSI significantly increased in the SH group, suggesting that high dose soy isoflavones may cause abnormal lipid accumulation in the hepatopancreas, inducing oxidative stress. Thus, high dose soy isoflavones may exert more potent antioxidant activity in the hepatopancreas rather than in muscle.

Furthermore, soy isoflavones significantly increase the content of collagen in muscle, which not only enhances the water-holding capacity of the meat but also further improves meat quality through the accumulation of collagen (Hagen et al., 2008). Although this study did not observe significant differences in centrifugal loss, a gradual downward trend in these values was noted. These findings suggest that soy isoflavones can be utilized as natural antioxidants to enhance the tenderness and quality of crab meat by inhibiting muscle oxidation and preserving protein structural stability, thereby highlighting their potential in aquaculture nutrition.

3.5. Gene expression

The effects of soy isoflavones on gene expression for gonadal development and protein metabolism was shown in Fig. 3 and Supplementary Fig. S1.

Fig. 3.

Effects of soy isoflavones on the expression of genes related to gonadal development and protein metabolism in male E. sinensis. Heatmap of the expression of genes; the values in red and blue indicate up/downregulation, respectively (expressed as log2-fold changes), and the size of the circle represents the degree of up/downregulation. Values are means (n = 6), the groups marked with different lowercase letters in the circles show significant differences (P < 0.05). dmc1: DNA meiotic recombinase 1; cul4: cullin 4; iag: insulin-like androgenic gland hormone; mtor: mammalian target of rapamycin; s6: ribosomal protein s6; s6k1: ribosomal protein s6 kinase-1; 4ebp1: eukaryotic translation initiation factor 4E-binding protein 1; eif4e: eukaryotic initiation factor 4E. C: control diet; SL: 33.14 mg/kg soy isoflavone diet; SM: 71.36 mg/kg soy isoflavone diet; SH: 373.26 mg/kg soy isoflavone diet. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.5.1. Related to gonadal development

In the groups supplemented with soy isoflavones, the iag gene expression in the accessory gonad was significantly upregulated (P < 0.05), with the highest expression observed in the SM group. The expression levels of dmc1, nanos, cul4, and iag genes in the testis showed no significant group differences. (P > 0.05). The expression of genes associated with testis development was examined in this study (Jiang et al., 2024). Dmc1 is a member of the RecA/Rad51 family and is expressed during the early stages of meiosis (Toshiyuki et al., 1996). The maternal-effect gene Nanos, first identified in Drosophila by Wang and Lehmann (1991), is essential to germline cytoplasm (Kadyrova et al., 2007). Cul4 is one of the core components of the Cullin-RING ubiquitin ligase complex (CRLs), which plays a role in regulating the cell cycle and other biological functions (Marín, 2009). These three genes exhibit progressively attenuated expression as E. sinensis testis develop. (Jiang et al., 2024). No significant intergroup differences emerged in the expression of these genes. These findings indicate that the testis may have fully developed at this stage of gonadal development and that the promoting effect of soy isoflavones was manifested primarily in the development of the accessory glands. The aforementioned AGI and GSI results further support this conclusion.

Secreted by accessory glands, the insulin-like androgenic gland hormone (IAG) critically regulates sex differentiation and maintains male sexual characteristics (Song et al., 2018). In male crustaceans, the neuropeptide hormones secreted by X-organ/sinus-gland (XO-SG) in the eyestalk, such as gonad-inhibiting hormone (GIH), directly inhibit IAG gene expression (Guo et al., 2019). Additionally, silencing of the IAG gene causes testicular atrophy and impaired sperm development (Chang et al., 2024). These findings not only provide empirical validation for our theoretical hypothesis but also suggest that soy isoflavones, which act as phytoestrogens, may regulate gonadal development by influencing the “eyestalk-accessory gonad-testis” endocrine axis. However, further studies are required to clarify the precise mechanisms of action. Moreover, the significant quadratic effect observed suggests that excessive supplementation may trigger negative regulatory responses, indicating the need to maintain supplementation levels within the current experimental dosage range.

3.5.2. Related to protein metabolism

In muscle, mtor, s6 and eif4e gene expression levels significantly upregulated with increasing levels of soy isoflavones supplementation (P < 0.05) and were significantly higher compared with the control group. s6k1 gene expression was significantly upregulated by 71.36 and 373.26 mg/kg supplementation (P < 0.05), whereas 4ebp1 expression was downregulated (P < 0.05). Soy isoflavones activate mTOR (mammalian target of rapamycin) through estrogen receptor-mediated signaling (Shao et al., 2024). The mTOR signaling pathway promotes protein synthesis by phosphorylating S6K1 (ribosomal protein s6 kinase-1) and 4EBP1 (eukaryotic translation initiation factor 4E-binding protein 1) (Wang & Proud, 2006). Activation of S6K1 stimulates phosphorylation of S6 (ribosomal protein s6), thereby enhancing protein translation (Kim & Guan, 2011). Moreover, the phosphorylation of 4EBP1 dissociates EIF4E (eukaryotic initiation factor 4E), initiating the translation process (Wang et al., 2006). This study found that soy isoflavones significantly increased mtor, s6k1, s6, and eif4e gene expression in muscle but decreased 4ebp1 expression. These findings indicate soy isoflavones could enhance protein translation and deposition of male crabs by modulating the expression of genes related to the mTOR pathway, thereby increasing the meat yield.

3.6. Fatty acid composition

Fig. 4 and Supplementary Table S3 presented soy isoflavone effects on muscle fatty acid composition in male E. sinensis. The fatty acid composition of male crab muscle was significantly altered by soy isoflavones. Specifically, dietary soy isoflavones significantly increased the relative levels of C17:0, C20:1n-9, C18:3n-3, C20:2, C20:4n-6, and C22:6n-3 in muscles (P < 0.05) but significantly decreased the relative levels of C14:0, C16:0, C18:1n-9 and C18:2n-6 (P < 0.05). The relative levels of C15:0 and C18:0 significantly increased in the SL and SM groups (P < 0.05), whereas the relative level of C16:1n-9 significantly decreased (P < 0.05). The relative levels of C17:1 and C22:5n-3 significantly reduced in the SM and SH groups (P < 0.05), and the relative level of C20:0 increased dramatically in the SL and SH groups (P < 0.05). Additionally, the relative levels of C20:5n-3 and C20:3n-3 significantly increased in the SL and SM groups, respectively (P < 0.05). Overall, dietary supplementation with soy isoflavones significantly increased the relative levels of PUFA, HUFA, and n-3 HUFA in muscle, as well as the DHA/EPA ratio (P < 0.05). The relative level of SFA significantly decreased in the SM and SH groups (P < 0.05), with the lowest relative level of PUFA observed in the SM group (P < 0.05).

Fig. 4.

Effects of soy isoflavones on the fatty acid composition of muscle in male E. sinensis. Values are the average value (n = 6). Stacked bar chart (A) and radar chart (B) of fatty acid composition. Σ SFA: total amount of saturated fatty acids, including C14:0, C15:0, C16:0, 17:0, C18:0, C20:0, and C23:0; Σ MUFA: total amount of monounsaturated fatty acids, including C16:1, C17:1, C18:1n-9, C20:1, C22:1n-9, and C24:1; Σ PUFA: total amount of polyunsaturated fatty acids, including C18:2n-6, C18:3n-3, C20:2, C20:3n-3, C20:4n-6, C20:5n-3, C22:5n-3, C22:6n-3; Σ HUFA: total amount of highly unsaturated fatty acids, including C20:3n-3, C20:4n-6, C20:5n-3, C22:5n-3, and C22:6n-3; n-3 HUFA: total amount of n-3 highly unsaturated fatty acids, including C20:3n-3, C20:5n-3, C22:5n-3, C22:6n-3; DHA: docosahexaenoic acid (C22:6n-3); EPA: eicosapentaenoic acid (C20:5n-3); DHA/EPA: C22:6n-3/C20:5n-3 ratio. C: control diet; SL: 33.14 mg/kg soy isoflavone diet; SM: 71.36 mg/kg soy isoflavone diet; SH: 373.26 mg/kg soy isoflavone diet.

Higher fatty acid value was observed in male E. sinensis muscle compared to gonads and hepatopancreas (Wu et al., 2020). In this study, the relative levels of C16:0, C18:0, C18:1n-9, C18:2n-6, C20:4n-6 (ARA), C20:5n-3 (EPA), and C22:6n-3 (DHA) in the muscle of male crabs were relatively high, which similar findings were reported in other studies (Wang et al., 2022). Polyunsaturated fatty acids (PUFAs), especially long-chain PUFAs (such as ARA, EPA, and DHA), are the most significant nutritional indicators of crabs (Song et al., 2023). ARA plays a vital role in nervous system development, cognitive function, and immune system enhancement (Tallima & Ridi, 2018). EPA and DHA are beneficial for reducing the risk of hypertension, cardiovascular disease, and inflammation (Chen & Liu, 2020). Specifically, the high DHA/EPA ratio fatty acids of crab serve as a core indicator of their nutritional quality (Wang, Wang, et al., 2021). Furthermore, the thermal degradation of PUFAs generates flavor-enhancing ketones and aldehydes that concurrently enhance the edible quality of crab meat (Wu et al., 2020). Soy isoflavones significantly increased the relative levels of ARA, DHA and ΣPUFAs in muscle, and EPA significantly increased when 33.14 mg/kg was supplemented. These findings suggest that soy isoflavones could stimulate the endogenous biosynthesis of PUFAs in E. sinensis. Similarly, soy isoflavones increase the relative levels of HUFAs, n-3 HUFAs and DHA/EPA ratio, which confer multiple health benefits to humans (Bu et al., 2022). This further enhances the bioactivity of crab meat in human nutritional applications, particularly in promoting neurological health and inhibiting the development of chronic diseases. Therefore, soy isoflavones enhances the health value of aquatic products through lipid nutrition fortification strategies. However, the metabolic homeostasis of fatty acids is affected by multiple regulatory factors, including gene expression regulation, enzyme activity modification, dietary nutrient regulation, and intestinal and microbial metabolic activities. It is imperative to conduct systematic investigations into the regulatory effects of soy isoflavones on fatty acid composition in E. sinensis, which will provide critical insights for elucidating nutritional modulation pathways and developing functional feeds with optimized lipid profiles.

3.7. Muscle flavor evaluation

3.7.1. The contents of FAAs and nucleotides

Fig. 5 and Supplementary Table S4 presented the effects of soy isoflavones on FAAs and nucleotides contents in male E. sinensis muscle. In diets supplemented with soy isoflavones, Lys content significantly increased (P < 0.05), whereas Cys content significantly decreased (P < 0.05). The contents of His, Ile, Leu and Val in the SL and SM groups significantly decreased (P < 0.05), whereas IMP content significantly increased (P < 0.05, Fig. 5D). Met content was significantly lower in the SL and SH groups (P < 0.05). In the SM and SH groups, Glu and Pro contents significantly increased (P < 0.05), and Glu content in the SH group was significantly higher than in other groups (P < 0.05). The contents of Asp, Thr, Ala, and AMP significantly increased in the SH group (P < 0.05), whereas GMP content significantly decreased (P < 0.05, Fig. 5C). Phe content significantly reduced in the SL group but significantly increased in the SH group (P < 0.05). Gly, Ser, Arg, and Tyr contents had no significant differences (P > 0.05).

Fig. 5.

Effects of soy isoflavones on the FAAs and nucleotides contents of muscle in male E. sinensis. Values are the average value (n = 3), and different letters indicate significant differences (P < 0.05). (A): Bubble chart (left) and radar chart (right) of the contents of FAAs. Essential free amino acids (TEAA, including Arg, His, Ile, Leu, Lys, Met, Phe, Thr and Val), tasty free amino acids (TTAA, including Ala, Asp, Glu, Gly, Pro and Ser), umami free amino acids (UFFA, including Asp and Glu), sweetish free amino acids (SFFA, including Thr, Ala, Gly, Pro and Ser) and bitter free amino acids (BFFA, including Arg, His, Ile, Leu, Lys, Met, Phe, Val and Tyr). (B) AMP: adenosine monophosphate; (C) GMP: guanosine monophosphate; (D) IMP: inosine monophosphate. C: control diet; SL: 33.14 mg/kg soy isoflavone diet; SM: 71.36 mg/kg soy isoflavone diet; SH: 373.26 mg/kg soy isoflavone diet.

The flavor of crab meat is formed mainly by the interaction of nonvolatile taste-active compounds, like FAAs and nucleotides. The taste properties of FAAs are associated with side chain hydrophobicity and volume (Kawai et al., 2012). Aspartic (Asp) and glutamic (Glu) acids present umami tastes, whereas alanine (Ala), threonine (Thr), proline (Pro), glycine (Gly), and serine (Ser) acids present sweet tastes. In addition to Thr, these FAAs are also considered as they influence the overall flavor profile of meat. In this study, supplementation with 71.36 and 373.26 mg/kg soy isoflavones markedly enhanced the proportion of tasty and umami-free amino acids while reducing the proportion of bitter-free amino acids. Taste nucleotides also influence the flavor of crab meat. GMP and IMP have strong umami tastes, and the taste properties of AMP are related to its concentration. When its concentration is less than 100 mg/100 g, AMP presents a sweet taste, whereas exceeding this concentration results in a diminished sweet taste and an enhanced umami taste (Linder & Ackman, 2010). Supplementation with soy isoflavones increased the concentrations of AMP and IMP. These results indicate that soy isoflavones effectively enhanced the delicious taste of crab meat.

3.7.2. TAV and EUC

Supplementary Table S5 presented the TAVs of muscle FAAs and nucleotides, with a TAV > 1 suggesting the substance made an essential contribution to flavor. As shown in Fig. 6, the SM and SH groups showed significant elevation in EUC (P < 0.05), with the highest value of 10.07 g MSG/100 g observed in the SM group. In other words, per gram of crab meat (wet weight) had umami intensity equivalent to 0.10 g of MSG. According to Mantel analysis, the increase in EUC was mainly resulted from the elevated content of umami-free amino acids, especially glutamic acid (Glu). Moreover, the evaluation of crab meat flavor should be combined with the results of TAV and ECU. If TAV >1, the substance significantly contributes to the flavor of crab meat. The results revealed that the TAVs of Glu, Ala, Gly, Arg, His, GMP and IMP were > 1. Notably, Gly and Arg are inherently present at high concentrations in crab meat, and the concentrations of Gly, Arg and GMP were not significantly different among all the groups. These findings suggest that soy isoflavones enhance the umami taste primarily by increasing the concentrations of Glu, Ala, and IMP, while decreasing the concentration of His, thereby weakening the bitter taste.

Fig. 6.

Effects of soy isoflavones on the EUC contents of muscle in male E. sinensis. Means (n = 3) and standard errors (vertical bars) are shown for the values, and groups marked with different lowercase letters show significant differences (P < 0.05). Adj. R² = adjusted R square. (A) EUC: equivalent umami concentration. (B) The Mantel test was performed to examine the relationship between EUC and the contents of umami-free amino acids and nucleotides in muscle. The Pearson coefficient value and positive and negative correlations are represented by the size and color of squares in the matrix, and the color and thickness of lines outside the matrix indicate statistical significance and the strength of the correlation between EUC and other variables, respectively. C: control diet; SL: 33.14 mg/kg soy isoflavone diet; SM: 71.36 mg/kg soy isoflavone diet; SH: 373.26 mg/kg soy isoflavone diet.

EUC is used to measure the umami flavor intensity generated through the synergistic action of umami components. In this study, the EUC value increased significantly in crab meat supplemented with 71.36 and 373.26 mg/kg of soy isoflavones. Moreover, Mantel analysis showed a significant positive correlation between EUC and umami amino acids, particularly glutamic acid (Glu). Compared with nucleotides, soy isoflavones presented more pronounced increases in the concentrations of Asp and Glu in crab meat. Therefore, in short, soy isoflavones may enhance the umami flavor quality of male crabs by reshaping the composition of FAAs, particularly through enriching umami amino acids. Notably, there is currently no direct evidence elucidating the relationships between soy isoflavones and FAAs and nucleotides, and the specific regulatory mechanisms involved require further investigation.

4. Conclusion

Dietary supplementation with 71.36 mg/kg soy isoflavones can significantly enhance gonadal development and muscle quality in male E. sinensis, while concurrently optimizing their flavor characteristics. Soy isoflavones increase the levels of PUFAs and HUFAs in crab meat. They may promote nutrient deposition in muscle by activating the mTOR signaling pathway, thereby enhancing the nutritional and health value. Moreover, altering the properties of muscle fibers improves the textural firmness and tenderness of the muscles. Notably, soy isoflavones significantly increased the proportion of umami amino acids (especially glutamic acid) to increase the umami intensity of the muscles. These comprehensive improvements in organoleptic quality enhance the palatability and market competitiveness of crab meat (See Fig. 7). However, a high dose of 373.26 mg/kg may induce negative health effects, such as lipid accumulation in the hepatopancreas. Therefore, this study contributes sustainable aquaculture and premium aquatic food provision, and its application can effectively enhance the economic value of aquaculture and the health benefits for consumers.

Fig. 7.

Effects of soy isoflavones on the gonadal development and muscle quality in adult male E. sinensis. Soy isoflavones may regulate the gonadal development of male crabs through the “eyestalk-accessory gonad-testis” endocrine axis. Additionally, they could increase the beneficial fatty acids content in muscles, and promote nutrient deposition by activating the mTOR pathway, consequently improving muscle nutritional quality. Moreover, soy isoflavones also flavor characteristics by enhancing muscle antioxidant capacity, optimizing muscle fiber characteristics, and regulating the composition of FAAs and nucleotides. Some of the materials in the figure come from the following online website: https://gdp.renlab.cn/#/.

CRediT authorship contribution statement

Long He: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Kaiqi Huang: Investigation, Formal analysis, Data curation. Xiaodan Wang: Methodology, Investigation, Conceptualization. Jianguang Qin: Writing – review & editing, Methodology, Investigation. Erchao Li: Writing – review & editing, Supervision, Resources, Conceptualization. Liqiao Chen: Supervision, Resources, Methodology, Investigation, Conceptualization.

Declaration of competing interest

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

Data availability

Data will be made available on request.