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. 2025 Apr 12;27:102460. doi: 10.1016/j.fochx.2025.102460

Enzymolysis pretreatment followed by fermentation is a novel method to prepare shrimp sauce with high quality from by-products in crayfish (Procambarus clarkii)

Bin Peng a,1, Sijie Xu a,1, Huimin Ma a,1, Chengwei Yu a, Mingming Hu a, Bizhen Zhong a, Zongcai Tu a,b,c, Jinlin Li a,c,⁎
PMCID: PMC12063136  PMID: 40351495

Abstract

A rapid fermentation process after enzymolysis pretreatment was applied to prepare shrimp sauce using crayfish heads. The optimal fermentation process through orthogonal experiments was obtained: 35 °C, 25 % koji addition, and 5 % salt addition. During fermentation, the content of amino acid nitrogen reached its maximum value of 7.00 mg/mL at 20 days. The content of volatile base nitrogen was increased to 281.64 mg/100 mL at 30 days. A total of 32 fatty acids were detected in the shrimp sauce, and the oleic acid and pentadecanoic acid existed during 0–9 days of fermentation. A total of 16 free amino acids were detected in shrimp sauce, mainly glutamic acid, leucine, aspartic acid, lysine, and alanine. Flavor and essential amino acids accounted for 51.09 % and 42.79 % at 30 days, respectively. Enzymolysis pretreatment followed by fermentation is a feasible method to prepare high-quality shrimp sauce rich in amino acids and fatty acids from crayfish heads.

Keywords: Crayfish heads, Shrimp sauce, Enzymolysis, Fermentation, Quality

Graphical abstract

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Highlights

  • •

    Enzymolysis followed by fermentation is a novel method to prepare shrimp sauce.

  • •

    Enzymolysis and fermentation of shrimp sauce were optimized.

  • •

    32 fatty acids and 16 free amino acids were detected in the shrimp sauce.

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    High-quality shrimp sauce rich in FAAs and FAs was prepared from crayfish heads.

1. Introduction

Red swamp crayfish (Procambarus clarkii), a freshwater aquatic product, is highly adaptable and widely distributed. Recently, the crayfish industry has grown and processing technology has continued to evolve (Xu et al., 2024). The aquaculture volume of crayfish in 2021 accounted for 8.27 % of the total freshwater aquaculture production in China, ranking sixth in China's freshwater aquaculture species (Gu et al., 2023). The application of crayfish by-product resources is also a problem. There are a lot of available substances in the waste crayfish by-products, and the crayfish head contains rich amino acids, astaxanthin, and protein (Chen, Xu, et al., 2024). The essential amino acids in the crude protein account for over 45 %, which is an excellent protein source (Zhang, Jiang, et al., 2024). At present, there are still many problems and challenges in the recovery and utilization of crayfish heads.

The preparation of high-quality aquatic condiment shrimp sauce is one of the ways to use crayfish heads. There are two main methods of making shrimp sauce, including the traditional fermentation process and the rapid fermentation process. Traditional fermented aquatic products suffer from open-process contamination risks, inconsistent quality, lengthy production (months-years), high salt (> 20 %), inefficient resource utilization, and environmental pollution from saline wastewater and odors (Liu et al., 2021; Yuan et al., 2024). In the rapid fermentation process, a series of methods such as modern biochemistry, microbiology, and advanced brewing technology combined with traditional fermentation technology can shorten the fermentation cycle (Wang et al., 2020). Among them, the commonly used methods are yeast fermentation, enzyme, and heat preservation fermentation. The principle of the enzyme fermentation method is that since the fermentation of aquatic condiments is mainly the function of protease, the addition of exogenous protease to promote protein hydrolysis can effectively improve the fermentation speed. Currently, the commonly used proteases include neutral protease, trypsin, and papain. Compared with other processes, the enzyme system of seed yeast fermentation is more comprehensive, including protease, lipase, amylase, and saccharification enzymes (Pereira Da et al., 2024). It can not only effectively shorten the fermentation time, but also obtain high-quality products, resulting in better flavor and taste of fermented aquatic condiments than other technologies. Although the fermentation period can be shortened by adding enzymes, the enzyme species is single and the cost is high (Chen, Wang, et al., 2024). Bacterial fermentation not only enhances product flavor but also improves nutritional and functional properties (Singh et al., 2024). Combined with enzymatic hydrolysis, the integrated approach addresses key limitations of conventional fermentation, including prolonged processing times, low yields, and suboptimal hygiene standards (Liu et al., 2021). However, it enables efficient and controllable industrial-scale condiment production.

The physicochemical properties of shrimp sauce have a great influence on its quality. A wide variety of microorganisms are present in the fermentation process and contribute to complex biochemical changes, resulting in a great diversity in product properties. Some chemical properties (such as amino acids) are highly influenced by metabolic activity in microorganisms (Gao, Bao, et al., 2023). Shrimp sauce, as a kind of aquatic seasoning, contains fatty acids, amino acids, and other nutrients. Amino acid nitrogen (AAN) is an important standard for flavoring classification, and trichloroacetic acid (TCA) soluble peptide is the embodiment of the protein hydrolysis degree. Fatty acids and amino acids are not only nutrients in shrimp sauce but also important flavor precursors. However, the optimum enzymatic hydrolysis and fermentation process of crayfish heads and the changes in physicochemical properties (AAN, volatile base nitrogen, fatty acids, and free amino acids) during fermentation are still unclear.

In this study, crayfish heads were used to prepare high-quality shrimp sauce by enzymolysis and then fermentation. The optimum enzymatic hydrolysis and fermentation condition of shrimp sauce from crayfish heads and the change of physicochemical properties during fermentation were investigated.

2. Materials and methods

2.1. Sample pretreatment

Crayfish were purchased from JiuJiang Kairui Ecological Agriculture Development Co.LTD. Alkaline protease, neutral protease, papain, and flavor protease were purchased from Beijing Solaibao Technology Co., LTD. Crayfish heads were collected and then washed and crushed. Soybean meal, wheat bran, salt, and sugar were purchased from Rainbow Market in Nanchang. Lactobacillus plantarum, Lactobacillus sake, Bacillus subtilis, Yeast, and Aspergillus oryzae 3.042 were purchased from the Shanghai Microbiological Preservation Center. Aspergillus oryzae CICC 2339 was purchased from the China Industrial Microbiological Preservation Management Center. Other chemicals (analytical grade) used in this study were purchased from Xilong Scientific Co., Ltd., Shantou, China.

2.2. Enzymolysis experiment

Five grams of crayfish heads and 15 g of water were mixed, and then 1 % protease (papain, alkaline protease, flavor protease, and neutral protease) was added to the mixture. The enzymolysis was carried out at 55 °C for 200 min. After enzymolysis, the enzyme was deactivated in a boiling water bath for 10 min. The sample was centrifuged at 4800 rpm for 10 min, and the supernatant was collected to measure the soluble protein.

Single-factor experiment: Five grams of crayfish pulp and 15 g of water were mixed. 0 %, 1 %, 2 %, 4 %, 8 %, and 16 % papain was added to the above mixture at 45, 50, 55, 55, 60, 65, 70, 75, and 80 °C for 0, 50, 100, 200, and 400 min.

Response Surface Methodology (RSM) experiment: According to the results of single-factor experiments, three-factor and three-level response surface optimization experiments (enzymolysis time, 25, 50, 75 min; temperature 75, 65, 55 °C; and enzyme dosage, 4, 8, 12 %) were carried out using Design Expert 8.0 software and Box-Behnken Center. The selection of the condition range referred to the parameter adjacent to the optimal value of the single factor optimization condition as the optimization parameter.

2.3. Determination of soluble protein content

The determination of soluble protein content was referred to the method of Chen, Jing, et al. (2023) with a slight modification. Biuret reagent configuration: 1.5 g copper sulfate and 6.0 g potassium sodium tartrate dissolved in 500 mL water, then 300 mL 10 % NaOH solution and water were added to a plastic bottle of 1.0 L. The standard solution of bovine serum albumin (BSA) was prepared with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mg/mL, which was mixed with the standard solution (biuret reagent = 1:4) at room temperature for 30 min. The absorption value at 540 nm in the above sample was measured to draw a standard curve.

2.4. Fermentation experiment

Fermentation was performed under the optimal conditions of the above enzymatic optimization experiments. About 500 g of crushed shrimp heads were taken and then 8.0 % papain was added. After the reaction, the enzyme was deactivated in a 100 °C water bath for 10 min.

Fermentation single factor test: The enzymic solution of shrimp head fermented by different strains was used to select the best fermentation strains based on AAN. The method of lactic acid bacteria (Lactobacillus sake, Lactobacillus plantarum) fermentation from enzymatic hydrolysate was detected by referring to Wang et al. (2024) method with slight modification. 30 g of enzymatic hydrolysate was taken, and 5 % lactic acid bacteria suspension was added, bacteria concentration of about 1 × 108 cfu/mL, 3 % glucose, and 6 % salt, was cultured at 37 °C. The yeast fermentation enzymatic hydrolysate was carried out according to the method of Tan et al. (2025) with slight modification. 30 g enzymatic hydrolysate was taken, 5 % yeast suspension was added, the bacterial concentration was about 1 × 108 cfu/mL, 3 % glucose, 6 % salt, and cultured at 30 °C. The enzymatic hydrolysate method of rice koji (Aspergillus oryzae CICC 2339/3.042) fermentation was referred to Puspitasari et al. (2024). 30 g of enzymatic hydrolysate was taken, 12 % rice koji and 15 % salt were added, and cultured at 30 °C. The fermentation enzymatic hydrolysate of Bacillus subtilis and Bacillus amylolyticus was slightly modified according to the method of Chen, Xin, et al. (2023). 30 g enzymatic hydrolysate and 5 % Bacillus suspension were added, and the concentration of Bacillus was about 1 × 108 fu/mL. After 3 days of fermentation, 14 % salt was added and cultured at 37 °C. The change of AAN content in fermentation broth under different conditions was detected.

Fermentation orthogonal optimization: According to the results of the single-factor experiments, three conditions (A: Temperature, B: Koji addition, C: Salt addition) and three levels (1,2,3) for the highest AAN in fermentation solution were selected, and the content of AAN was used as the index to design L9 (33) by SPSSAU software. The orthogonal test further optimized the enzymolysis factors, and the levels of orthogonal test factors are shown in Table 2.

Table 2.

Orthogonal experiment results, range analysis (R) and variance analysis (ANOVA) on indicator parameters obtained from L9 (33).

level A: Temperature B: Koji addition C: Salt addition
1 25 15 5
2 30 20 10
3 35 25 15
No. A B C Amino acid nitrogen mg/mL
1 1 1 1 3.19
2 1 2 3 3.20
3 1 3 2 3.83
4 2 1 3 2.86
5 2 2 2 3.60
6 2 3 1 4.38
7 3 1 2 2.95
8 3 2 1 4.24
9 3 3 3 4.05
K1 1.023 0.899 1.182
K2 1.084 1.104 1.038
K3 1.124 1.227 1.011
k1 0.341 0.300 0.394
k2 0.361 0.368 0.346
k3 0.375 0.409 0.337
R 0.034 0.109 0.057
Order B > C > A
Excellent level A3 B3 C1
Excellent A3B3C1
Source Type III Sum of Squares Df. Mean Square F.
Corrected Model 0.077 6 0.013 56.664
Intercept 3.478 1 3.478 15,402.27
C 0.017 2 0.008 37.256
A 0.005 2 0.003 11.5
B 0.055 2 0.027 121.237
Error 0.005 20 0
Total 3.56 27
Corrected Total 0.081 26
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A. R Squared = 0.944(Adjusted R Squared = 0.928)

2.5. Measurement of pH, total acid, and amino acid nitrogen

The pH, total acid, and AAN were determined by the acidometer method Determination of Amino acid Nitrogen in Food Safety National Standard in GB5009.235–2016.

2.6. TCA soluble peptide examination

TCA soluble peptide in the samples was examined by referring to Yang et al. (2023) with slight modification. Accurately 2.00 g of shrimp sauce sample and 1 mL 5 % Trichloroacetic acid (TCA) solution were added into homogenizer for 3 min. Then the mixture was refrigerated at 4 °C for 1.0 h and centrifuged at 4 °C and 5000 r/min for 10 min. The content of TCA soluble peptide was determined by the biuret method. The standard curve was drawn with bovine serum protein as the standard substance.

2.7. Total volatile base nitrogen determination

Total volatile base nitrogen (TVB-N) was tested by referring to GB 5009.228–2016 determination of volatile base nitrogen in food under the national standard for food safety.

2.8. Fatty acid composition analyses

The determination of fatty acid composition was modified based on Sivakanthan et al. (2024). The oil sample was taken in a glass bottle, added with 1.0 mL n-hexane and 100 μL 4 % Naoh-methanol solution, oscillated and mixed, placed in a 37 °C water bath for methyl esterification reaction for 35 min, centrifuge 0.5 mL n-hexane layer and 0.22 μm organic film, and injected for analysis.

The column was a DBWAX fused quartz capillary column (30 m × 0.25 mm × 0.25 μm). The inlet temperature was 250 °C. The pressure was 24.52 psi; The total flow rate was 29.4 mL/min. Constant pressure without shunt injection; The sample size was 1.0 μL. The carrier gas was He (99.99 %); Column pressure was 24.52 psi; Programmed temperature rise: 50 °C for 1.0 min, 25 °C/min to 200 °C, 3 °C/min to 230, held for 18 min.

2.9. Free amino acid assay

The determination of free amino acid (FAA) assay has been partially modified based on a published report (Li et al., 2023). Eight mL of samples were absorbed into a centrifuge tube and centrifuged at 3000 rpm for 5.0 min. Accurately 1.0 mL of supernatant was absorbed into another centrifuge tube, and 9.0 mL of 2 % sulfosalicylic acid was added to mix well, leaving for 15 min. The above mixture was centrifuged at 3000 rpm for 20 min, the supernatant was collected to pass 0.22 μm film.

2.10. Statistical analysis

All experiments were conducted in triplicate, and the results were expressed as mean ± standard deviation. Origin 2021 was used to plot the experimental data, and SPSS 16.0 statistical software was used to conduct a one-way analysis of variance (ANOVA) on the data. Duncan test was used to analyze the significance difference, and p < 0.05 indicated a significant difference.

3. Results and discussions

3.1. Optimization of enzymolysis conditions

3.1.1. Single-factor experiment in enzymolysis

The results of soluble protein in enzymatic hydrolysate of crayfish heads with different proteases are shown in Fig. 1(A). The content of soluble protein in enzymatic hydrolysate obtained by papain (916.99 ± 35.97 μg/mL) was significantly higher than that of neutral protease (814.16 ± 46.89 μg/mL), flavor protease (538.78 ± 29.68 μg/mL), and alkaline protease (732.03 ± 176.94 μg/mL) (p < 0.05). Therefore, papain was selected to optimize the conditions of subsequent experiments. The soluble protein content in the enzymatic hydrolysate increased with the increase of reaction temperature at 40–65 °C, and reached the highest (7.13 ± 0.16 mg/mL) at 65 °C (Fig. 1B). The soluble protein content decreased significantly as the temperature rose to 70 °C, indicating that 70 °C was no longer the appropriate enzymolysis for papain. The contents of soluble protein in the enzymatic hydrolysate increased slightly at 75 °C and 80 °C, but they were still lower than that at 65 °C, suggesting that high temperature might promote the dissolution of protein. Therefore, the optimum temperature for enzymatic hydrolysis of papain was 65 °C. As shown in Fig. 1(C), the soluble protein content increased significantly (p < 0.05) from 2.71 ± 0.17 μg/mL to 6.26 ± 0.21 μg/mL as the papain dosage increased from 0 % to 4 %. When the amount of enzyme was greater than 4 %, the soluble protein content in the enzymatic hydrolysate did not change significantly (p > 0.05). The soluble protein growth curve tended to be horizontal as the amount of enzyme was up to 8 %, which might be that the content of the substrate was limited. As the amount of protease reached the optimal concentration, increasing the amount of protease would not help the enzymatic hydrolysis. Therefore, the optimal enzyme dosage of papain was 8 %. As depicted in Fig. 1(D), there was no significant difference in soluble protein content (p > 0.05) at 50–200 min of enzymolysis time. The soluble protein content increased significantly (p < 0.05) as the enzymolysis time reached 400 min. Considering the cost management in industrial production, 50 min was selected as the favorable time for enzymatic hydrolysis. In conclusion, the optimal enzymolysis temperature was 65 °C, the optimal enzymolysis amount was 8 %, and the optimal enzymolysis time was 50 min.

Fig. 1.

Fig. 1

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Soluble protein content in enzymatic hydrolysis from crayfish heads under different conditions. (A) Protease type; (B) Temperature; (C) Enzyme dosage; (D) Reaction time.

3.1.2. Response surface methodology in enzymolysis

Regression analysis was carried out on the 17 groups of data in Table 1 using the Design Expert 12.0 software, and then the equation and the response surface regression model were established. As shown in Table 1, the enzymolysis temperature, enzyme addition, and enzymolysis time were treated as variables. The regression equation of soluble protein content was 8.05–0.0043 A + 0.0260B + 0.2590C-0.0666AB-0.0637 AC-0.0376 BCE-0.3808A2–0.3316B2–0.1435C2. Further variance analysis was performed on the regression equation. The coefficient of determination (R2) represented the similarity between the measured value and the estimated value, and the R2 was 0.8972, indicating that 89.72 % of the measured value agreed with the estimated value. This result indicated that a high degree of similarity between the measured and estimated values was built (Zhang, Li, et al., 2023). The Adjusted coefficient (Adjusted R2) represented the effective metric fit, and the adjusted coefficient (Adjusted R2) of the regression equation was 0.7651, indicating that the model could explain 76.51 % of the change in response value. The model demonstrated approximately 23.5 % unexplained variability, with a significant discrepancy (Δ R2 = 0.1321) between the R2 and adjusted R2 values. Both observations indicate that the model may contain redundant variables or inadequately capture critical nonlinear relationships. For instance, microbial communities exhibit nonlinear behavioral patterns while transitioning from the lag to the stationary phase. The standard quadratic polynomial approach in RSM might fail to characterize these complex dynamics. Furthermore, the current model formulation omitted key substrate parameters, including concentration, crystallinity, and particle size characteristics, while neglecting the effects of metabolic byproduct accumulation during fermentation. These unaccounted factors substantially contributed to the observed variability gap. The adjusted R2 penalizes redundant predictors, revealing unexplained variations arising from unmeasured microbial dynamics and substrate heterogeneity, ultimately resulting in a significantly reduced adjusted R2 value. Adeq Precision referred to the standard deviation ratio of expected product performance to its performance fluctuation (Fig. S1). Generally, only the Adeq Precision is greater than 4 can the results be accurate (Hu et al., 2024). In this experiment, the Adeq Precision was 6.3039, indicating a successful model design and credible experimental results were built.

Table 1.

The optimization results of response surface methodology in enzymatic hydrolysis of crayfish heads.

No. A: Temperature(°C) B: Enzyme dosage (%) C: Time (min) Soluble protein mg/mL
1 65 8 50 8.33
2 65 4 25 7.25
3 65 8 50 8.12
4 55 4 50 7.29
5 55 8 75 7.75
6 55 12 50 7.53
7 65 8 50 7.92
8 75 8 75 7.76
9 65 4 75 7.89
10 65 12 75 7.81
11 65 8 50 8.04
12 75 8 25 7.42
13 75 4 50 7.27
14 55 8 25 7.15
15 65 12 25 7.32
16 75 12 50 7.24
17 65 8 50 7.81
Std. Dev. 0.1743 R2 0.8972 Std. Dev.
Mean 7.6400 Adjusted R2 0.7651 Mean
C.V. % 2.2800 Predicted R2 0.4625 C.V. %
Adeq Precision 6.3039 Adeq Precision
Factor Sum of squares Df. Mean Square F. P
Model 1.86 9 0.2063 6.79 0.0097
A-Te 0.0002 1 0.0002 0.005 0.9458
B-Enzyme dosage 0.0054 1 0.0054 0.1786 0.6852
C-Time 0.5365 1 0.5365 17.67 0.004
AB 0.0177 1 0.0177 0.5833 0.47
AC 0.0162 1 0.0162 0.5337 0.4888
BC 0.0057 1 0.0057 0.1864 0.679
A2 0.6105 1 0.6105 20.1 0.0029
B2 0.463 1 0.463 15.24 0.0059
C2 0.0867 1 0.0867 2.86 0.1349
Residual 0.2126 7 0.0304
Lack of fit 0.054 3 0.018 0.4542 0.7286
Pure error 0.1586 4 0.0396
Cor total 2.07 16
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The variance analysis and significance test of the model are shown in Table 2. According to the model, the Pmodel was 0.0097, which was less than 0.05, indicating that this model was significant, the model F value was only 0.97 % probability of interference. The missing item = 0.729, which was greater than 0.05, indicating that the response surface model was good, and the response values of each factor value could be functionally adjusted by this regression model (Chang et al., 2024). In the primary term of the regression equation, enzymolysis time and temperature had significant effects on the content of soluble protein, and the p values of A2 and B2 were less than 0.05, indicating that temperature and temperature, enzyme dosage and enzyme dosage had a significant influence.

The response surface plot (Fig. 2) exhibits a distinct peak within the experimental range, indicating an optimal point for soluble protein extraction. As the temperature was between 60 and 70 °C and the amount of enzyme addition was between 6 and 10 %, the content of soluble protein appeared to be the highest (Fig. 2A). As the time was greater than 45 min and the temperature appeared at the highest point between 60 and 70 °C, the soluble protein content increased with the increase of enzymolysis time, and finally it was flat (Fig. 2B). As the time was greater than 45 min, the amount of enzyme added appeared at the highest point between 6 and 10 % (Fig. 2C). According to the quadratic regression equation model and the response surface curve, the maximum enzymolysis conditions were 64 °C of temperature, 8.00 % of enzyme dosage, and 73 min of enzymatic hydrolysis time.

Fig. 2.

Fig. 2

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Three-dimensional analysis of response surface methodology.

The optimized condition was a temperature of 64 °C, an enzyme amount of 7.979 %, and an enzymatic hydrolysis time of 73 min. Under the optimal conditions, the optimal soluble protein concentration was 8.165 mg/mL. The soluble protein concentration in the experimental result was 8.143 mg/mL, which was consistent with the prediction results of the optimal response surface conditions. Therefore, the optimal enzymolysis conditions were as follows: 64 °C of temperature, 8.00 % of enzyme amount, and 73 min of enzymolysis time. Under this condition, the soluble protein content was 8.143 mg/mL.

3.2. Optimization of fermentation reactive conditions

3.2.1. Single factor experiment of fermentation

The content of AAN in fermentation broth obtained from enzymatic hydrolysate of different strains is shown in Fig. 3 (A). The content of AAN in the fermentation broth obtained from lactic acid bacteria, Bacillus, and Yeast was less than 1.00 mg/mL. The content of AAN in the fermentation broth of Aspergillus oryzae (greater than 3.00 mg/mL) was significantly higher than that of other bacteria (p < 0.05). The total acid content in the fermentation broth obtained from the enzymatic hydrolysate of different strains is shown in Fig. 3 (B). The total acid content of the fermentation broth obtained by Lactobacillus plantarum was the highest (70 g/L), which might be due to the metabolism of organic acid produced by Lactobacillus plantarum during fermentation. The total acid content of the fermentation broth obtained by Yeast 2 and two Aspergillus oryzae was lower than that of Lactobacillus plantarum, and higher than that of the other three strains, ranging from 16.50 to 22.00 g/L. The total acid content of the fermentation broth obtained by the other three strains was lower than 15.00 g/L. The content of AAN in Lactobacillus plantarum fermentation broth was the lowest (0.50 mg/mL). It might be due to the low proteolytic capacity of Lactobacillus plantarum, which was unable to efficiently convert and metabolize nutrients in the enzymatic hydrolysate (Satılmış et al., 2023). The content of AAN in the fermentation broth of Lactobacillus sake and Lactobacillus plantarum was low (0.70 mg/mL). The total acid content was low (13.40 g/L), which was much lower than the total acid content in Lactobacillus plantarum fermentation broth, possibly due to its low acid production capacity. The contents of AAN and total acid in the fermentation broth of the two strains of Bacillus were low (0.60 mg/mL, 0.80 mg/mL, AAN; 12.60 g/L, 9.40 g/L, total acid). These two Bacillus played little role in the enzymatic hydrolysate, which might be that the fermentation conditions were not suitable for the growth. The content of AAN after fermentation of the two yeasts (0.8. mg/mL, 0.70 mg/mL) was not high, which was similar to that of Bacillus. The total acid content was 8.60 g/L and 22.0 g/L, respectively. The total acid content of Yeast 2 was higher than that of the other 6 species except for Lactobacillus plantarum. Lactobacillus plantarum is a kind of lactic acid bacteria, that belongs to edible probiotics, and it is widely used in industrial production, food fermentation, and medical care (Ajibola et al., 2023). The metabolites of Lactobacillus plantarum contain many organic acids, such as phenyllactic acid, lactic acid, and butyric acid (Liu, Du, et al., 2024). Lactobacillus sake is also a kind of lactic acid bacteria, which is one of the important strains in the edible lactic acid bacteria catalog (Yu et al., 2022). Lactobacillus plantarum is commonly used in food fermentation, which can change the flavor and taste of food, and can also produce bacteriocin. Therefore, Lactobacillus sake is widely used in the fermentation of meat products, dairy products, and other foods. Bacillus subtilis fermentation can improve the nutritional value and biological activity of food, such as increasing the phenolic substances in food (Xu et al., 2023). In the fermentation process, yeast can synthesize a large number of enzymes, proteins, and other substances. It also produces esters, advanced alcohols, and other compounds, increasing the flavor complexity of products, and thereby improving product flavor (Singh et al., 2024). Aspergillus oryzae 3.042 is commonly found in the production of domestic soy sauce and it can produce highly active glycosidase, cellulase, and esterase (Zhao et al., 2024). The results indicate that the fermentation broth of Aspergillus oryzae 2339 contains a higher amino acid nitrogen content compared to other strains. Therefore, Aspergillus oryzae 2339 was selected for further fermentation experiments.

Fig. 3.

Fig. 3

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Single factor experiment results of fermentation. Amino acid nitrogen (A) and total acid content (B) after fermentation by different bacterias and fungus. Effects of (C) Salt addition; (D) Aspergillus oryzae addition; (E) Reaction time on the amino acid nitrogen of shrimp sauce from by-products in crayfish during fermentation.

The effect of salt addition on the content of AAN during fermentation is shown in Fig. 3 (C). The content of AAN first increased and then decreased with the increase in salt content. The content of AAN reached the maximum at 10 % salt content and then decreased with the increase in salt content. Adding salt to fermentation broth was mainly the use of salt to inhibit the growth of harmful microorganisms and prevent the fermentation broth from producing odor during fermentation. The increase of salt content would inhibit the effect of Aspergillus oryzae on the fermentation solution as the salt content was greater than 10 %. However, the content of AAN was also lower as the salt addition was lower than 10 %. It might be that when the salt content was low, the microbial growth could not be effectively inhibited, then AAN was consumed (Wang, Huang, et al., 2023). Therefore, the amount of salt addition was about 10 %.

The effect of the amount of koji on the content of AAN in fermentation solution is shown in Fig. 3 (D). The content of AAN increased with the increase of the amount of koji. The increase in the amount of Koji had no significant effect on the content of AAN (p > 0.05) at 20 % of the amount of Koji. As soybean was contained in Aspergillus oryzae koji, plant protein would be introduced, and the content of AAN would increase correspondingly with the increase of koji amount. The protein content in the enzymatic hydrolysate was limited, and the increase in the amount of koji would not accelerate the fermentation process of the fermentation solution. At the same time, the increase in the amount of curvature reduced the fluidity of the fermentation liquid and affected the fermentation process. Therefore, combining the above factors, the optimal amount of koji should be about 20 %.

Temperature is very important for fermented products. The temperature affects the growth of microorganisms during fermentation, thus affecting the metabolism of microorganisms and directly determining the quality of products (Wang, Gai, et al., 2023). The optimal growth temperature of Aspergillus oryzae was 30 °C, and the AAN content of fermentation liquid obtained at different temperatures was shown in Fig. 3 (E). The content of AAN was the highest (2.66 ± 0.07 mg/mL) at 30 °C, which was in line with the optimal growth temperature of Aspergillus oryzae. It was not the optimal temperature for Aspergillus oryzae as the temperature was below or above 30 °C. Therefore, 30 °C was chosen as the optimal fermentation temperature.

3.2.2. Fermentation orthogonal experiment

Based on the results of the single factor experiment, L9 (33) orthogonal test was designed using SPSSAU to further optimize each factor, and the results are shown in Table 2. Data analysis software SPSS was used to further analyze the orthogonal experiment results. The order of influence of the three factors on the content of AAN in fermentation liquid was B (the amount of yeast addition) > C (the amount of salt addition) > A (temperature). The optimal conditions for fermentation of shrimp head enzymatic hydrolysate by Aspergillus oryzae were A3B3C1: the temperature was 35 °C, the amount of koji was 25 %, and the amount of salt was 5 %. In single-factor experiments, with other factors held constant, the optimal temperature might be 30 °C and inoculum size 20 %. But significant interactions among multiple factors can occur. Orthogonal experiments account for these, finding globally optimal conditions under combined effects and giving more accurate results. For example, when temperature, inoculum size, and salt concentration are considered together, 35 °C, 25 %, and 5 % prove better. However, future research should prioritize pilot-scale validation to investigate the adaptability of the optimized conditions in scaled production systems. Pilot-scale implementation still encounters critical scalability challenges, particularly in maintaining batch consistency and meeting energy demands. Concurrently, economic feasibility concerns persist, notably regarding the cost-efficiency of enzyme utilization and microbial strain maintenance.

3.3. Changes in physicochemical properties during fermentation of shrimp sauce

3.3.1. Changes in pH, total acid, and amino acid nitrogen

Acidity can reflect the fermentation quality of shrimp sauce and it is the result of various biochemical reactions during fermentation. The maps and pH changes in shrimp sauce samples prepared at different fermentation times are shown in Fig. 4 (A) and Fig. 4 (B), respectively. The pH value of shrimp sauce decreased rapidly at 0–9 days, stabilized at 9–15 days, and then increased slightly at 15–30 days during fermentation. During fermentation, a large amount of organic acids were produced, and then the pH value in the early stage dropped significantly (p < 0.05). The microbial acid production rate slowed down, and it tended to be stable. In addition, during fermentation, amino acids would be decarboxylated and deammoniated and other biochemical reactions would produce slightly alkaline substances such as volatile base nitrogen, resulting in a slight increase in pH value (Liu, Deng, et al., 2024). Total acids in shrimp sauce included organic acids, and fatty acids, which were precursors of important components of shrimp flavor. As shown in Fig. 4 (C), the total acid content in shrimp sauce showed an overall rising trend, and the rising speed slowed down with the extension of fermentation time, and became stable after 15 days of fermentation.

Fig. 4.

Fig. 4

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Changes in physicochemical indexes of shrimp sauce from by-products in crayfish during fermentation: pH (A); total acid (B); amino acid nitrogen (C); TCA (D); and TVB-N content (E).

AAN is an important criterion to evaluate the quality of shrimp sauce, which can reflect the degree of protein utilization in microbial shrimp sauce. The change of AAN content in shrimp sauce is shown in Fig. 4 (D). During 0–20 days of fermentation, the content of AAN gradually increased to 7.00 mg/mL at 20 days, reaching the national secondary fish sauce standard, and then decreased slightly. The increase in AAN content in the early stage was due to the enzyme produced by Aspergillus oryzae acting on the protein to degrade it, increasing AAN content. After 20 days, the decrease in AAN results from microbial consumption of free amino acids as a nitrogen source, declining protease activity due to substrate changes which slows protein degradation, and amino acid depletion through deamination into ammonia and organic acids (Ritter et al., 2023). On day 25, the amino acid nitrogen content was decreased, reaching the national tertiary fish sauce standard.

3.3.2. Changes in TCA soluble peptide content

TCA soluble peptide is mainly derived from the degradation of protein, which can reflect the hydrolysis of protein in shrimp sauce. As shown in Fig. 4 (E), TCA soluble peptide content in shrimp sauce increased first and then decreased. The content of TCA soluble peptide increased in the early stage, indicating that the protein in the shrimp sauce was hydrolyzed during fermentation (Zhou et al., 2025), and the content decreased in the later stage. It may be due to the restriction of protein content, coupled with the use of microorganisms for further decomposition.

3.3.3. Changes of volatile basic nitrogen content

The change of TVB-N content in the fermentation process of shrimp sauce is shown in Fig. 4 (F). The content of TVB-N increased with time, from 68.95 mg/mL to 281.64 mg/mL. TVB-N was mainly derived from ammonia and trimethylamines produced by the decomposition of proteins into amino acids. During fermentation, the protein was continuously broken down due to protease production by Aspergillus oryzae. Therefore, the content of TVB-N was also increasing during fermentation. The TVB-N value of such products was generally required not to exceed 250 mg/mL. For the first 25 days, the TVB-N values of the enzymatic hydrolysate remained below the 250.00 mg/mL threshold, reaching 203.96 mg/mL on day 25. On day 30, it shot up to 281.64 mg/mL, going over the standard. The increase in TVB-N after 25 days might be due to bacterial proliferation. Initially, adding salt and Aspergillus oryzae inhibits spoilage bacteria from using proteins. Later, halotolerant bacteria gradually multiply over time. They decompose proteins, producing ammonia and trimethylamine, thus spoiling fish muscle and increasing TVB-N (Zhang, Jiang, et al., 2024; Zhang, Yu, et al., 2024).

Therefore, 25 days of fermentation for shrimp sauce is better when the volatile basic nitrogen is used as the criterion. Compared with traditional fermentation methods, enzymolysis pretreatment followed by fermentation can be completed within 25 days by selecting efficient strains and optimizing factors such as temperature and salt concentration. This novel process has improved fermentation efficiency, shortened production time, reduced energy, and lowered costs.

3.3.4. Changes in fatty acid composition and content

Fatty acids are pivotal precursors for flavor in shrimp paste fermentation. Their oxidative metabolism yields small molecular compounds that contribute significantly to the flavor. Also, research indicates fish oil diet fatty acids uniquely reduce pro-tumor macrophages (Liu et al., 2020). As shown in Table S1, a total of 32 fatty acids were detected in shrimp sauce, the distribution range was C8–24, including 15 saturated fatty acids (SFA), 8 monounsaturated fatty acids (MUFA), and 9 polyunsaturated fatty acids (PUFA). Shrimp sauce exhibited the highest SFA content (44.49 % to 61.15 %), with no significant changes in total SFA during fermentation (p > 0.05). Palmitic acid and stearic acid accounted for more than 40 % of the total contents. Palmitic acid could reduce the content of serum cholesterol in the human body, while stearic acid could inhibit the absorption of cholesterol and regulate the production of cholic acid (Zhu et al., 2023). The content of MUFA in shrimp sauce was between 19.03 % and 28.54 %, which showed a decreasing trend during fermentation. The content of oleic acid in MUFA was the highest (13.13 % to 20.55 %), which showed a trend of first increasing and then decreasing during fermentation. Palmitoleic acid could be oxidized to produce alcohols (Gao, Liu, et al., 2023). PUFA has unique physiological functions, which could improve cardiovascular diseases, inflammatory responses, and other diseases (Spaggiari et al., 2024). PUFA could be oxidized and degraded in food processing, thus forming small molecule compounds with unique flavors (Zhang, Li, et al., 2023). The content of PUFA in shrimp sauce, mainly linoleic acid and linolenic acid, increased first and then decreased during fermentation, accounting for 19.81 % to 30.24 %. The content of linoleic acid was the highest (15.37 % to 26.54 %) in PUFA, and it rose first and then decreased during fermentation. Linoleic acid could be oxidized to produce a variety of flavor substances, such as hexaldehyde and 1-octene-3-ol, which were very important for the formation of food flavor (Liu et al., 2023). The degradation of linolenic acid could produce volatile substances such as enal and dienal, and their contents gradually decreased during fermentation. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in PUFA also had unique physiological functions for the human body. EPA/DHA improved the development of the nervous and visual systems, and reduce the incidence of Alzheimer's disease and cardiovascular diseases (Kousparou et al., 2023; Liu et al., 2020). In short, in shrimp paste, SFA mainly palmitic and stearic acids) showed stable levels due to oxidation resistance. MUFA decreased overall, with oleic acid rising and then falling and palmitoleic acid oxidizing into alcohols. PUFA peaked then declined as linoleic and α-linolenic acids oxidized readily.

3.3.5. Changes in composition and content of free amino acids

FAAs are important flavor precursors in fermented condiments, as well as important indexes for evaluating their umami and quality (Han et al., 2024). The changes in FAA content in the fermentation process of shrimp sauce are shown in Table S2. A total of 16 kinds of amino acids were detected in the shrimp sauce, including 6 flavor amino acids and 6 essential amino acids. During fermentation, the total free amino acid (TFAA) content initially increased and then decreased. It might be that Aspergillus oryzae secreted protein-metabolizing enzymes, initiating proteolysis and promoting TFAA production and preservation. As proteins were depleted, free amino acids were also broken down and utilized (Han et al., 2024). TFAA reached a maximum (30.35 mg/mL) at 25 days and then decreased slightly during fermentation. The FAAs were mainly glutamic acid, aspartic acid, alanine, leucine, valine, and lysine. The content of glutamic acid was the highest in FAAs, reaching 7.29 mg/mL. Glutamic acid was a typical umami amino acid, which could provide umami flavor for shrimp sauce. In addition to glutamic acid, flavor amino acids were aspartic acid, alanine, glycine, tyrosine, and phenylalanine. The contents of aspartic acid, glycine, alanine, and phenylalanine exceeded 1.0 mg/mL at the 15–30 days of fermentation, and the content of aspartic acid reached 4.00 mg/mL at 25 days. Flavorful amino acids were abundant in shrimp sauce, accounting for more than half of the amino acid content, which showed a trend of first increasing and then decreasing during fermentation and reaching the maximum value (16.66 mg/mL) at 25 days. A total of 6 essential amino acids were detected in shrimp sauce, namely valine, methionine, isoleucine, leucine, phenylalanine, and lysine. The content of essential amino acids accounted for more than one-third of the total amino acids, which first increased and then decreased during fermentation and reached the maximum value of 11.84 mg/mL at 20 days. The highest content of essential amino acids was leucine, followed by lysine and valine.

4. Conclusion

In conclusion, the enzymatic hydrolysis pretreatment and fermentation process of shrimp sauce from crayfish heads were studied, and the changes of AAN, TCA soluble peptide, volatile base nitrogen, fatty acids, and FAAs during fermentation were investigated. The optimal enzymolysis conditions were as follows: temperature 64 °C, enzyme amount 8.00 %, and enzymolysis time 73 min. Under this condition, soluble protein content was 8.143 mg/mL. The optimal fermentation process was as follows: 35 °C of the temperature, 25 % of the amount of Aspergillus oryzae, and 5 % of the amount of salt. During fermentation, pH in shrimp sauce decreased first then slightly increased, and finally became acidic. The total acid content increased first and then stabilized, reaching about 70 g/L. The content of AAN increased with the extension of fermentation time and finally tended to be stable. The content of volatile basic nitrogen increased with the extension of fermentation time. A total of 32 fatty acids were detected in the shrimp sauce, most of which were SFA. The content of MUFA decreased slightly and the content of PUFA did not change much during fermentation. A total of 16 amino acids were detected in shrimp sauce, and their contents increased with time, reached the maximum at 25 days then slightly decreased. Shrimp sauce was rich in flavor and essential amino acids. For quality control, the optimum fermentation time of an enzymatic solution made by Aspergillus oryzae from crayfish heads was within 25 days. In summary, this enzyme-bacteria synergy facilitates shrimp sauce production while enhancing flavor and nutrition, ensuring efficiency and safety.

CRediT authorship contribution statement

Bin Peng: Writing – original draft, Project administration, Methodology. Sijie Xu: Resources, Project administration. Huimin Ma: Investigation, Conceptualization. Chengwei Yu: Writing – review & editing, Data curation. Mingming Hu: Investigation, Formal analysis. Bizhen Zhong: Formal analysis, Data curation, Conceptualization. Zongcai Tu: Resources, Project administration. Jinlin Li: Validation, Software, Project administration.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. All authors declare no competing financial interest.

Acknowledgments

This work was supported by the Project of High-value Comprehensive Utilization Research Center for Agricultural Products of Jiangxi Normal University supported by the Administration Committee of Qinzhou Port Area, China (Guangxi) Pilot Free Trade Zone, the earmarked fund for CARS (CARS-45), the Youth Fund Project of Natural Science Foundation of Jiangxi Province (20242BAB20327, 20232BAB215060), and Zhejiang Provincial Postdoctoral Research Project in 2023 (319030).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2025.102460.

Appendix A. Supplementary data

Supplementary material

mmc1.docx (69.1KB, docx)

Data availability

Data will be made available on request.

References

  1. Ajibola O.O., Thomas R., Bakare B.F. Selected fermented indigenous vegetables and fruits from Malaysia as potential sources of natural probiotics for improving gut health. Food Science and Human Wellness. 2023;12(5):1493–1509. doi: 10.1016/j.fshw.2023.02.011. [DOI] [Google Scholar]
  2. Chang G.H., Liu Y., Luo Z.H., Ni K., Zhang P.F., Zhou T.…Wang X. Response surface methodology to optimize the sterilization process of slightly acidic electrolyzed water for Chinese shrimp (Fenneropenaeus chinensis) and to investigate its effect on shrimp quality. Food Chemistry: X. 2024;21 doi: 10.1016/j.fochx.2024.101180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen B.M., Xu X.Q., Chen Y.J., Xie H.K., Zhang T., Mao X.Z. Red swamp crayfish (Procambarus clarkii) as a growing food source: Opportunities and challenges in comprehensive research and utilization. Foods. 2024;13(23) doi: 10.3390/foods13233780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen J., Wang W.W., Jin J.H., Li H., Chen F., Fei Y.…Wang Y.B. Characterization of the flavor profile and dynamic changes in Chinese traditional fish sauce (Yu-Lu) based on electronic nose, SPME-GC-MS and HS-GC-IMS. Food Research International. 2024;192 doi: 10.1016/j.foodres.2024.114772. [DOI] [PubMed] [Google Scholar]
  5. Chen Y.D., Xin Q.L., Pan L., Wang B. Improved recombinant expression of maltogenic α-amylase AmyM in Bacillus subtilis by optimizing its secretion and NADPH production. Fermentation. 2023;9(5) doi: 10.3390/fermentation9050475. [DOI] [Google Scholar]
  6. Chen Y.H., Jing H.Z., Xiong S.B., Manyande A., Du H.Y. Comparative study on hydrolysis, physicochemical and antioxidant properties in simulated digestion system between cooked pork and fish meat. Foods. 2023;12(9) doi: 10.3390/foods12091757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gao P.X., Bao Y.X., Wang S., Lei L.M., Wang B.C., Xiao L., Liang D. Mechanism of palmitoleic acid oxidation into volatile compounds during heating. Flavour and Fragrance Journal. 2023;38(2):95–107. doi: 10.1002/ffj.3728. [DOI] [Google Scholar]
  8. Gao R.C., Liu H.J., Li Y., Liu H.Y., Zhou Y., Yuan L. Correlation between dominant bacterial community and non-volatile organic compounds during the fermentation of shrimp sauces. Rabie. 2023;12(1):233–241. doi: 10.1016/j.fshw.2022.07.002. [DOI] [Google Scholar]
  9. Gu Y.L., Zhu L., Wang X.R., Li H., Hou L.B., Kong X.H. Research progress of pattern recognition receptors in red swamp crayfish (Procambarus clarkii) Fish & Shellfish Immunology. 2023;141 doi: 10.1016/j.fsi.2023.109028. [DOI] [PubMed] [Google Scholar]
  10. Han J.R., Jiang J.L., Zhao X., Zhao X.L., Kong T., Li P., Gu Q. Comparative analysis of key precursors and metabolites involved in flavor formation of different rapid-fermented Chinese fish sauces based on untargeted metabolomics analysis. Food Chemistry. 2024;433 doi: 10.1016/j.foodchem.2023.136998. [DOI] [PubMed] [Google Scholar]
  11. Hu G.H., Chen X.W., Cao Z.L., Yang L.Z. Optimization design of cotton-straw-fiber-modified asphalt mixture performance based on response surface methodology. Buildings. 2024;14(11) doi: 10.3390/buildings14113670. [DOI] [Google Scholar]
  12. Kousparou C., Fyrilla M., Stephanou A., Patrikios I. DHA/EPA (Omega-3) and LA/GLA (Omega-6) as bioactive molecules in neurodegenerative diseases. International Journal of Molecular Sciences. 2023;24(13) doi: 10.3390/ijms241310717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Li C.J., Tu Z.C., Liu W.Y., Wu C.L., Hu Y.M., Wang H. Flavor substances of low-valued red swamp crayfish (Procambarus clarkii) hydrolysates derived from double enzymatic systems. Food Research International. 2023;165 doi: 10.1016/j.foodres.2023.112461. [DOI] [PubMed] [Google Scholar]
  14. Liu J.G., Lin C.X., Zhang W., Yang Q., Meng J., He L.P.…Zeng X.F. Exploring the bacterial community for starters in traditional high-salt fermented Chinese fish (Suanyu) Food Chemistry. 2021;358 doi: 10.1016/j.foodchem.2021.129863. [DOI] [PubMed] [Google Scholar]
  15. Liu L., Zhao Y.H., Lu S.X., Liu Y.H., Xu X.X., Zeng M.Y. Metabolomics investigation on the volatile and non-volatile composition in enzymatic hydrolysates of Pacific oyster (Crassostrea gigas) Food Chemistry: X. 2023;17 doi: 10.1016/j.fochx.2023.100569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Liu L.L., Jin R., Hao J.Q., Zeng J., Yin D., Yi Y.M., Li B. Consumption of the fish oil high-fat diet uncouples obesity and mammary tumor growth through induction of reactive oxygen species in protumor macrophages. Cancer Research. 2020;80(12):2564–2574. doi: 10.1158/0008-5472.can-19-3184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu M., Deng N., Li H., Hou X.Y., Zhang B., Wang J.H. Characterization and comparison of flavors in fresh and aged fermented peppers: Impact of different varieties. Food Research International. 2024;182 doi: 10.1016/j.foodres.2024.114187. [DOI] [PubMed] [Google Scholar]
  18. Liu Y.C., Du S., Sun L., Li Y.Y., Liu M.J., Sun P.B., Wang Z.J. Volatile metabolomics and metagenomics reveal the effects of lactic acid bacteria on alfalfa silage quality, microbial communities, and volatile organic compounds. Communications Biology. 2024;7(1) doi: 10.1038/s42003-024-07083-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pereira Da S.A., Souza C.P.L., Franson R.C.B., Ferreira T.F., Amaral P.F.F. From Agri-food wastes to enzyme production: A systematic review with method Ordinatio. Waste and Biomass Valorization. 2024;15(10):5843–5870. doi: 10.1007/s12649-024-02565-6. [DOI] [Google Scholar]
  20. Puspitasari C., Pinsirodom P., Wattanachaisaereekul S. Effect of solid-state fermentation using aspergillus oryzae and aspergillus Niger on bitter and bioactive compounds of Moringa oleifera seed flour. LWT. 2024;207 doi: 10.1016/j.lwt.2024.116616. [DOI] [Google Scholar]
  21. Ritter S., Gastl M., Becker T. Impact of germination on the protein solubility and antinutritive compounds of Lupinus angustifolius and Vicia faba in the production of protein-rich legume-based beverages. Journal of Agricultural and Food Chemistry. 2023;71(23):9080–9096. doi: 10.1021/acs.jafc.3c01249. [DOI] [PubMed] [Google Scholar]
  22. Satılmış M.K., Öztürk H.İ., Demirci T., Denktaş B., Akın N. Revealing the proteolytic characteristics of Lactobacillus, Lacticaseibacillus, and Lactiplantibacillus isolates by in vitro and in situ perspectives. Food Bioscience. 2023;55 doi: 10.1016/j.fbio.2023.103086. [DOI] [Google Scholar]
  23. Singh S., Kumar Sharma P., Chaturvedi S., Kumar P., Deepak Nannaware A., Kalra A., Kumar Rout P. Biocatalyst for the synthesis of natural flavouring compounds as food additives: Bridging the gap for a more sustainable industrial future. Food Chemistry. 2024;435 doi: 10.1016/j.foodchem.2023.137217. [DOI] [PubMed] [Google Scholar]
  24. Sivakanthan S., Fawzia S., Mundree S., Madhujith T., Karim A. Investigation of the influence of minor components and fatty acid profile of oil on properties of beeswax and stearic acid-based oleogels. Food Research International. 2024;184 doi: 10.1016/j.foodres.2024.114213. [DOI] [PubMed] [Google Scholar]
  25. Spaggiari R., Angelini S., Di Vincenzo A., Scaglione G., Morrone S., Finello V., Passaro A. Ceramides as emerging players in cardiovascular disease: Focus on their pathogenetic effects and regulation by diet. Advances in Nutrition. 2024;15(7) doi: 10.1016/j.advnut.2024.100252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tan X., Wen L., Li Y., Zhang Q., Tang S., Sheng Y., Lai C. Distinct effects of dilute acid prehydrolysate inhibitors on enzymatic hydrolysis and yeast fermentation. Bioprocess and Biosystems Engineering. 2025;48(1):133–145. doi: 10.1007/s00449-024-03098-x. [DOI] [PubMed] [Google Scholar]
  27. Wang J., Huang X.H., Zhang Y.Y., Li S.J., Dong X.P., Qin L. Effect of sodium salt on meat products and reduction sodium strategies-A review. Meat Science. 2023;205 doi: 10.1016/j.meatsci.2023.109296. [DOI] [PubMed] [Google Scholar]
  28. Wang L., Wang J., Wang P., Liu C., Li X., Chang J., Zhu Q. Effect of corn straw treated with Lactobacillus plantarum and cellulase on ruminal fermentation and microbiota of Hu sheep. Fermentation. 2024;10(8) doi: 10.3390/fermentation10080402. [DOI] [Google Scholar]
  29. Wang Y.Q., Li C.S., Zhao Y.Q., Li L.H., Yang X.Q., Wu Y.Y.…Yang S.L. Novel insight into the formation mechanism of volatile flavor in Chinese fish sauce (Yu-lu) based on molecular sensory and metagenomics analyses. Food Chemistry. 2020;323 doi: 10.1016/j.foodchem.2020.126839. [DOI] [PubMed] [Google Scholar]
  30. Wang Y.R., Gai J.S., Hou Q.C., Zhao H.J., Shan C.H., Guo Z. Ultra-high-depth macrogenomic sequencing revealed differences in microbial composition and function between high temperature and medium–high temperature Daqu. World Journal of Microbiology and Biotechnology. 2023;39(12) doi: 10.1007/s11274-023-03772-4. [DOI] [PubMed] [Google Scholar]
  31. Xu R.P., Tian T., Hu B., Zhang Z.Q., Liu J., Yu D.H., Xu H.S. Effect of solid-state fermentation with Bacillus pumilus on the nutritional value, anti-nutritional factors and antioxidant activity of faba bean (Vicia faba L.) meal. LWT. 2023;185 doi: 10.1016/j.lwt.2023.115117. [DOI] [Google Scholar]
  32. Xu W.S., Yang Q.F., Li D., Liu X.Y., Yang P.H., Song L., Zhou D.Y. Elucidating the effects of precooked treatments on the quality attributes of red swamp crayfish (Procambarus clarkia): Insights from water boiling vs. microwaving. Food Chemistry: X. 2024;23 doi: 10.1016/j.fochx.2024.101692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yang B.B., Liu Y.L., Sang Y.X., Sun J.L. Bacterial diversity and physicochemical properties of low-salt shrimp sauce fermented at different temperatures. LWT. 2023;187 doi: 10.1016/j.lwt.2023.115277. [DOI] [Google Scholar]
  34. Yu L., Chen Y., Duan H., Qiao N., Wang G., Zhao J.…Chen W. Latilactobacillus sakei: A candidate probiotic with a key role in food fermentations and health promotion. Critical Reviews in Food Science and Nutrition. 2022;64(4):978–995. doi: 10.1080/10408398.2022.2111402. [DOI] [PubMed] [Google Scholar]
  35. Yuan L., Sun L., Xiong Z., Zhang Q., Jin W., Gao R. Preparation and mechanistic exploration of fermented shrimp surimi gel utilizing enterococcus lactis S-15. Food Chemistry: X. 2024;22 doi: 10.1016/j.fochx.2024.101314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhang L., Jiang X.D., Wu X.G., Wang X.C., Zhang R.Y. Dietary supplementation with synthetic astaxanthin and DHA interactively regulates physiological metabolism to improve the color and odor quality of ovaries in adult female Eriocheir sinensis. Food Chemistry. 2024;430 doi: 10.1016/j.foodchem.2023.137020. [DOI] [PubMed] [Google Scholar]
  37. Zhang L., Yu Y., Tan C., Nie S., Wen Q., Tu Z. Exploration of changes in sensory, physicochemical properties and microbial metabolic activities of grass carp meat with five thermal processing treatments during refrigerated storage. Food Chemistry: X. 2024;23 doi: 10.1016/j.fochx.2024.101662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhang Y.Z., Li Y.Q., Ren X.X., Zhang X., Wu Z., F., & Liu, L. L. The positive correlation of antioxidant activity and prebiotic effect about oat phenolic compounds. Food Chemistry. 2023;402 doi: 10.1016/j.foodchem.2022.134231. [DOI] [PubMed] [Google Scholar]
  39. Zhao C., Zhang Y.X., Li S.S., Lin J.Y., Lin W.F., Li W.X., Luo L.X. Impacts of aspergillus oryzae 3.042 on the flavor formation pathway in Cantonese soy sauce koji. Food Chemistry. 2024;441 doi: 10.1016/j.foodchem.2024.138396. [DOI] [PubMed] [Google Scholar]
  40. Zhou Y., Wu J.X., Monto A.R., Yuan L., Gao R.C. Elevated levels of branched chain fatty acids in low-salt fish sauce by co-fermentation: Flavor improvement and metabolism analysis. Journal of the Science of Food and Agriculture. 2025;105(1):305–314. doi: 10.1002/jsfa.13829. [DOI] [PubMed] [Google Scholar]
  41. Zhu L., Fang S.Z., Liu W.W., Zhang H., Zhang Y.Q., Xie Z.H., Yu L.L. The triacylglycerol structure and composition of a human milk fat substitute affect the absorption of fatty acids and calcium, lipid metabolism and bile acid metabolism in newly-weaned Sprague-Dawley rats. Food & Function. 2023;14(16):7574–7585. doi: 10.1039/d2fo01800d. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Supplementary material

mmc1.docx (69.1KB, docx)

Data Availability Statement

Data will be made available on request.

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