Effects of thermal processing methods on the edible quality, nutrition, and metabolites of shrimp of Metapenaeus ensis

Abstract

Thermal processing significantly impacts the quality and characteristics of shrimp meat. In this study, we employed essential physicochemical composition determination, texture analysis, sensory evaluation, an electronic tongue assay, and UHPLC-Q Exactive-MS/MS metabolomics to investigate changes in the edible quality and metabolites of the meat of Metapenaeus ensis meat subjected to different thermal processing procedures (air frying, boiling, frying, drying). A total of 79 key differentially abundant metabolites affecting the eating quality of shrimp were identified. The different metabolites were significantly correlated with the color, meat quality, pH value, and nutritional components of the shrimp (p < 0.05). The primary metabolites affecting shrimp flavour were AMP, D-pyroglutamic acid, succinic acid, and benzoic acid. The flavour richness and sensory evaluation scores of shrimp e-tongues subjected to air frying were the highest. Purines and heterocyclic amines produced in the boiling treatment group are the least harmful and healthiest heat treatment methods. Drying treatment is the most nutritious heat treatment method for ensuring flavour. This study clarified the molecular mechanism of the quality change in shrimp meat after heat treatment. This study provides a theoretical basis for optimizing and selecting Metapenaeus ensis processing technology.

Graphical Abstract

Introduction

Metapenaeus ensis is nutrient-rich, and optimizing its processing methods is crucial for its wider dissemination (Liao et al., 2023). Most research on Metapenaeus ensis focuses on basic biology, aquaculture models, and related disciplines (Y. D. Li et al., 2023). Currently, research on the nutritional changes in shrimp meat under various heat treatment methods remains scarce, and the underlying molecular mechanisms still require further exploration. Therefore, it is necessary to conduct scientific research on the processing technology of Metapenaeus ensis.

Different heat treatment methods are crucial factors influencing the flavour and quality of seafood (Tian et al., 2023). Heat treatment significantly affects the quality and flavour of shrimp meat, as it can promote maturation, improve texture, and enhance flavour. However, it can also lead to losing nutrients such as protein, fat, and amino acids. Additionally, different heat treatments have varying effects on the quality of shrimp meat because of their distinct heat transfer media and rates (Hu et al., 2021). Currently, the common heat treatment methods for shrimp include frying, grilling, microwaving, steaming, boiling, etc. Among these methods, steaming and boiling are the most widely adopted because of their benefits, such as easy cooking, effective nutrient retention, authentic flavour, and healthiness (Nieva-Echevarría et al., 2017). Although frying can improve the flavour and taste of shrimp, the process of frying subjects shrimp and edible oil to severe heat and mass transfer. This results in a series of complex chemical reactions, including the production of harmful substances such as acrylamide, trans fatty acids and polycyclic aromatic hydrocarbons, which is detrimental to human health (Khan et al., 2021). As society develops and ideas progress, people's demand for processed food has gradually shifted towards healthier and more nutritious options. Therefore, selecting the appropriate shrimp hot processing technology is necessary to produce high-quality shrimp products.

The physicochemical changes triggered by alterations in moisture and protein contents during the thermal processing of meat not only result in modifications to color, texture, and flavour but also influence other nutritional components (R. Wang et al., 2019). For example, the oxidative denaturation of crucial lipids and proteins in meat alters its nutritional profile, influencing consumer preferences (AlFaris et al., 2021; H. Chen et al., 2023a). These complex processes necessitate various analytical methods for evaluation. Metabolomics-based approaches can facilitate the establishment of connections between pertinent food attributes and small-molecule metabolites (Qiu et al., 2016). Furthermore, explori00000ng the metabolic mechanisms of different metabolites related to food quality and providing a theoretical basis for the selection of appropriate processing methods for Metapenaeus ensis will help improve the competitiveness of new shrimp products on the market. Research indicates that the characteristic taste compounds of heated shrimp (Bai et al., 2022), including succinic acid, AMP, GMP, IMP, aspartic acid, and glutamate, change significantly. Additionally, adding nucleotides and related compounds to water can improve the flavour of shrimp (R. J. Zhang et al., 2018b). The electronic tongue device evaluation method offers advantages such as low cost, fast detection speed, and high accuracy in qualitative and quantitative analysis (Xu et al., 2022). Liang used the electronic tongue technique to analyse the flavour quality of four thermally processed and raw shrimp types. The electronic tongue could discriminate well between different shrimp samples, with samples from direct roasting or steaming + roasting being more acceptable (Liang et al., 2022).

In conclusion, this study aims to investigate the effects of boiling, air frying, drying, and frying on the edible quality, nutrition, and metabolites of Metapenaeus ensis. By utilizing the metabolomics approach, we established a connection between shrimp meat quality indicators and their associated small molecular metabolites. This enabled us to identify differential metabolites associated with food quality and nutrition, elucidating the molecular mechanisms underlying the changes in shrimp meat quality and nutrition after thermal processing. This research provides theoretical guidance for consumers in choosing suitable processing methods, lays the foundation for establishing quality control techniques during the thermal processing of seafood, and offers new insights for the safe consumption and industrial development of shrimp.

Materials and methods

Materials

Fresh Metapenaeus ensis were purchased from the local seafood market in Kunming, Yunnan Province, stored with oxygenated ice, and transported to the laboratory within 30 min.

Sample preparation

The heating parameters and sensory evaluation results of shrimp meat subjected to different heat treatment methods are summarized. Finally, the optimal parameter settings for different heat treatment methods for shrimp were determined in the following experiments (Table S1).

Live shrimp (19 ± 1.3 g) purchased from the market were narcotized in a mixture of ice and water for 10 min. The thickest part of the middle of the shrimp was taken, and shrimp segments of similar size (approximately 3 cm) were cut, washed with purified water, and placed on ice for backup. The average gram weight of the shrimp segments was 9 g, and they were randomly divided into five groups of 30 each and subjected to five different heat treatments: the raw sample group, boiling group, air frying group, frying group, and drying group. All heat treatment experimental groups were prepared in triplicate.

Figure 1 and Table 1 show the preparation process of the shrimp sample. After the sample has cooled, the intact shrimp segments are first taken out to measure color difference and texture indicators. Then, the shrimp segments are crushed, packaged in a plastic bag, and stored in a −80 °C refrigerator for the subsequent testing of other indicators. Before testing, the meat sample (from the self-sealed bag) is thawed at room temperature (20 °C) for 1 h.

Fig. 1

Flowchart of the heat treatment method for the samples. R: raw shrimp; B: boiling; A: air frying; D: drying; F: frying

Table 1 Sample Preparation methods and conditions

Determination of basic nutrient composition and pH value

Moisture: direct drying method (GB 5009.3–2016); ash: high-temperature combustion method (GB 5009.4–2016); crude protein: Kjeldahl nitrogen determination method (GB 5009.5–2016); crude fat: Soxhlet extraction method (GB 5009.6–2016); pH: methods specified (GB 5009.237–2016). Each indicator is repeated four times, and the three stable data are retained. The average of these values is then taken as the final result.

Color characterization

The colorimetric determination of shrimp meat was carried out via a CM-5 (KONICA MINOLTA, Japan) benchtop spectrophotometer. After calibration with a prescribed whiteboard, the L*, a*, and b* values were recorded via a pair of medium-aperture reflectors placed in front of the new pair of shrimp flesh for determination. Each indicator was measured four times.

Textural property analysis (TPA)

Intact cooked samples were taken and measured via a TMS-PRO texture analyser (FTC, USA) and a 75 mm disc probe, with the test mode set to texture multifaceted profiling (TPA) mode. For compression testing, the shrimp samples were placed parallel to each other on the test platform with a force sensing range of 100 N, a pretest speed of 1 mm/s, two cycles, a test speed of 1 mm/s, a downwards compression distance of 20 mm, and a time interval of 5 s for the measurements, and the experiment was repeated four times.

Electronic tongue analysis

Using a taste analysis system (TS-5000 Z, INSENT, Japan), 30 g of sample with 150 ml of pure water was poured into a blender and mixed for 30 s. The sample was then poured into a centrifuge tube and centrifuged at 4000 rpm for 10 min, and the supernatant was collected for testing. The acidity, sweetness, bitterness, saltiness, freshness, richness, and astringency of the samples were determined. Each sample was repeated four times, three stable datasets were retained, and the average was taken as the final test result.

Sensory evaluation

Sensory evaluation was performed according to Rui's method (Liang et al., 2022) with slight modifications. Five attributes were developed and defined according to the Chinese standard GB 37062, 2018: texture, appearance, flavour, taste, and color. Fifteen food science undergraduates who received professional training conducted the sensory evaluation. Each sample was scored on a 9-point happiness scale ranging from 1 (very disliked) to 9 (very liked).

Metabolomic analysisvia UHPLC-Q Exactive MS/MS

Main chemical reagents and laboratory equipment

The main chemicals used for metabolite extraction, methanol, acetonitrile, and formic acid, were purchased from Fisher (Thermo Fisher Scientific, USA). The tissue breaker (JXFSTPRP-24, Josin), chromatographic column (T3 column (100 mm * 2.1 mm, 1.8 µm), Waters), ultrahigh-performance liquid chromatography (Vanquish Flex UHPLC, Thermo), and high-resolution mass spectrometry (Q Exactive, Thermo) were purchased from Fisher.

Metabolite extraction and chromatographic and mass spectrometric conditions

Samples were thawed on ice. Metabolites were extracted using 50% methanol buffer. Briefly, 100 mg of the sample was extracted with 1 ml of pre-cooled 50% methanol, vortexed for 1 min, and incubated at room temperature for 10 min. The extraction mixture was then stored overnight at −20 °C. The supernatants were transferred to new 96-well plates after centrifugation at 4,000 g for 20 min. Samples were stored at −80 °C before the LC–MS analysis. In addition, pooled QC samples were also prepared by combining 10 μL of each of the extraction mixtures of each other.

All chromatographic separations were first performed on a Vanquish Flex UHPLC system (Thermo Fisher Scientific, Bremen, Germany). An ACQUITY UPLC T3 column (100 mm * 2.1 mm, 1.8 µm, Waters, Milford, USA) was used for the reversed-phase separation. The temperature of the column oven was maintained at 35 °C. The flow rate was 0.4 ml/min, and the mobile phase consisted of solvent A (water, 0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid). Gradient elution conditions were set as follows: 0 ~ 0.5 min, 5% B; 0.5 ~ 7 min, 5% to 100% B; 7 ~ 8 min, 100% B; 8 ~ 8.1 min, 100% to 5% B; 8.1 ~ 10 min, 5% B.

A Q-Exactive high-resolution tandem mass spectrometer (Thermo Scientific) was used to detect metabolites eluted from the column. Both positive and negative ion modes were used on the Q-Exactive. Precursor spectra (70–1050 m/z) were collected at 70,000 resolution to hit an AGC target of 3e6. The maximum injection time was set to 100 ms. A top 3 configuration for data acquisition was set in DDA mode. Fragment spectra were collected at 17,500 resolution to hit an AGC target of 1e5 with a maximum injection time of 80 ms. To assess the stability of the LC–MS throughout the whole acquisition, a quality control sample (pool of all samples) was acquired after every 10 samples.

Data processing

The raw data were converted to readable mzXML via Proteowizard's MSConvert software, peaks were extracted via XCMS software, and quality control of peak extraction was performed. The extracted compounds were annotated with summed ions via CAMERA and then identified at the first level via metaX software. The first level of mass spectrometry information was used for identification, and the second level was used to match the in-house standard database. Candidate compounds were annotated via HMDB, KEGG, and other databases. The annotations explained the physicochemical properties and biological functions of the metabolites. MetaX software was used for metabolite quantification and differential screening.

Statistical analyses

SPSS 25.0 software and Origin 2021 Pro software were used to analyse and visualize the meat quality of the shrimp. The results of the experiment are expressed as the means ± standard deviations (means ± SDs) and were statistically analysed via Duncan's multiple extreme difference test (p < 0.05). Metabonomics was performed via Origin 2021 Pro software and the website https://www.omicstudio.cn/tool. The volcanic diagram, PCA and PLS-DA model, Venn diagram, and thermal diagram were analysed. https://www.bioinformatics.com.cn, an online data analysis and visualization platform, plotted the enrichment map of pathways and correlation coefficient matrix.

Results and discussion

Quality analysis of Metapenaeus ensis subjected to different heat treatments

In general, the quality of shrimp meat is evaluated by measuring the basic nutritional components, color differences, texture indices, and sensory evaluation (Zhou et al., 2022). In this study, the quality of the shrimp meat included physical and nutritional indices (Fig. 2). The physical indices were determined by measuring the texture and color differences of the shrimp meat. Additionally, the basic nutritional composition and pH were also determined.

Fig. 2

Graphical representation of basic food quality analysis. A Effects of different thermal processing methods on the pH of shrimp meat; B Effects of different thermal processing methods on the basic nutrient content of shrimp meat; C-F Effects of different thermal processing methods on the color and textural properties of shrimp meat: color characterization (C), hardness (D), springiness (E), and chewiness (F). Each indicator is repeated four times, and the three stable data are retained. The average of these values is then taken as the final result. All the results are presented as the means ± SDs. The error bars indicate the standard deviation. Different letters indicate significant differences (p < 0.05)

Analysis of nutritional indices of Metapenaeus ensis from different heat treatment processes

pH is an essential indicator to measure the quality of aquatic products (Zhou et al., 2022). The pH value of the raw shrimp meat group was 7.01, which increased to 7.21, 7.48, 7.25, and 7.29 after air frying, boiling, drying, and frying, respectively. Significantly greater pH values were observed in the four heat treatments than in the raw meat (p < 0.05) (Fig. 2A). The pH of a food is determined by the free carboxyl and amino groups of the small-molecule compounds it contains, as well as some large-molecule compounds such as proteins and nucleic acids (Sumer et al., 2023). When muscle proteins are heated, some peptide chains open and are broken down into small molecules of amines with fewer acidic groups, increasing the pH compared with that of raw meat (Ferreira et al., 2024). Therefore, in this experiment, the pH levels of the different groups after undergoing thermal processing were elevated compared to the raw sample.

The basic nutrient composition is an essential indicator of the edible quality of shrimp meat (Yang et al., 2023). Metapenaeus ensis has a high amount of crude protein, with levels reaching 22.67 g/100 g, and a crude fat content of 0.70 g/100 g (Fig. 2B). This demonstrates that shrimp meat is a high-protein, low-fat food that can serve as a vital source of animal protein in the diet of the population. Compared with those of fresh shrimp meat, the crude protein, and crude fat contents after thermal processing were significantly greater (p < 0.05), especially the intergroup differences between the air frying and drying treatment groups. This occurred because with increasing heat treatment temperature, water gradually dissipates, nutrients such as crude protein and crude fat are enriched, and their relative contents increase.

Analysis of physical indices of Metapenaeus ensis from different heat treatment processes

The market value of shrimp is dependent mainly on the visual appeal of their body color (Liang et al., 2022). The results revealed significant color differences due to differences in the heating medium and the shrimp temperature after heat treatment. Compared with those of the control group, the L* value, a* value, and b* value of the shrimp meat after heat treatment were significantly greater (p < 0.05) (Fig. 2C). The increase in the L* value was related to the brightness of the shrimp meat. Compared with those of the control group, the L* values of the four thermal processing methods were highest in the boiling group, followed by those of the frying group, and were similar to those of the air-frying and drying groups. The increased a* and b* values were visually represented by the red–orange and yellowish color of the shrimp. The a* and b* values of the heat-treated samples were the highest in the air-frying and frying groups and similar to those of the boiling and drying groups, and the change in a* values was related mainly to the content of free astaxanthin. The principal pigment substance in shrimp provides reddish-orange pigmentation to shrimp tissues (Yanar et al., 2004). Changes in b* values are associated with the degree of lipid oxidation in shrimp (H. J. Li et al., 2021). This may be attributed to the fact that cooking oil was added to both the air frying group and the frying group, resulting in a more reddish-orange and yellowish appearance for both groups compared to other heat treatment groups.

The texture characteristics of meat are crucial for consumers’ eating experience (Ilic et al., 2022). These properties encompass hardness, springiness, and chewiness. Figure 2D-F displays the measured values of hardness, springiness, and chewiness of the meat of Metapenaeus ensis meat following various thermal processing methods. Generally, higher levels of hardness and springiness in the meat correspond to increased chewing capacity requirements and greater chewiness. These three factors are positively correlated with each other (Y. Wang et al., 2023). In this study, the hardness (Fig. 2D) and springiness (Fig. 2E) of the samples in the air frying and drying treatment groups were significantly greater than those in the control group. This can be attributed to the high heating temperature and long heating time in the air frying and drying treatments, which caused the surface temperature of the shrimp meat to increase rapidly, the water to evaporate, and a dry layer to appear on the surface of the shrimp meat, and the formation of a hard shell. As a result, the crushing force requirement was greater, and the chewiness (Fig. 2F) was significantly (p < 0.05) greater than those of the other treatment groups.

Comprehensive sensory evaluation of Metapenaeus ensis under different heat treatments

The comprehensive sensory evaluation consists of electronic tongue measurement and sensory evaluation.

Electronic tongue analysis of Metapenaeus ensis subjected to different heat treatments

The electronic tongue can be used to objectively analyse the overall flavour quality of the samples being tested, and it can also be used to determine the flavour differences among the samples (L. L. Chen et al., 2023b). In the electronic tongue principal component analysis of shrimp meat subjected to different heat treatments of Metapenaeus ensis, the contributions of the first principal component (PC1) and the second principal component (PC2) were 1.8% and 96.9%, respectively (Fig. 3A), indicating that the electronic tongue could clearly distinguish the different treatments of shrimp flavour in terms of the overall flavour. The flavour characteristics of the raw shrimp meat and four types of thermally processed shrimp meat were analysed via the electronic tongue technique (Fig. 3B), and the sourness, bitterness, and bitter aftertaste of the shrimp meat from the different processing methods were below the relative zero value. Therefore, it is not easy to perceive these flavours of shrimp meat in the body. The effective taste indices of shrimp meat freshness, richness, sweetness, and saltiness were compelling and consistent with previous findings (Liang et al., 2022). This richness is the fresh flavour aftertaste, reflecting the persistence of the sample’s fresh flavour, also known as fresh flavour persistence. Compared with that of raw shrimp, the most impressive salty taste of shrimp was in the drying treatment group, the best fresh taste was in the raw sample treatment group, the most incredible sweet taste was in the boiling treatment group, and the most significant fresh taste aftertaste richness index was in the air frying treatment group.

Fig. 3

E-tongue and sensory evaluation plots of the meat of Metapenaeus ensis subjected to different thermal treatments. A PCA of the e-tongue; B Taste values of the e-tongue; C Sensory evaluation radar plot. Each sample was repeated four times, three stable datasets were retained, and the average was calculated to determine the final test result

Sensory evaluation of Metapenaeus ensis subjected to different heat treatments

The shrimp samples subjected to different thermal processing methods were evaluated according to the sensory criteria, and a radar chart of the sensory evaluation is shown in Fig. 3C. In terms of color and appearance, the shrimp samples from the boiling group presented the highest scores, which is consistent with the conclusion that the boiling group presented the highest L* value in the color characterization analysis, where an increase in the L* value was visually manifested as whitening of the shrimp meat. On the other hand, the boiling group had the highest moisture content in terms of basic nutrient composition, which resulted in the shrimp in the boiling group being full and plump, with a powdery-white color. In terms of taste and flavour, the air frying treatment group had the highest score, which was also consistent with the results of the e-tongue measurements. The fresh aftertaste, i.e., the longest-lasting freshness, was relatively highest in the air frying group. In terms of taste, the boiling group had the highest score, which was consistent with the results of the texture measurements. This was probably due to its moderate firmness, chewiness, and high elasticity, resulting in a firm, elastic-tasting meat. Overall, the shrimp samples from the boiling and air-frying groups presented the highest sensory scores.

Metabolomic analysis of different heat treatments of Metapenaeus ensis

Multivariate statistical analysis of metabolites

UHPLC-Q Exactive-MS/MS metabonomics technology was used to analyse the metabolites of Metapenaeus ensis subjected to different heat treatments. A total of 7392 metabolite ions were extracted, and 5146 and 2246 metabolites were identified as having positive and negative patterns, respectively. In the volcano map, except for the boiled-raw sample control group, the metabolites in the raw control and heat treatment control groups were significantly upregulated (Fig. 4A-E).

Fig. 4

Multidimensional statistical analysis of the metabolites. A-E Volcano plots of raw samples versus different thermal processing treatments of shrimp meat (A: air-frying samples vs. raw samples; B boiling samples vs. raw samples; C drying samples vs. raw samples; D frying samples vs. raw samples; E bar charts comparing the overall trend of changes). F-I PLS‒DA score plots. J-M PLS-DA scores 200 permutation tests (F, J: air-frying vs. raw samples; G, K: boiling vs. raw samples; H, L: drying vs. raw samples; I, M: frying vs. raw samples)

The metabolic analysis results were visualized via principal component analysis (PCA) (Figure S1). The QC samples were clustered together, indicating that the instrumental analysis system had good stability and reproducibility. Therefore, the identified metabolite differences could reflect compositional differences between shrimp meat samples subjected to different thermal processing methods. Owing to the unsupervised downscaling analysis method, a clear separation between the raw sample group and the four heat treatment groups was observed. Nevertheless, the different processing methods could not be well separated.

Therefore, the discriminant statistical analysis method PLS-DA (partial least squares discriminant analysis) was further improved to eliminate the influence of unrelated factors in the experimental data. Compared with PCA, PLS-DA revealed that the difference in samples within the parallel sample group decreased, the difference between groups increased significantly, and the treatment groups were completely separated (Fig. 4F-I). In addition, 7 cross-verifications and 200 response sequence tests were performed on the PLS-DA model to avoid overfitting and random effects to improve the prediction ability. R² and Q² are the intercept values of the regression line, the Y-axis R² is the sum of the variances the model can explain, and Q² is the model's prediction ability. When the response ranking test is used, it is generally required that Q² is less than zero. The closer it is to 1, the better the model fit and the more stable and reliable it is. Compared with that of the original sample, the Q² intercept of the four heat treatment groups was less than 0, and R² was close to 1 (Fig. 4J-M). In conclusion, our results show good predictability and reliability, which can be used for further analyses.

Selection and identification of differentially abundant metabolites (DMs)

VIP > 2 and p < 0.05 were considered DMs. Finally, 33, 29, 42, and 32 differentially abundant metabolites were screened and identified in these groups, respectively. The above four datasets were collated, and 79 differentially abundant metabolites were identified, as shown in Table 2. Compared with HMDB, the secondary classification of these 79 metabolites was divided into six major groups, namely, 36 organic acids and their derivatives (45.6%), 9 nucleotides and their analogues (11.4%), 12 organic heterocyclic compounds (15.2%), 9 lipids and lipid-like molecules (11.4%), and 13 other compounds (16.5%) (Fig. 5A). These 36 organic acids and their derivatives were further subdivided according to the classification of the official HMDB website: 11 amino acids, 21 short peptides, and 4 organic acids.

Table 2 Differential metabolites and their expression trends in the meat of Metapenaeus ensis subjected to different thermal processing methods (A/R: air frying Vs. raw; B/R: boiling Vs. raw; D/R: drying Vs. raw; F/R: frying Vs. raw)

Fig. 5

Correlation analysis of differentially abundant metabolites in the meat of Metapenaeus ensis subjected to different thermal treatments. A Classification of differentially abundant metabolites in the HMDB; B Venn diagram of differentially abundant metabolites; C Visualization of heatmaps of differentially abundant metabolites; D Pathway enrichment analysis of differentially abundant metabolites

Wayne's plot of the differentially abundant metabolites of the different heat treatment groups compared with the raw samples revealed that the drying group had the highest number of differentially abundant metabolites and that the boiling group had the lowest number of differentially abundant metabolites compared with the raw samples (Fig. 5B). Additionally, a visual heatmap analysis was conducted on the differentially abundant metabolites of the shrimp meat from Metapenaeus ensis (Fig. 5C). If the content of a metabolite was higher than the mean content of a sample, it was indicated in pink. Otherwise, the content was noted in green. The darker the color is, the more the metabolite content differs from the mean. We found that raw and cooked shrimp meat could be distinguished and that the boiling group was significantly different from the other treatment groups.

Analysis of metabolic enrichment pathways of DMs

Metabolic pathway analysis is an effective method for analysing direct links between metabolites (Tsouka et al., 2023). As shown in Fig. 5D, we found that the 79 different metabolites were distributed in 18 metabolic pathways, including 8 amino acid metabolic pathways, 5 carbohydrate metabolic pathways, 2 cofactor and vitamin metabolic pathways, 2 nucleotide metabolic pathways, and 1 transporter metabolic pathway. Six metabolic pathways were significantly enriched (p < 0.05), including alanine, aspartate, and glutamate metabolism; butyrate metabolism; purine metabolism; glutathione metabolism; phenylalanine biosynthesis; tyrosine and tryptophan biosynthesis; and cysteine and methionine metabolism, four of amino acid metabolic pathways. These results indicate that amino acid metabolism plays a vital role in the metabolic effects of various thermal processing methods on the metabolism of Metapenaeus ensis.

Analysis of significantly different metabolites (SDMs) associated with the thermal processing quality of Metapenaeus ensis

The smaller the p value was, the more significant the differentially abundant metabolite was, and the greater the P value was, the greater the intensity of the definite influence of the differentially abundant metabolites on the samples between the groups (Shen et al., 2016). On this basis, the SDMs with VIP > 2 and p < 0.01 were further screened, and 61 differentially abundant metabolites were screened (Table S2). The correlation between shrimp meat quality and SDMs was analysed (Fig. 6), and the relative contents of different metabolites in shrimp meat were calculated via the area normalization method (Fig. 7) to evaluate the effects of various heat treatments on shrimp meat quality and metabolites.

Fig. 6

Correlation analysis of shrimp meat quality parameters and SDMs. A Correlation analysis between shrimp meat quality and amino acid content; B Correlation analysis between shrimp meat quality and nucleotides and lipids; C Correlation analysis between shrimp meat quality and small peptides; D Correlation analysis between shrimp meat quality and organic heterocyclic compounds; E Correlation analysis between shrimp meat quality, organic acids, and other SDMs

Fig. 7

Relative contents of SDMs in shrimp meat subjected to different heat treatment methods. A Relative content of amino acid SDMs in shrimp meat; B Relative contents of nucleotide and lipid SDMs in shrimp meat; C Relative content of small peptide SDMs in shrimp meat; D Relative content of the organic heterocyclic compound SDMs in shrimp meat; E Relative contents of organic acids and other SDMs in shrimp meat

In Fig. 6, texture indices were significantly correlated with pH, color, and nutritional indices (p < 0.05). The chewiness of the texture index was significantly positively correlated with the nutritional indices of crude protein and ash (p < 0.05) because shrimp is a food with high protein content and low fat content, and its high protein content leads to increased chewiness. The moisture content, crude protein content, and crude fat content were significantly negatively correlated with the ash content, which is consistent with the analysis results in Fig. 2B. The results revealed that different metabolites were significantly associated with the color, meat quality, pH value, and nutritional composition of shrimp meat (p < 0.05). Next, we discuss the potential metabolites that specifically affects the quality of shrimp meat.

Amino acids

Amino acids are the basic building blocks of proteins, which have various nutritional functions and essentially affect the flavour of foods (Sun et al., 2023). Ten significantly different metabolites in the amino acid class were screened in the Metapenaeus ensis samples (Fig. 7A).

D-Pyroglutamic acid and L-5-oxoproline are the two metabolites with the highest relative contents of amino acids. L-5-oxoproline is an alias of D-pyroglutamic acid, but the two differ in configuration. D-pyroglutamic acid is an intermediate in the synthesis of many amino acids and proteins. It is a differentially abundant metabolite rich in the glutathione metabolic pathway. Relevant studies have shown that D-pyroglutamic acid is a compound with high fresh taste intensity (L. Y. Zhang et al., 2018). Given that this substance has the highest relative content of amino acids, it is speculated that it is a vital metabolite affecting the fresh taste of shrimp meat. Heat treatment increased the content of D-pyroglutamic acid in the shrimp, and the contents of groups A and D were the highest. The longer the heat treatment time was, the greater the contribution to the flavour, indicating that the heat treatment time had a more significant effect on the flavour. N- (1-Deoxy-1-fructosyl) proline and N- (1-Deoxy-1-fructosyl) valine are stable compounds formed by the reaction of glucose with amino acids or amino groups of proteins, participating in the Maillard reaction and primarily influencing the flavour of food (Yan et al., 2023). This study identifies N- (1-deoxy-1-fructosyl) proline and N- (1-deoxy-1-fructosyl) valine as common SDMs in addition to boiling methods, indicating that these two substances are the key metabolites affecting the flavour of the three heat treatment methods of air frying, frying, and drying.

Nucleotides

Nucleotides are closely related to meat flavour (Jin et al., 2023). Four metabolites of nucleotides and their derivatives were screened from Metapenaeus ensis samples (Fig. 7B).

5'-Adenosine monophosphate (AMP) is the metabolite with the highest relative content of nucleotides. AMP is a critical flavour nucleotide, and its content significantly affects the flavour of shrimp meat (Qiu et al., 2016); it can enhance flavour, inhibit bitterness in aquatic products, and improve taste (W. Li et al., 2014). After heat treatment, the relative content of AMP in each treatment group was significantly greater than that in the raw sample, indicating that AMP is the critical metabolite affecting the flavour and taste of shrimp meat after heat treatment.

Adenylosuccinic acid belongs to a class of organic compounds called purine nucleoside monophosphates. It is an intermediate product of the mutual transformation of the purine nucleotides inosine monophosphate (IMP) and adenosine monophosphate (AMP) (Andres-Hernando et al., 2021). The degradation of macromolecular nucleotides in shrimp meat changed the freshness of the shrimp meat to varying degrees. This study revealed that the freshness of shrimp meat subjected to air frying was the greatest, and its relative content of adenylosuccinic acid was the highest. Therefore, it was speculated that this substance is the critical metabolite affecting the freshness of shrimp meat.

Lipids

During the pretreatment of shrimp meat in this study, the shrimp head and back digestive tract (shrimp line) were removed. The lipids in shrimp are distributed throughout the digestive tract (Xie et al., 2021). Therefore, fewer differential lipid metabolites were detected in this study, and 7 significantly different lipid metabolites were detected (Fig. 7B).

Ethyl 2-furanacrylate, which is correlated with meat quality, is positively correlated with fat content (Fig. 6) and is a fatty acyl (FA), the main component of lipids with complex structures. Moreover, as a vital biomolecule, it participates in lipid metabolism in vivo and has a variety of critical physiological functions in organisms (Patel et al., 2022). Compared with that of the raw samples, the fat content of the shrimp after heat treatment tended to increase overall, and there were some differences between the different treatment methods.

Four metabolites, L-Propionyl carnitine, 3-Hydroxy hexadecyl carnitine, Acylcarnitine 4:0, and Acylcarnitine 7:0, are acylcarnitines (acars). Acylcarnitine is an ester that combines carnitine with fatty acid or amino acid metabolites. The mitochondria are rich in muscle and brain and participate in several critical metabolic processes in the human body. It can take acetyl groups from cells and assist in energy transfer (Wei et al., 2023). Acylcarnitines are the metabolites related to the greatest number of lipids, and their content also increased significantly after heat treatment. In conclusion, acylcarnitines and ethyl 2-furanacrylate can be considered important metabolites affecting lipids in shrimp meat subjected to different heat treatments.

Small peptides

In addition to the production of amino acids, the accumulation of small peptides, totaling up to 17 species, was observed during the different heat treatments of the shrimp meat (Fig. 7C).

Small peptides generally refer to oligopeptides composed of more than two amino acids. Some small-molecule peptides containing Glu are related to the freshness of meat. (Mora et al., 2008). In this study, gamma-Glu Cys is a precursor of glutathione (GSH), which can effectively improve the flavour and quality of food. The relative content of small peptides after cooking was significantly greater than that of raw samples, which indicated that thermal processing affects small peptides to a large extent, thus affecting their characteristic flavour.

Heterocyclic compounds

Heterocyclic compounds are considered essential sources of characteristic flavours in meat (Luo et al., 2023). Twelve SDMs of heterocyclic compounds were screened from shrimp samples (Fig. 7D).

Hypoxanthine is a purine organic compound. Xanthine and hypoxanthine are intermediate products produced in the degradation process. Because high temperatures can degrade purines, different heat treatment methods specifically impact the content of purine substances in aquatic products (Cao et al., 2024). The reduction in hypoxanthine was greatest after boiling. This finding is consistent with previous research results. Cooking can significantly reduce the purine content, possibly because purines are dissolved in cooked soup (Sabolová et al., 2023).

The three SDMs, 4,6-dihydroxy-2-quinoline carboxylic acid, levofloxacin, and xanthurenic acid, are all quinoline carboxylic acids, which are heterocyclic aromatic amines (HAAs). HAAs are carcinogenic and mutagenic compounds produced in foods with high protein contents after long-term high-temperature processing (Teng et al., 2022). During the heating process of aquatic products, the amount and type of heterocyclic amines change with changes in heating methods and heating conditions. Among the different heat treatment methods used in this study, the content of heterocyclic amines produced by drying and air frying was the highest, and the relative content was significantly different from that of the raw sample group. The second is frying, and boiling produces the least amount. Therefore, the greater the number of heterocyclic amines, the higher the content, the higher the heating temperature, and the longer the heating time. These findings indicate that the production of heterocyclic amines can be controlled by controlling the processing time and temperature and that boiling is the healthiest and least nutritious thermal processing method.

Organic acids and other compounds

In addition to the above metabolites, four organic acids and seven other compounds were detected (Fig. 7E). Succinic acid is the metabolite with the highest content of organic acids, whereas benzoic acid and choline are the metabolites with the highest content of other compounds.

Organic acids in meat products help to stabilize and improve quality and flavour (Y. Q. Wang et al., 2022). It has been reported that succinic acid is a potential flavour factor of pufferfish and that the flavour of shrimp meat significantly contributes to flavour (Zhang et al., 2019). In this study, the amount of succinic acid in each heat treatment group was found to be significantly higher than that in the raw shrimp, indicating that succinic acid plays a significant role in the flavour profile of shrimp meat. Additionally, among the organic acids analyzed, only 2-hydroxyglutaric acid exhibited a notable decrease in concentration. 2-Hydroxyglutaric acid is considered an abnormal metabolite in organisms, and its excessive accumulation may lead to cancer and other diseases (M. M. Zhang et al., 2018a). After heat treatment, only the relative content of the boiled group decreased significantly, indicating that the boiled group was healthier than the other heat treatment methods. Among other compounds, choline is considered an essential vitamin, serving as the source of methyl for many biochemical reactions in human metabolism. The human body needs choline to produce acetylcholine (Hawley et al., 2022). However, in this study, compared with that in fresh shrimp meat, the relative content of choline in cooked shrimp meat decreased, indicating that heat treatment is not conducive to preserving vitamins. Benzoic acid, (2RS, 5RS)—(E) −2- (2-phenylethyl) −1,3-dioxan-5-ol, benzoylecgonine, and carboaniline are all benzene compounds. Compared with those of the original sample, the relative content of each treatment group increased, and the benzene compounds presented an aromatic odor (Chen et al., 2020). In conclusion, the key metabolites affecting the flavour of shrimp meat after heat treatment are succinic acid and benzoic acid.

Conclusion

The findings demonstrated notable variations in the physicochemical properties and edible qualities of the shrimp exposed to diverse heat treatments. These treatments improved the overall visual quality of the shrimp meat. Post-heat treatment, the levels of crude protein, crude fat, and pH were significantly increased compared to fresh shrimp. Furthermore, the shrimp in the air-fried group exhibited a more abundant taste and flavor, achieving the highest overall sensory evaluation score, along with excellent acceptability and affinity. Different metabolites were significantly correlated with the color, meat quality, pH value, and nutritional composition of shrimp meat (p < 0.05), and d-pyroglutamic acid is a vital metabolite affecting the taste of shrimp meat. The key metabolites affecting flavour were amp, n-(1-deoxy-1-fructosyl) proline, n-(1-deoxy-1-fructosyl) valine, succinic acid, and benzoic acid. Compared with the other heat treatment groups, the boiling treatment group presented the lowest relative contents of harmful purines, heterocyclic amine metabolites, and the abnormal metabolite 2-hydroxyglutaric acid, which is a healthy thermal processing method. This study elucidated the changes in the edible quality and metabolites of shrimp meat under different heat treatment conditions, offering a theoretical basis for improving the production technology and quality of shrimp products and providing theoretical guidance for future research.