TPA analysis
Texture is an important quality attribute of meat. Changes in texture during heat treatment are directly associated with tissue state, sensory quality, and physical structure of obtained meat products (Wang et al. 2020). Figure 1 shows changes in hardness (Fig. 1a) and chewiness (Fig. 1b) of Antarctic krill meat treated at different temperatures and holding times. Both hardness and chewiness increased at higher holding times and temperatures; the higher the temperature, the shorter the time to reach the equilibrium value. A more significant increase in both hardness and chewiness was observed in samples heated at 95°C for 10 min compared to 95°C for 8 min.
Heat treatment is often accompanied by denaturation and coagulation of myofibrillar proteins, contraction and exposure of hydrophobic regions of the myofibrillar structure, and significant loss of water between myofibrils, which leads to an increase in hardness of krill meat (Straadt et al. 2007). Interestingly, krill meat samples heated at 85°C for 10 min had higher hardness and chewiness values than those treated at 95°C for 10 min. This could be linked to protein degradation induced by heating, which caused severe tissue damage when treated at 95°C for 10 min, and the higher temperature might have contributed to further break and relax myofibrillar fibers, leading to a decrease in hardness and chewiness, which is consistent with the results of Jiang et al. (2018). In addition, chewiness of krill meat treated at 55°C for 4 min was higher than that of samples treated at 65°C for 4 min; thus, moderate protein denaturation might have occurred in samples treated at 65°C for 4 min, which led to changes in texture.
Cooking loss
Cooking loss is an important factor in heat-treated meat, and includes water loss and leakage of lipids, peptides, and other nutrients, resulting in decreased quality (Purslow et al. 2016). Cooking loss rate of krill meat heated at different temperatures and holding times are shown in Fig. 2. Cooking loss of krill meat showed an upward trend with increasing holding times, in which values initially increased and then decreased. Considering the same holding time, higher cooking loss rates were observed at higher temperatures, which could be due to the fact that proteins on the surface of krill meat undergo rapid denaturation, and the muscle tissue rapidly contracts, thus resulting in rapid juice loss. In addition, cooking loss of krill meat peaked in samples treated at 95°C for 8 min (up to 43%), while cooking loss in samples treated at 95°C for 10 min was lower than of samples treated at 95°C for 8 min, which was likely due to almost complete denaturation of proteins after prolonged heating; moreover, the reduced degree of conversion of water molecules between immobilized and free states might have amplified aggregation of denatured proteins found on the surface of krill meat with myofibrils, which might have prevented water transfer and decelerated cooking loss (Haghighi et al. 2021). Niamnuy et al. (2008) showed that cooking loss rates of white shrimp treated in boiling water at 100°C for 7 min was within the range of 8–21%, which were significantly lower than those of krill meat found in the present study, which might be related to the lower myofibrillar protein content of Antarctic krill meat.
Proton dynamics by LF-NMR
Moisture content and water state are attributes that are closely related to meat texture. Proton spin-spin relaxation times (T2) can explain mobility and degrees of freedom of water molecules, thus reflecting water dynamic properties such as diffusion and state (Zhu et al. 2021). LF-NMR is a commonly used to characterize hydrogen protons in different matrices.
The effect of heat treatment on hydrogen proton peaks and relative individual water content of krill meat is shown in Fig. 3 and Tables 1 and 2. Three hydrogen proton peaks were observed in untreated krill meat (sample A) and all heat-treated treatment samples: T21 peak (0–10 ms) indicated water associated with macromolecules, i.e., bound water; T22 peak (10–100 ms) indicated immobile water located between fine and thick filaments of the myofibrillar protein network; T23 peak (100–1000 ms) was attributed to free water in myofibril lattice (Ling et al. 2020). Changes in T21 peak values did not significantly differ for heated krill samples compared to untreated samples, which could be explained by the stable degree of binding of water to macromolecules of myofibrils during heat treatment. In contrast to untreated krill meat (sample A), T22 and T23 relaxation times of all heat-treated samples both gradually decreased at higher temperatures and longer holding times.
Table 1
T2 relaxation times and peak areas in Antarctic krill muscle after treatment at different temperatures.
Sample | Heat treatment | T21 (ms) | T22 (ms) | T23 (ms) | A21 (g− 1) | A22 (g− 1) | A23 (g− 1) |
A | Untreated | 0.26 ± 0.04a | 27.36 ± 0.00a | 401.09 ± 15.89a | 233.89 ± 10.25a | 2799.46 ± 41.66a | 442.42 ± 3.51d |
B | 55°C/10 min | 0.26 ± 0.22a | 20.27 ± 0.80b | 230.17 ± 9.12b | 297.18 ± 63.24a | 2680.82 ± 16.56b | 405.43 ± 8.81e |
C | 65°C/10 min | 0.27 ± 0.22a | 16.08 ± 0.65c | 214.73 ± 8.51bc | 243.90 ± 33.76a | 2555.46 ± 14.92c | 456.02 ± 0.61c |
D | 75°C/10 min | 0.15 ± 0.12a | 14.32 ± 0.57d | 200.33 ± 7.93cd | 292.32 ± 50.66a | 2246.01 ± 11.23d | 597.77 ± 6.71b |
E | 85°C/10 min | 0.33 ± 0.37a | 12.75 ± 0.53e | 186.89 ± 7.40d | 294.25 ± 8.62a | 2187.11 ± 8.99e | 620.92 ± 4.19a |
A: untreated sample; B: 55°C/10 min; C: 65°C/10 min; D: 75°C/10 min; E: 85°C/10 min. Data are presented as means ± SD (n = 3). Different superscript letters within the same column indicate significant differences at P < 0.05. |
Table 2
T2 relaxation times and peak areas in Antarctic krill muscle after treatment for different holding times.
Sample | Heat treatment | T21 (ms) | T22 (ms) | T23 (ms) | A21 (g− 1) | A22 (g− 1) | A23 (g− 1) |
A | Untreated | 0.26 ± 0.04a | 27.36 ± 0.00a | 401.09 ± 15.89a | 233.89 ± 10.25a | 2799.46 ± 41.66a | 442.42 ± 3.51b |
F | 95°C/2 min | 0.55 ± 0.17a | 19.80 ± 0.80b | 224.90 ± 9.12b | 207.47 ± 25.98a | 2703.54 ± 26.64b | 271.50 ± 6.97e |
G | 95°C/4 min | 0.26 ± 0.10a | 15.00 ± 0.61c | 187.20 ± 15.34c | 246.34 ± 32.10a | 2425.71 ± 15.20c | 414.32 ± 3.30c |
H | 95°C/6 min | 0.35 ± 0.30a | 13.36 ± 0.53d | 174.36 ± 6.91c | 254.52 ± 57.62a | 2408.15 ± 10.38c | 434.31 ± 4.51b |
I | 95°C/8 min | 0.33 ± 0.17a | 11.90 ± 0.00e | 166.65 ± 11.56cd | 222.74 ± 25.88a | 2314.86 ± 6.90d | 369.03 ± 5.59d |
J | 95°C/10 min | 0.42 ± 0.09a | 11.36 ± 0.46e | 151.75 ± 6.01d | 258.95 ± 34.57a | 2303.79 ± 12.35d | 459.02 ± 7.00a |
A: untreated; G: 95°C/4 min; F: 95°C/2 min; H: 95°C/6 min; I: 95°C/8 min; J: 95°C/10 min. Data are presented as means ± SD (n = 3). Different superscript letters within the same column indicate significant differences at P < 0.05. |
Figure 3a and Table 1 show T2 relaxation times and peak regions of krill meat at different heating temperatures. Compared with sample A, T22 and T23 in samples B, C, D, and E decreased gradually with the increase in temperature. T22 and T23 decreased from 27.36 ms and 440.47 ms in sample A, and to 12.75 ms and 186.89 ms in group E. Heated krill meat samples undergo protein denaturation and muscle contraction, which affects the degrees of freedom of water molecules which are then restrained and of reduced mobility, leading to peak blueshift; these observations are consistent with those of Sun et al. (2020). In contrast to the untreated sample (sample A), T22 of samples F, G, H, I, and J decreased significantly with prolonged holding times, hence immobile water was more affected. In addition, T23 of samples G, H, I, and J did not change significantly, which could possibly be due to the fact that the degrees of freedom of water molecules had reached equilibrium in samples treated at 95°C for 4 min and did not change significantly with prolonged heating (Fig. 3b and Table 2).
Tables 1 and 2 show peak areas corresponding to each peak thus representing the water relative content. A21 peak areas of samples A, B, C, D, E, and F, G, H, I, J changed only slightly in heated samples, indicating that heating did not significantly change the content of bound water in krill meat. Compared with untreated samples A, A22 peak area in samples B, C, D, and E decreased significantly at higher temperatures, thus indicating a possible decrease in the content of immobile water between fibers induced by heating, which caused myofibrils to contract and led to protein structure disruption, thereby resulting in water release from within the myofibril into the inter-myofibrillar space and, consequently, in water loss (Xia et al. 2018). The A22 peak area in samples F, G, H, I, and J decreased with prolonged heating time. Overall, the A23 peak area in samples B, C, D, E, and F, G, H, I, J showed an upward trend, indicated that protein was progressively degraded and denatured at increasing temperatures and holding times, resulting in gradual conversion of immobile water into free water. Moreover, the A23 peak area in samples treated at 95°C for 8 min was lower than that in samples treated at 95°C for 10 min; this may be linked to severe muscle damage caused by heating at 95°C for 8 min, resulting in the mobilization of free water from the myofibrillar lattice, which likely resulted higher cooking loss, thus corroborating the results of cooking loss experiments (Fig. 2).
MRI analysis
MRI is a fast, direct, and non-destructive technique that enables the determination of water distribution in foods as well as the visualization of changes in the internal structures induced by heat treatment (Ezeanaka et al. 2019). Pseudo-colored images enable a more readily visualization of changes in water mobility and distribution of samples after different heat treatments.
Figure 4a reflects changes in proton density after heat treatment of krill meat. Bright areas in the untreated sample A corresponded to tissues emitting high signal intensities (Bouhrara et al. 2011), thus indicating that untreated krill meat had the highest water content. With increasing temperatures, the size of bright areas in samples B, C, D, and E gradually decreased, and red-colored areas gradually changed to blue, indicating a decrease in proton density and an increase in water loss in these krill meat samples. In addition, samples F, G, H, I, and J showed pseudo-colored images when treated at 95°C for 2, 4, 6, 8, and 10 min, in which red-colored areas decreased with increasing heating time, whereas red-colored areas in samples treated at 95°C for 8 min were smaller compared to samples treated at 95°C for 10 min, which is consistent with cooking loss results. Figure 4b and 4c show the corresponding signal intensities in pseudo-colored images. MRI intensities of samples B, C, D, E, and samples F, G, H, I, and J decreased significantly with increasing temperatures and holding times; MRI signal intensities of samples I and J were consistent with pseudo-colored images (Song et al. 2021). Collectively, these observations confirmed that heat treatment significantly affected water distribution in krill meat.
Microstructure of heated krill meat
Changes in the state of water molecules and water holding capacity are usually related to meat microstructure (Yu et al. 2021). SEM micrographs of microstructural changes in krill meat samples after heat treatment are shown in Fig. 5. The untreated sample A had dense and intact tissue with smooth and flat muscle fiber planes. The microstructure differed significantly after heat treatment compared to untreated samples. Microstructure of samples B, C, D, E, and J showed after heating at different temperatures for 10 min. In contrast, samples B, C, D, E, and J heated for 10 min showed disordered structures and corrugated folded regions, which became increasingly disordered at increasing temperatures. Samples F, G, H, I, and J showed microstructural changes when heated at 95°C for 2, 4, 6, 8, 10 min; thus, with prolonged heating times, protein denaturation occurred and muscle tissue contracted, thus resulting in the formation of corrugated folded structures; treatment for 6 min led to the formation of the densest corrugated folded structures. As holding time extended, corrugated folded structures are destroyed, forming a large area of fractured and disordered structure. Liu et al. (2019) observed that fibrous bundles in white shrimp submitted to heat treatment were significantly larger and more regular. In contrast, in the present study the aggregation of corrugated folded structures formed by krill meat and the lower myofibrillar protein content hindered the formation of water-entrapped structures, which may explain why Antarctic krill meat had higher water loss rate than other shrimp meat.
Intrinsic fluorescence analysis
Changes in muscle tissue microstructure are usually associated with changes in the structure of major proteins. Heat treatment leads to protein denaturation, which alters protein conformation (tertiary structure) mainly due to changes in the location and microenvironment of aromatic amino acid residues. Aromatic amino acid residues can absorb ultraviolet (UV) radiation to fluorescence, and tryptophan (Trp) has a high molar extinction coefficient and is sensitive to the surrounding microenvironment. Thus, changes in the tertiary structure of proteins can be determined by assessing (λmax) and fluorescence intensity changes in aromatic amino acid residues within the target protein molecule (Shi et al. 2020).
Fluorescence spectra of krill myofibrillar proteins submitted to different temperatures are shown in Fig. 6a. The maximum emission wavelength (λmax) of untreated krill myofibrils (sample A) was 337.5 nm, which is consistent with the findings of Li et al. (2021b). At increasing temperatures, λmax of myofibrillar proteins in samples A, B, C, D, and E red-shifted from 337.5 nm to 341.5 nm, which could be linked to the gradual exposure of Trp sides chains to the aqueous solution, thus increasing the polarity of the surrounding environment. Fluorescence intensity of Trp residues decreases significantly at temperatures above 55°C. Considering that heat treatment changed the microstructure of the protein-peptide chain, myofibrillar protein unfolded, and Trp residues were exposed on the surface, which enhanced the intermolecular interaction, resulting in reaggregation of molecules, which further led to more Trp residues being embedded in hydrophobic regions of the protein. This caused a decrease in endogenous fluorescence intensity (Sun et al. 2013), whereas reaggregation of molecules caused an increase in muscle hardness. As shown in Fig. 6b, samples A, F, G, H, I, and J showed a significant decrease in fluorescence intensity of Trp residues as λmax redshifted from 337.5 nm to 342.5 nm with extended heating time at 95°C. The decrease in intrinsic fluorescence intensity and the λmax redshift indicate that the heat treatment caused significant changes in the tertiary structure of myofibrillar protein molecules. However, fluorescence intensity in samples treated at 95°C for 8 min was lower than that of samples treated for 10 min, which showed a slight blueshift; this could probably be explained by a transient contraction of the Trp side chain portion induced by heating, followed by a gradual stretching and exposure until the conformation reached stability, which is consistent with the findings of Lefèvre et al. (2007).
Changes in protein secondary structure
Changes in tertiary structure of proteins are determined by changes in secondary structure. FT-IR spectra have been widely used for the characterization of changes in protein structure, mainly focusing on the amide I band (1600–1700 cm− 1) (Ovissipour et al. 2017), which was applied to further investigate change in the structure of krill myofibrillar proteins. Figure 7 shows the percentage of secondary structures in Antarctic krill myofibrillar proteins calculated from the amide I in obtained FT-IR spectra of samples submitted to different heat treatments. The content of α-helix motifs decreased whereas the content of β-sheets increased at increasing temperatures (Fig. 7a), thus indicating that heating induced protein denaturation, unfolding α-helix deconvolution, and conversion to the β-sheet structure. At lower temperatures, the content of α-helix significant decreased due to the disruption of hydrogen bonds and hydrophobic bonds of the internal structure of the protein-peptide chain maintaining myofibrillar protein structure (Gao et al. 2021); however, at increasing temperatures, the myofibrillar protein structure underwent more significant changes, muscle structure decomposition, and water loss from inner myofibrils, thus leading to an increase in cooking loss, which is in agreement with MRI results. Moreover, the increase in the relative content of β-sheets indicated an increase in the number of hydrogen bonds between protein molecules and in the degree of aggregation between protein molecules at higher temperatures, which led to a significant increase in hardness. Interestingly, the β-sheet content increased in a non-linear manner, which is in agreement with the findings of Xu et al. (2010).
In addition, the content of α-helix and β-sheet in samples F, G, H, I, and J did not change significantly (Fig. 7b) with prolonged heating times, probably because after treatment at 95°C for 2 min, the α- helix structure was completely deconvoluted and transformed into β-sheets, and more β-turned structures were transformed into random coiled structures, which increased randomness. Collectively, prolonged heating times increased randomness of myofibrillar protein structure, and ordered structures gradually changed into an irregular folded structure at higher temperatures (Wijayanti et al. 2014).
SDS-PAGE analysis
In the present study, changes in protein aggregation, degradation, and solubility in krill myofibrillar proteins were evaluated by SDS-PAGE, and results are shown in Fig. 8. Myofibrillar proteins in untreated krill meat (lane A) yielded three major protein bands in SDS-PAGE, which corresponded to myosin heavy chain (MHC) (200 kDa), actin (43 kDa), and troponin T (37 kDa) (Jiang et al. 2014). B, C, D, and E lanes corresponded to samples heated at 55, 65, 75, 85°C for 10 min, while lanes F, G, H, I, and J corresponded to samples heated at 95°C for 2, 4, 6, 8, and 10 min. With heat treatment, protein composition of lanes B, C, D, E, and J changed significantly; at temperatures above 75°C, the MHC band in lanes D and E had reduced intensity, and was more degraded in lane J (95°C/10 min). When heated at 95°C for 4 min, degradation of MHC was more pronounced in lanes G, H, I, and J, probably because higher temperatures accelerated protein degradation. Degradation of MHC disrupted the ordered protein structure of krill meat, leading to increased loss moisture and increased cooking loss, which is consistent with results of cooking loss experiments (Fig. 2). After heat treatment, lanes D, E, G, H, I, and J only the bands corresponding to actin and troponin could be observed. These results indicated that thermostability of actin is significantly higher compared to myosin (Qi et al. 2018). Moreover, troponin bands in lanes B, C, D, and E and lanes F, G, H, I, and J were only slightly decreased, which suggests that troponin is also heat stable, as suggested by Hu et al. (2017).
Due to changes in the dynamic balance of acid-based groups on the surface of proteins caused by the degradation of myofibrillar protein, the formation of disulfide bonds between denatured myofibrillar protein may further promote cross-linking between protein molecules (Ko et al. 2007), resulting in increased muscle contraction, water loss, and hardness.