3.1. Comparison between Manually and Mechanically Separated Fresh Flesh
In Table 1
, the physico-chemical parameters and fatty acid (FA) composition of manually and mechanically separated flesh, are compared. No significant differences were observed for water content and pH. On the contrary, the color parameter L* showed a significant variation; indeed, after mechanical separation, the flesh appeared darker. Secci et al. [17
] detected a similar color variation in horse mackerel after mechanical separation. The initial TBARs value was similar to that found by Sundararajan et al. [18
] in frozen shrimp. However, a significant increase of this parameter was observed, indicating that the mechanical separation process promoted lipid oxidation; similar results were detected by Secci et al. [3
] in two different fish species.
The fat content of the flesh varied from 2.8% to 3%. This value is in agreement with literature results [7
] and did not change according to the different separation method.
Regarding lipid classes, no significant differences were observed between the manually and mechanically separated fresh flesh. It must be noted that DAG, EST and TAG were the most abundant lipid classes in both types of mantis flesh, evidencing already a hydrolytic process of lipids. Concerning the total FA composition (Supplementary material—Table S1
), it remained practically unchanged in both manually and mechanically separated fresh flesh, even though mechanical separation led to higher oxidation which was reflected in a significant decrease of some unsaturated FA (C16:1 n-7 and C20:3); similar results were obtained by Secci et al. [17
] on horse mackerel flesh. The main FA were oleic acid (C18:1 n-9, 16.9 ± 0.2% of total FA) and palmitic acid (C16:0, 16.4 ± 0.5% of total FA), followed by palmitoleic acid (C16:1 n-7, 14.6 ± 0.4% of total FA) and nervonic acid (C24:1 n-9, 14.6 ± 0.5% of total FA). Eicosapentaenoic (C20:5 n-3, EPA) and docosahexaenoic (C22:6 n-3, DHA) acids were present as 5.26 ± 0.36% and 0.88 ± 0.02% of total FA, respectively. Among FA categories, monounsaturated FA (MUFA) were the more abundant, followed by saturated FA (SFA) and PUFA. In particular, SFA represented about 30% of total lipids and were mainly composed by C16:0 and C18:0; in the case of MUFA, C18:1 n-9 and C16:1 n-7 represented about 60% of this FA class. On the other hand, PUFA accounted for 13% of total lipids and, while more than 90% was constituted by PUFA n-3 (in particular EPA and DHA), only 2.8% was represented by PUFA n-6. These values are within the ranges of FA percentage distribution reported by Mili et al. [7
] for S. mantis
fished in Tunisian waters in different seasons, as well as those found by Passi et al. [19
] for Mediterranean mantis shrimp. In fact, it is well known that FA composition of fish lipids can be affected by species, genetic, physiological, morphological, dietary, seasonal and environmental factors, among others [20
]. The PUFA n-6/PUFA n-3 ratio, suitable index to compare the nutritional value of food, was around 3.6 and no significant differences were observed with respect to the separation process used. According to Simopoulos [22
], a low PUFA n-6/PUFA n-3 ratio (< 4) is desirable for a healthy human diet. This result confirms the importance of Mediterranean mantis shrimp as a rich dietary source of PUFA n-3.
3.2. Variation of Quality Indices during Frozen Storage
During the storage at three different temperatures, pH varied from 6.49 to 6.71, while moisture content ranged from 85.59% to 86.62% (pH and moisture data not reported); in both cases, no significant differences were observed across storage at the different temperature conditions.
shows the evolution of the colorimetric parameters of luminosity L* (Figure 1
A) and red index a* (Figure 1
B) measured in the mechanically separated S. mantis
flesh during frozen storage at the three selected temperatures.
Evolution of color during storage can be associated with structural changes [23
], as well as variations in pigments concentrations and their oxidative status [24
]. While at the temperatures of −18 °C and −26 °C the L* values were roughly constant (37–42) throughout the 12-months storage, the sample stored at −10 °C showed a significant increase during the entire storage period, reaching values of 56. This parameter was significantly influenced by storage temperature, time and their interaction (Table 2
). By contrast, the red index was significantly affected only by storage time and by the interaction between time and temperature. Although some significant variations were observed among samples during storage, there was not a clear trend and values remained between 5 and 7.
Sundararajan et al. [18
] observed an increase in a* value for peeled frozen shrimp stored at –21 °C for 180 days, while no significant changes in L* values were observed. These authors suggested that the decrease in a* values could be mainly attributed to the degradation of astaxanthin and lipid oxidation.
To the best of our knowledge, there are no previous reports about the storage of mechanically separated flesh obtained from crustaceans, hence it is impossible to directly compare our results. Changes in the mechanically separated fish flesh obtained from horse mackerel [16
], as well as from gilted sea bream, sea bass and rainbow trout [3
], were evaluated during frozen storage, showing that color variations depended on the species considered. Shrimp flesh is highly perishable and normally high product quality can be obtained when immediately frozen after capture [25
]. Generally, results showed that the main color differences occurred during processing rather than during storage and that white flesh led to lower changes, proving to be more suited for the development of fish processed products [3
]. However, color fading, lipid oxidation, protein denaturation, and dehydration can occur during the frozen storage of shrimp and other crustaceans [25
]. Color variations observed in the sample stored at −10 °C may be related to enzymatic and non-enzymatic reactions that result in degradation of myofibrillar proteins and disorganization of myofibrils. Chéret et al. [23
] and Torres et al. [26
] observed a similar change upon high hydrostatic pressure processing of sea bass fillets and horse mackerel, respectively.
reports the TBARs values found in the S. mantis
flesh during the frozen storage, which varied from 1.4 to 2.4 mg MDA/kg for all the considered period, without significant variations in all storage conditions. Despite the secondary lipid oxidation initially induced by the mechanical separation process, TBARs did not show a steady increase during storage as expected, being thus in disagreement with data reported by various authors for oxidative stability of crustacean flesh and minced fish during frozen storage [3
]. Sundararajan et al. [18
] found a value of 0.47 mg MDA/kg in shrimp that increased progressively during frozen storage up to 2.96 after 180 days. Tsironi et al. [25
] observed an increased rate of TBARs formation with increasing storage temperature in frozen shrimp. However, in the present research, after the initial increase of TBARs during processing, no further oxidation was detected by means of this index.
Besides the data dispersion observed, other factors could have also contributed too, such as the type of packaging, the presence and amount of lipophilic (such as vitamin E) and enzymatic (i.e., GSH) antioxidants in mantis shrimp [19
]. On the other hand, aldehydes deriving from lipid oxidation could have also interacted with other matrix components (such as proteins, amines and peptides) [30
], thus leading to the formation of compounds (i.e., Schiff bases) that cannot be determined as TBARs. In fact, lipid and protein oxidations can occur independently or in parallel, but they can also interact with each other [30
reports the distribution of the main lipid classes (expressed as % of total lipids) and the main total FA classes (expressed as % of total FA) in mechanically separated S. mantis
flesh, as related to storage conditions. The total fat content (% on flesh) was significantly affected only by storage time (St); however, no significant differences were observed among all the determined values.
Concerning the distribution of the main lipid classes, FFA was found to be influenced by both storage temperature and time, increasing from 9% up to around 40% in samples stored at −10 °C after 6 months. Similarly, MAG rose by increasing storage time and storage temperature, while TAG and DAG content showed the opposite trend. These results evidence the occurrence of lipid hydrolysis during frozen storage, being more intense at storage temperatures above −26 °C. The accumulation of FFA in frozen marine species is related to some extent with lack of acceptability. FFA, in fact, are known to cause deterioration of seafood products through their interaction with proteins and have been reported to exert a great effect on lipid oxidation development [26
]. FFA have also been shown to oxidize faster than higher molecular-weight lipids, i.e., TAG and phospholipids, due to their higher accessibility caused by their lower steric hindrance to oxygen and other prooxidant molecules [27
Regarding the total FA composition (expressed as % of total FA), significant differences were observed in all FA classes as related to both storage temperature and the interaction between time and temperature (St T), except for PUFA n-6 and the n-6/n-3 ratio. Among the single FA (Table S2
), docosapentaenoic acid (DPA) was noticeably affected by the storage temperature. On the other hand, storage time did not show any significant effect on the main FA classes, but some single FA (such as linolenic acid) varied to a relevant extent depending on the time of storage. However, it must be noted that there was not a clear trend of the concentration of most single FA with respect to the temperature and time of storage (Table S2
), which could depend on a dynamic equilibrium between their accumulation and conversion into other compounds (i.e., oxidized fatty acids, volatile compounds).
In general, the impact of the storage method and duration on FA content varies according to the fish species and seems to greatly depend on their total lipid content. In fact, Rudy et al. [31
] observed that the effect of storage conditions was greatest in fish species whose lipid content was around 10–19%, while species with lower lipid content (< 10%), like S. mantis
(2.9–3.1%) in the present study, are usually less affected. The lipid content relates to the taxonomic classification, environment (freshwater or marine), season and/or geographic location (warm or cool waters), and lipid storage; all these factors influence the FA content of fish tissue and their susceptibility to degradation under diverse storage conditions. Although long-chain PUFA are usually more prone to oxidation, Rudy et al. [31
] observed that FA degradation was more a function of fish species rather than FA type, suggesting species-specific FA dynamics during storage, probably related to the total lipid content in the fish species. In our work, as well as in that of Rudy et al. [31
], no specific FA or FA class consistently and preferentially underwent a change in quantity with increasingly poor handling and storage conditions, even though a decrease in the amount of some FA was observed over time. Besides the influence of lipid content on FA alterations in marine species, they may also depend on other factors like size, sex, diet, season, state when captured, microbial load, genuineness, presence of natural antioxidants, tissue type, number of lipases and their location in cells [31
shows the concentration of some selected compounds present in the S. mantis
flesh during storage and analyzed by 1
HR-NMR. In Table 4
, results of multivariate analysis show that all parameters were significantly influenced by storage time, temperature, and their interaction. TMA-O breakdown can occur via bacterial enzymes that release TMA [32
], or by the activity of trimethylamine oxide demethylase (TMAOase) that leads to the formation of DMA and formaldehyde [33
]. The production of TMA during refrigerated storage is considered an index of fish freshness as it is strongly correlated with microbial spoilage and it is characterized by a pungent, often associated with the typical “fishy” smell of seafood undergoing spoilage [34
]. During frozen storage, bacterial activity should be absent; however, as mentioned earlier, TMA has also been reported to be a product of enzymatic degradation of TMA-O.
In the present study, TMA-O (Figure 3
A) decreased rapidly in the first month and then remained fairly constant in samples stored at −18 °C and −26 °C. In the sample stored at −10 °C, instead, lower values were observed during the rest of the storage; in fact, at the end of the 12-month storage period, TMAO was half as much the initial value. In parallel to the decrease of TMAO, both TMA (Figure 3
B) and DMA (Figure 3
C) increased in samples stored at −18 and −10 °C, while roughly the same values were observed in the sample at −26 °C. In particular, TMA increased from the 4th month in both samples stored at −10 °C and −18 °C proportionally to the storage temperature. DMA started to increase after the first month for samples stored at –10 °C, whereas in samples kept at −18° C it rose just after 4 months and to a lower extent.
Sotelo et al. [35
] found an increase of TMA during storage at −5 °C, but not at −12 °C. These authors suggested that some residual bacterial activity could still be found at temperatures slightly below zero. However, the TMA increase observed at −10 °C and −18 °C in the present study is probably related to enzymatic degradation. According to García-Soto et al. [27
], the formation of TMA during frozen storage of crustaceans can also be due to biochemical breakdown of proteins and non-protein nitrogen (NPN) compounds.
Free amino acids in fish are the main components of non-protein nitrogen and, since some of them are precursors of aromatic components, they are directly responsible for the development of flavor and taste during cooking [36
]. Amino acids have also been used as quality indices for various fish and crustacean species [37
]. Some of them are precursors of biogenic amines obtained by decarboxylation, which are very important from the toxicity standpoint, and as quality control indices for fish spoilage.
During storage, changes in amino acids are caused by muscle autolysis and the concentration of the single components depends on a dynamic balance between their production and destruction, this balance being associated with muscle enzymes [37
In the present study, starting from the 4th month, lysine (Figure 3
D) and alanine (Figure 3
F) began to increase more rapidly in sample at −10 °C and to a lesser extent in samples stored at −18 °C. In samples stored at –26 °C, by contrast, it remained constant. These results indicate a high level of proteolysis at storage temperatures above −26 °C. On the contrary, sarcosine (Figure 3
E) displayed a variable trend. After a rapid decrease in the first months, it started to increase slowly in all samples until the 8th month and, thereafter, it decreased notably in all three samples.
3.3. Data Correlation
The Pearson correlation matrix of all data obtained during frozen storage of samples is reported in Table 5
. Luminosity showed high positive or negative correlation with the majority of the other tested parameters, proving to be a valid indirect parameter for the quality determination of frozen flesh. By contrast, the red index (a*) was not correlated to any other parameters.
The use of TBARs for the determination of the oxidation level during storage only showed a significant correlation with TMA-O, confirming its low suitability for the discrimination of samples as described above.
The concentration of TMA-O, TMA and DMA were highly correlated to the content of lysine and alanine indicating that these components are strictly related to the protein breakdown occurring during shelf-life. They showed also highly significant correlation to some lipid classes, in particular FFA, MAG and TAG. This may indicate that the variation of all these indexes during storage at –10 °C is related to the same cause, probably a residual enzymatic activity. The relative content of the different FA classes instead did not show significant correlation with other quality parameters.
PCA was developed considering all parameters evaluated in this study and the score plot is reported in Figure 4
. Table S3
reports the contribution of the variables to each component. Along PC1 (45.58%), samples stored at −10 °C after 6 and 12 months are clearly separated from the rest. Both samples stored at −26 °C and sample stored for 6 months at −18 °C were very close to the initial sample (0), while after 12 months of storage at −18 °C a separation occurred along the PC2 (33.79%), confirming the faster degradation rate due to increased storage temperature.
The loading plot of the variables shows that the discrimination is related mainly to the transformation of TMA-O in TMA and DMA and to lipolysis leading to the release of FFA and MAG from TAG. The different FA classes were separated mainly along PC2. The colorimetric parameter of lightness (L*) showed a higher influence on the PC1 compared to the red parameter (a*). Among the considered amino acids, alanine and lysine concentration were highly correlated to the quality degradation, while sarcosine, being close to zero, showed a weak influence.
These results confirm that the quality of mechanically separated mantis flesh subjected to proper industrial and domestic frozen storage is preserved, thus representing a suitable processing and storage technology for obtaining a valuable alternative source of n-3 and n-6 PUFA and essential amino acids for the development of innovative fish-based products addressed for human consumption. Attention should be paid in particular to avoiding the abuse of storage temperature (−10 °C) as it was demonstrated that it promoted an extensive lipid and protein degradation, due to both hydrolytic and oxidative reactions which affected the overall product quality. Whenever the frozen chain is abused, degraded frozen stored mechanically separated mantis flesh could be instead utilized for non-food sectors (animal feeding, pet food, pharmaceutical, cosmetic, etc.), thus contributing in any case to increasing the sustainability and the economic value of the overall food chain.