Red Shrimp Are a Rich Source of Nutritionally Vital Lipophilic Compounds: A Comparative Study among Edible Flesh and Processing Waste

This study was aimed at comparatively analyzing the sterols, tocopherols and fatty acids from edible flesh and processing waste obtained from three shrimp species, utilizing rapid liquid chromatography (LC)-atmospheric-pressure chemical ionization (APCI)-tandem mass spectrometry (MS/MS) and gas chromatography-mass spectrometry (GC-MS). Results revealed the presence of significantly (p < 0.05) high proportions of health-beneficial omega-3 (n3) polyunsaturated fatty acids (PUFAs) in Argentine red shrimp (34.3% in waste and 38.2% in the flesh), compared to black tiger shrimp (16.5–24.2%) and whiteleg shrimp (13.2–22.6%). Among sterols, cholesterol was found most dominant, accounting in the range 349.4 (white shrimp flesh) to 559.3 µg/g fresh weight (FW) (black shrimp waste). Surprisingly, waste was found to contain a substantially higher amount of α-tocopherol, for instance, 21.7 µg/g FW in edible flesh and 35.3 µg/g FW in the waste of black tiger shrimp. The correlation analysis indicated that shrimp with low total contents of lipids might have higher proportions of health-beneficial long-chain (LC)-n3-PUFAs eicosapentaenoic (EPA) and docosahexaenoic acid (DHA). The fat quality indices, including the high ratios of hypocholesterolemic (h)/hypercholesterolemic (H) fatty acids, and lowest values of the atherogenic index (AI) and thrombogenic index (TI) indicated the health-beneficial potential associated with fat intake from red shrimp. Overall, a significant amount of health-beneficial compounds in edible flesh of studied shrimp confers its extraordinary nutritional benefits. Moreover, considering the richness of processing waste with these compounds, their valorization can be prompted.


Introduction
Shrimp are the most economically vital and globally traded commodity among crustaceans and all fish products [1]. According to the FAO statistics, 9.4 million tons of crustaceans (live weight), worth USD 69.3 billion were produced in 2018 [1]. Among them, whiteleg shrimp (Penaeus vannamei) alone accounted for 4.9 million tons of production (52.9% of total crustacean production) [1]. However, the marine capture production is dominated by Argentine red shrimp (Pleoticus muelleri) which accounted for 256 thousand tons of production (4% of the total 6 million tons of marine capture Table 1. List of shrimp samples procured for the study and the amount of waste generated from each species. Authentic standards of fatty acids methyl esters (FAMEs, 37 mix CRM47885), tocols (mix of α-, β-, γ-and δ-tocopherol, and α-, β-, γ-, and δ-tocotrienol), and sterols, including 24α-ethyl cholesterol, 24α-methyl cholesterol, cholesterol, and 5-α-cholestan-3β-ol (internal standard) were obtained from Merck Ltd., Seoul, South Korea. All organic solvents used for extraction were of high-pressure liquid chromatography (HPLC) grade obtained from Daejung Chemicals & Metals Co., Ltd., Korea.

Extraction of Major Lipophilic Compounds
The major lipophilic compounds, including fatty acids, tocols, and sterols, were simultaneously extracted following the previous method [6][7][8] with minor modification. The details procedure is illustrated in Figure 1. The extracted crude lipids were aliquoted to three fractions, as illustrated in Figure 1 and utilized accordingly. Tocols were analyzed before hydrolysis as suggested by Cruz et al. [6]. Fraction 2 was hydrolyzed following the procedure of Cruz et al. [6] with minor modification (Figure 2A). The extracted crude lipids were converted to fatty acid methyl esters (FAMEs) ( Figure 2B) and analyzed by gas chromatograph-mass spectrometry (GC-MS).

Extraction of Major Lipophilic Compounds
The major lipophilic compounds, including fatty acids, tocols, and sterols, were simultaneously extracted following the previous method [6][7][8] with minor modification. The details procedure is illustrated in Figure 1. The extracted crude lipids were aliquoted to three fractions, as illustrated in Figure 1 and utilized accordingly. Tocols were analyzed before hydrolysis as suggested by Cruz et al. [6]. Fraction 2 was hydrolyzed following the procedure of Cruz et al. [6] with minor modification (Figure 2A). The extracted crude lipids were converted to fatty acid methyl esters (FAMEs) ( Figure  2B) and analyzed by gas chromatograph-mass spectrometry (GC-MS).

↓
Crude lipids congaing fatty acids, tocopherol, and sterols were recovered in 15 ml of methanol/methylene chloride (2:1, v/v) containing 0.1 % BHT, and aliquoted as follows: ↓  1 ml of non-saponified crude lipid sample was syringe filtered and transferred to a HPLC vial for liquid chromatography (LC)-atmospheric-pressure chemical ionization (APCI)-multiple reaction monitoring (MRM) based tandem mass spectrometry (MS/MS) of tocols  3 ml sample was hydrolyzed and utilized to quantify the sterols using LC-MRM-MS/MS  2 ml of sample was converted to fatty acid methyl esters (FAMEs) and analysed by gas chromatograph-mass spectrometry (GC-MS)

Analysis of Sterols, Tocols, and Fatty Acid Methyl Esters (FAMEs)
Sterols and tocols were quantified using liquid chromatography (LC)-atmospheric-pressure chemical ionization (APCI)-multiple reaction monitoring (MRM) based tandem mass spectrometry (MS/MS) studies as optimized recently [9]. The optimized instrumental parameters for the simultaneous analysis of sterols and tocols using LC-MRM-MS/MS are illustrated in Table 2. Similarly, the optimized values of collision energy (CE) and declustering potential (DP) of selected Q1 and Q3 MRM transitions for the simultaneous analysis of sterols and tocols using LC-MRM-MS/MS are given in Table 3.  2 ml aliquoted fraction of crude lipids were transferred into a 20 ml glass vial fitted with a Teflonlined screw cap and contents were evaporated to dryness using rotary evaporator at

Analysis of Sterols, Tocols, and Fatty Acid Methyl Esters (FAMEs)
Sterols and tocols were quantified using liquid chromatography (LC)-atmospheric-pressure chemical ionization (APCI)-multiple reaction monitoring (MRM) based tandem mass spectrometry (MS/MS) studies as optimized recently [9]. The optimized instrumental parameters for the simultaneous analysis of sterols and tocols using LC-MRM-MS/MS are illustrated in Table 2. Similarly, the optimized values of collision energy (CE) and declustering potential (DP) of selected Q1 and Q3 MRM transitions for the simultaneous analysis of sterols and tocols using LC-MRM-MS/MS are given in Table 3. Table 2. The optimized instrumental parameters for the simultaneous analysis of sterols and tocols using liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS/MS).  Fatty acid methyl esters (FAMEs) were analyzed by gas chromatography (GC)-mass spectrometry (MS) utilizing a GC-2010 Plus Gas Chromatograph (Shimadzu, Kyoto, Japan) equipped with a QP2010 SE GC-mass spectrophotometer and an HP-5 column (Agilent; 30 m, 0.250 µm thick, and 0.25 mm ID). The injector port and ion source were maintained at 250 and 260 • C, respectively. Helium was used as a carrier gas. The thermal program followed 120-260 • C in 28 min (5 • C/min linear gradient) and held for 10 min. The FAMEs were precisely identified by comparing their retention time and fragmentation pattern with authentic standards [10].

Statistical Analysis and Quality Control
The samples were extracted in triplicates and analyzed separately. The results were analyzed using IBM SPSS statistics (version 25) employing a one-way analysis of variance (ANOVA), and homogenous subsets were determined (considering a significance level of 0.05) to separate the mean values of edible flesh and processing waste of black (black tiger), white (whiteleg), red (Argentine red) shrimp.

Statistical Analysis and Quality Control
The samples were extracted in triplicates and analyzed separately. The results were analyzed using IBM SPSS statistics (version 25) employing a one-way analysis of variance (ANOVA), and homogenous subsets were determined (considering a significance level of 0.05) to separate the mean values of edible flesh and processing waste of black (black tiger), white (whiteleg), red (Argentine red) shrimp.
Shrimp are well known to contain a significant amount of EPA, DHA, and other health-beneficial fatty acids [15][16][17]. The unusual presence of high proportions of linoleic acid and the composition of other fatty acids of white shrimp waste (P. vannamei) observed in the present study agree with previous reports [15,16]. In contrast, Sriket et al. [17] observed only a slight difference for the linoleic acid contents among edible flesh of black tiger shrimp (P. monodon; 13.0%) and white shrimp (P. vannamei, 15.6%). In the present investigation, we observed significantly (p < 0.05) low proportions of linoleic acid in black tiger (3.46% in waste and 3.79% in edible flesh), compared to whiteleg shrimp (24.6% in waste and 20.1% in edible flesh). Interestingly, in the present study, we observed that linoleic acid is compensated by palmitic, palmitoleic (C16:1n7c; cis-9), stearic, and arachidonic acid in the black tiger shrimp. Meanwhile, in the Argentine red shrimp, linoleic acid is compensated by EPA (14.1% in waste and 15.8% in edible flesh) and DHA (17.2% in waste and 20.2% in edible flesh) (Table 4). Furthermore, with the highest occurrence of EPA and DHA, the highest total amount of n3-PUFAs was recorded in Argentine red shrimp (34.3% in waste and 38.2% in edible flesh), with the highest ratio of n3/n6 PUFAs (4.03 in waste and 5.65 in edible flesh) ( Table 5). Consumption of PUFAs in such proportions is highly beneficial to reduce the risk of CVD and many other chronic diseases [18]. Furthermore, among the studied shrimp, the lowest amount of total saturated fatty acids (SFAs) were recorded in Argentine red shrimp (25.2% in waste and 26.7% in edible flesh). Fats with the PUFAs/SFAs ratio of greater than 0.45 are recommended for human consumption to minimize the risk of CVD and other chronic diseases [12]. In the present study, PUFAs/SFAs ratios ranged from 0.89 (black shrimp waste) to 1.70 (red shrimp waste) ( Table 5), which falls within the recommendations. Moreover, the fats with lower AI and TI, and higher ratios of h/H fatty acids are recommended for minimizing the risk of CVD [11]. In the present study, the TI varied significantly among the studied shrimp species, and the lowest value of 0.18 was obtained in red shrimp (waste and edible flesh) (Table 5). Surprisingly, Rosa and Nunes [19] also recorded the TI of 0.18 in edible flesh of red shrimp (Aristeus antennatus) and Norway lobster (Nephrops norvegicus). In the present study, the AI ranged from 0.25 (white and red shrimp waste) to 0.42 (black shrimp waste) which is lower than reported from brown shrimp, Crangon crangon (AI of 1.34) [20]. Similarly, in the present investigation, the ratios of h/H fatty acids were recorded in the range of 2.03 (black shrimp waste) to 3.73 (white shrimp waste). Furthermore, considering the high ratios of h/H fatty acids, shrimp fats are health-beneficial, similar to duck meat (h/H = 3.5), marine fish fillets (h/H = 3.1), and common carp fillets (h/H = 3.4) [12].
Marine species, including shrimp, are an excellent source of health-beneficial LC-n3-PUFAs (especially EPA and DHA) [21]. Furthermore, among the studied shrimp species, Argentine red shrimp are the richest source of n3-LC-PUFAs with the lowest amount of SFAs. Moreover, head and carapace residues are found to contain a similar amounts of these health-beneficial fatty acids, which are discarded during the processing.

Sterols and Tocols Composition
In the present study, three major sterols and α-tocopherol were identified using rapid LC-APCI-MRM based MS/MS analysis (Figures 4 and 5). The representative LC-MRM-MS/MS chromatograms of sterols and α-tocopherol are given in Figure 4. Among waste and edible flesh of studied shrimp, cholesterol was found to be the most dominant, accounting for the range of 349.4 (white leg shrimp flesh) to 559.3 µg/g fresh weight (FW) (black shrimp waste). While other sterols, including 24α-ethyl cholesterol (β-sitosterol) and 24α-methyl cholesterol, were recorded in a small amount ( Figure 5).
Foods 2020, 9, x FOR PEER REVIEW 10 of 20 study, the AI ranged from 0.25 (white and red shrimp waste) to 0.42 (black shrimp waste) which is lower than reported from brown shrimp, Crangon crangon (AI of 1.34) [20]. Similarly, in the present investigation, the ratios of h/H fatty acids were recorded in the range of 2.03 (black shrimp waste) to 3.73 (white shrimp waste). Furthermore, considering the high ratios of h/H fatty acids, shrimp fats are health-beneficial, similar to duck meat (h/H = 3.5), marine fish fillets (h/H = 3.1), and common carp fillets (h/H = 3.4) [12]. Marine species, including shrimp, are an excellent source of health-beneficial LC-n3-PUFAs (especially EPA and DHA) [21]. Furthermore, among the studied shrimp species, Argentine red shrimp are the richest source of n3-LC-PUFAs with the lowest amount of SFAs. Moreover, head and carapace residues are found to contain a similar amounts of these health-beneficial fatty acids, which are discarded during the processing.

Sterols and Tocols Composition
In the present study, three major sterols and α-tocopherol were identified using rapid LC-APCI-MRM based MS/MS analysis (Figures 4 and 5). The representative LC-MRM-MS/MS chromatograms of sterols and α-tocopherol are given in Figure 4. Among waste and edible flesh of studied shrimp, cholesterol was found to be the most dominant, accounting for the range of 349.4 (white leg shrimp flesh) to 559.3 μg/g fresh weight (FW) (black shrimp waste). While other sterols, including 24α-ethyl cholesterol (β-sitosterol) and 24α-methyl cholesterol, were recorded in a small amount ( Figure 5).   Values are mean ± standard deviation of three replicate determinations. a The mean value is significantly (p < 0. 05) highest among the processing waste or edible flesh obtained from black (black tiger), white (whiteleg), red (Argentine red) shrimp.  Values are mean ± standard deviation of three replicate determinations. a The mean value is significantly (p < 0. 05) highest among the processing waste or edible flesh obtained from black shrimp (black tiger), white (whiteleg), red (Argentine red) shrimp.
Similar to the edible flesh of other animals, such as egg, pork, and fish [2,22], shrimp are well known to contain a significant amount of cholesterol [23]. Tsape et al. [23] recorded 1440 and 5210 µg/g FW of cholesterol in muscles and cephalothorax of Penaeus kerathurus, respectively. Turan at al. [20] recorded the 1730 µg/g FW of cholesterol in edible flesh of brown shrimp, C. crangon. In contrast, 608-724 and 578-689 µg/g FW of cholesterol was recorded in edible flesh of red shrimp (Aristeus antennatus) and pink shrimp (Parapenaeus longirostris), respectively [19]. In contrast, in the present study, we recorded 349.4 (white shrimp flesh) to 559.3 µg/g FW (white shrimp waste) of cholesterol. The results of previous studies and the current study indicate that a significant variation is existing for cholesterol content among shrimp species.
A diet rich in cholesterol is considered a negative nutritional aspect, as excess intake of cholesterol may increase the risk of developing cardiovascular diseases (CVD) [22]. Moreover, cholesterol is known for the initiation of pathophysiological angiogenesis [24]. However, in recent years, several countries have withdrawn the upper limit (300 mg/day) of daily cholesterol intake, considering only a slight effect of dietary cholesterol on plasma cholesterol and overall impact on the risk of CVD among the healthy population [22,25]. Moreover, the results of the present study indicate that consumption of 100 g edible flesh of studied shrimps may provide only 34.9-41.6 mg cholesterol, which is under safe limits. Besides, shrimp are rich in health-beneficial LC-n3-PUFA and α-tocopherol, a key cellular antioxidant.
The correlation coefficients (r) between major lipophilic compounds (fatty acids, sterols, and α-tocopherol) recorded in studied shrimp species are given in Table 6. Correlation analysis shows that EPA and DHA are negatively correlated (r = −0.920 and −0.776, respectively) with crude lipids. The significantly higher proportions of linoleic acid in white leg shrimp lipids correlated positively with 24α-ethyl cholesterol (r = 0.952) and 24α-methyl cholesterol (r = 0.983), and negatively with EPA (r = −0.622) and DHA (r = −0.548). These observations suggest that shrimp with low total contents of lipids may have higher proportions of EPA and DHA.