Article pubs.acs.org/JAFC
MALDI-TOF/TOF Mass Spectrometric Determination and Antioxidative Activity of Purified Phosphatidylcholine Fractions from Shrimp Species Li Zhou, Yan Wang, Xiaolin Wang, Yi Liang, Zheng Huang, and Xiaoxiong Zeng* College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China ABSTRACT: Purification, characterization, and antioxidative activity in vitro of shrimp phosphatidylcholines (PCs) were investigated. The molecular structures of shrimp PCs were determined by MALDI-TOF/TOF MS. The MS2 fragments produced from protonated PC precursors and sodiated PC precursors were identified. The specific fragments including [M + Na − trimethylamine]+, [M + Na − 205]+, [M + Na − RCOOH − trimethylamine]+, and [M + H − RCOOH − trimethylamine]+ could distinguish the precursor type to confirm PC molecular structures. The antioxidative activities of purified shrimp PC fractions were evaluated by assay of DPPH free radical scavenging activity, and their effects on the oxidative stability of camellia oil were measured by monitoring changes in the peroxide value assay during oxidation. The PC fractions from Penaeus chinesis and Macrobranchium nipponense showed stronger antioxidative activities than those of other species. All of the shrimp PCs at 0.2% (w/w) improved the oxidative stability of camellia oil significantly (P < 0.05) compared to controls. The experimental findings suggest that shrimp PCs might be a valuable source of natural antioxidants for edible oils or other food dispersions. KEYWORDS: shrimp PC, MALDI-TOF/TOF MS, identification, oxidative stability
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INTRODUCTION Phospholipids (PLs) are major constituents of cell membranes. They play important roles in membrane transport, biological signal transduction, and protein sorting.1,2 Structurally, PLs can be divided into glycerophospholipids and sphingolipids. Glycerophospholipids consist of a polar headgroup with a phosphate moiety and two fatty acyl residues linked to the glycerol backbone. The main classes are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS). Sphingolipids contain a backbone of sphingosine esterified with a single fatty acyl chain. Consequently, PLs have a large structural diversity due to variations in the degree of unsaturation and the length of the alkyl chain, as well as in the nature of the polar headgroup. Several methods are available for the separation of PL classes, such as thin-layer chromatography (TLC) and normal-phase high-performance liquid chromatography (HPLC). The determination of PL molecular species can be achieved by gas chromatography (GC) or HPLC. GC requires hydrolysis of PLs first and then derivatization, which often results in the loss of analyte. HPLC coupled with mass spectrometry (MS) is widely used to identify PL molecular structures.2,3 Soft ionization techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), have been widely employed for lipid analysis.4 Until now, ESI-MS is more often applied for the determination of intact PLs. However, MALDI-MS offers more rapid analysis.5 Furthermore, MALDI has additional advantages such as excellent sensitivity and high tolerance against salts and other impurities found in complex biological samples such as lipids, proteins, and oligosaccharides.6,7 Recently, MALDI-TOF MS has been increasingly used to determine PLs from various matrices.8−12 Unfortunately, most studies reported the possible assignments of m/z values for PL directly instead of providing a detailed © XXXX American Chemical Society
description of the structural analysis. Moreover, to our knowledge, several studies introduced the structure analysis of PLs by postsource decay (PSD) technique. Normally, the PSD fragmentation spectrum of PL sodiated adduct [M + Na]+ is shown, whereas that of the [M + H]+ is not found,6 or only one fragment ion at m/z 184 corresponding to choline headgroup is detected.13 However, measurements performed in MS/MS mode by MALDI-TOF/TOF usually produce protonated and sodiated precursors simultaneously. Thereby, two important questions, that is, how to distinguish the MS fragments produced by protonated precursors or sodiated precursors and how to identify the fatty acyl residues composition of PL, arise. Thus, there is growing interest in developing methods in detail for characterizing and identifying the molecular structures of PLs in biological samples. Furthermore, recent studies are increasingly focused on the nutriprevention of cardiovascular diseases and the minimization of their sequences. Intake of appropriate polyunsaturated fatty acid (PUFA) is a benefit for human health. Previous studies have demonstrated that PC is a better carrier of PUFA than triglycerides (TAG) according to the bioavailability;14,15 thus, they have received more and more attention. However, PUFArich PL may be susceptible to oxidation. Thereby, the oxidative stability of PL during food processing should be investigated. Minor amounts of PL added to edible oils are known to improve stability against oxidation.16,17 PC and PE are the two main PL classes in most food matrices, and PC usually accounts for a much higher proportion than PE. Meanwhile, significant studies on the capacity of shrimp PC as an antioxidant are limited. Therefore, the antioxidant properties of PC were Received: January 16, 2017 Accepted: January 23, 2017 Published: January 23, 2017 A
DOI: 10.1021/acs.jafc.7b00217 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 1. Chromatogram of PL classes identified in E. superba. Separation was performed using a Luna normal phase (150 mm × 3 mm, 3 μm) and a linear gradient ranging from CHCl3/CH3OH (88:12, v/v) to CHCl3/1 M aqueous formic acid (adjusted to pH 3 with triethylamine) (82.4:17.6, v/ v) at a flow rate of 0.5 mL/min. Detection was performed using an ELSD detector. TAG, triglyceride; PE, phosphatidylethanolamine; PC, phosphatidylcholine. v/v)] and solvent B [CHCl3/1 M aqueous formic acid (pH 3) (82.4:17.6, v/v)] with a 20 min gradient transition from 100% solvent A to 100% solvent B. Twenty microliters of sample dissolved in solvent A was injected. The flow rate of mobile phase was 0.5 mL/min. The drift tube temperature and the N2 flow rate of ELSD were set at 45 °C and 1.8 L/min, respectively. Peaks were identified by comparison of retention times with those of standards. PL Purification. According to the method of Zhou et al.,18 the TL extract (150 mg) was placed into a silica gel column, which was preconditioned with 10 mL of CHCl3. Then, the column was eluted with 250 mL of CHCl3 to remove neutral lipid (NLs) and further with 200 mL of CH3OH/1 M aqueous formic acid (pH 3) (98:2, v/v) resulting in the PL fraction. The eluate was evaporated and stored at −20 °C for PC purification. PC Purification. PC was separated from PL solutions by TLC on aluminum foil plates with silica gel 60 F254 (Merck), CHCl3/ CH3OH/25% aqueous ammonia (65:25:4, v/v/v) was used as mobile phase, and phosphomolybdic acid in ethanol (20:80, v/v) was used to visualize compounds. PC was identified by comparison with the retention factor (Rf) of standard PC. Fractions were collected and dissolved in CHCl3, and the supernatant was evaporated under vacuum. The purity of shrimp PCs was checked by HPLC-ELSD as described above. Identification of PC Molecular Species by MALDI-TOF/TOF MS. Sample analysis was performed on a MALDI-TOF/TOF MS instrument (Bruker Daltonics, Bremen, Germany) equipped with a 200 Hz tripled-frequency Nd:YAG pulsed laser with 355 nm. Positive ion spectra were acquired by reflectron mode. The extraction voltage was set at 20 kV. Shrimp PCs were premixed with corresponding matrix solution of DHB, and the mixture (1 μL) was transferred onto a gold-coated MALDI target. Mass spectra were acquired and processed using Flexanalysis, version 3.3 (Bruker Daltonics).19 Gas Chromatographic Analysis of Fatty Aacids. Fatty acid methyl esters were prepared according to the method of Ragonese et al.20 Quantitative analysis was performed on a 3400 Varian GC fitted with an SP 2560 column (0.25 mm × 100 m, 0.20 μm) and equipped with a flame ionization detector (FID). Each sample (1 μL) was injected with a run time of 65 min. The injector was set at 280 °C, the detector was set at 285 °C, and the column was set at 140 °C and raised to 220 °C at a rate of 5 °C/min; the total running time was 65 min. Peaks were identified by comparison of their retention times with those of standards (FAME mix C4−C24). GC data were normalized, and the percentage of fatty acid was calculated as the ratio of each identified peak area against the sum of all identified peak areas. Measurement of DPPH Free Radical Scavenging Activity. According to the method of Maqsood and Benjakul21 with minor modification, 2 mL of sample solution (10% w/v) was added to 2 mL of a freshly prepared DPPH solution (0.1 mM). The solution was mixed thoroughly and placed at 30 °C in the dark for 30 min. The absorbance of the resulting solution was measured at 517 nm using a V-1200 UV/visible spectrophotometer (Mapada Instruments, Shanghai, China). The solution was prepared with ethanol as blank and
investigated. As for PE, it will be another objective of our further study. This study set out to investigate the antioxidant capacities of shrimp PC in relationship to their structures and to investigate their influence in preventing the oxidative deterioration of camellia oil. First, four shrimp species, Euphausia superba, Macrobranchium nipponense, Macrobranchium rosenbergii, and Penaeus chinesis, were chosen. Among them, E. superba and P. chinesis represent marine shrimps and the other two species represent freshwater shrimps. Total lipids (TLs) were extracted with the use of pressurized liquid extraction (PLE), PLs were isolated from TLs by silica-based SPE, and PCs were purified by TLC. Then, the molecular structures of PCs were characterized by MALDI-TOF/TOF MS. Second, the antioxidant capacity of each shrimp PC was evaluated by assay of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, whereas the effect of PC addition on the oxidative stability of camellia oil was determined by measuring the peroxide value (Schaal oven method).
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MATERIALS AND METHODS
Materials. E. superba was provided by Dalian Marine Co., Ltd. (Dalian, China). M. nipponense, M. rosenbergii, and P. chinesis were obtained from local supermarkets (Nanjing, China). They were shelled to afford the meats and were stored at −20 °C prior to processing. Chemicals for extraction were of analytical grade, and those for HPLC analysis were of HPLC grade. Ethanol and n-hexane were provided by Hanbon Science and Technology (Jiangsu, China). Chloroform and methanol was purchased from J. T. Baker (Phillipsburg, NJ, USA). 2,5-Dihydroxybenzoic acid (DHB) and trifluoroacetic acid (TFA) were purchased from Fisher Scientific GmbH (Acros Organics, Germany). DPPH, Supelco 37 component FAME Mix C4−C24, and standard PC (ref: P3556) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sample Preparation. According to Zhou et al.,18 the meat of the above four shrimp species was ground under cryogenic conditions using a 6870 freezer/mill (Spex CertiPrep, Stanmore, UK). The obtained powder was further lyophilized (Telstar LyoQuest, HT-40 Beijer Electronics, Spain) to reach a moisture content of 5% and then stored at −20 °C. Pressurized Liquid Extraction. A Dionex PLE 350 (Dionex, Sunnyvale, CA, USA) was applied for TL extraction. The unit was an automated system with temperature, time, and pressure set at 120 °C, 10 min, and 10 MPa, respectively. The sample (1.0 g) was mixed with sand and placed in a stainless steel cell (10 mL). The solvent mixture employed was n-hexane/ethanol (2:1, v/v). The resulting extracts was vacuum-dried with a rotavapor (40 °C, 30 kPa). Quantification of PC by HPLC-ELSD. The sample was analyzed in an Agilent 1100 HPLC system equipped with a 2424 ELSD (Palo Alto, CA, USA) and a Luna normal-phase silica gel column (150 mm × 3 mm, 3 μm). The eluents were solvent A [CHCl3/CH3OH (88:12, B
DOI: 10.1021/acs.jafc.7b00217 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 2. Positive ion MALDI-TOF mass spectra of purified PC fractions from P. chinesis (A), M. nipponense (B), M. rosenbergii (C), and E. superba (D). The spectra were recorded with DHB as matrix. All peaks are marked by the corresponding m/z values.
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DPPH without antioxidant as control. The calculation of DPPH radical scavenging activity was done as follows:
RESULTS AND DISCUSSION
Separation of PLs and Purification of PC. TLs were first extracted by using the PLE method. Two classes (PE and PC) were identified in the four shrimp species. A representative chromatogram of PL classes of E. superba is shown in Figure 1. It indicated that PC was the predominant PL in the shrimp sample investigated. PL classes were identified and quantified by comparison of retention times and peak areas with standards. PC contents were expressed in milligrams per milligram of TLs. E. superba PC, M. nipponense PC, M. rosenbergii PC, and P. chinesis PC were 29.4, 25.53, 26.26, and 26.16%, respectively. PLs were separated from NLs by column chromatography, and PC was then purified by TLC. The purity of each shrimp PC fraction was >98% detected by HPLCELSD. Molecular Species of PCs. The molecular mass peaks for PC in the four shrimp species were detected by positive MALDI-TOF/TOF MS analysis (Figure 2). All spectra were recorded in the presence of DHB. So far, DHB is the most established and the best characterized MALDI matrix in the lipid analysis.22 The positive ion MALDI spectra of PC contained two precursor ions, [PC + H]+ and [PC + Na]+. The two ions were
DPPH radical scavenging activity (%) = [1 − (A1 − A 2 )/A 0] × 100
A0 is the absorbance of the blank solution (DPPH in ethanol, 0.1 mM), A1 is the absorbance of the sample, and A2 is the absorbance of sample solution in ethanol. The EC50 value (mg/mL) was calculated as the concentration at which the DPPH radical scavenging activity was 50%. Oxidative Stability of Camellia Oil Enriched with PCs. The oxidative stability of shrimp PCs in camellia oil was evaluated using the Schaal oven method. Purified shrimp PCs (0.05, 0.1, 0.15, and 0.2%) and tocopherol (0.02%) were added into 30 g of camellia oil, respectively. All of the samples were incubated at 60 °C monitored with time, and their oxidative stabilities were investigated by peroxide value. Each sample (0.3 mL) was added to 1.5 mL of CHCl3/CH3OH (2:1, v/v) and then vigorously mixed by a vortex appatus for 10 s. Subsequently, the samples were centrifuged for 10 min at 4000g, and 0.2 mL of the organic layer was extracted and added to 2.8 mL of methanol/butanol solution (2:1, v/v), followed by the addition of 15 μL of 3.94 M ammonium thiocyanate and 15 μL of a ferrous iron solution. Finally, the absorbance of the sample was measured by V1200 UV/visible spectrophotometer at 510 nm. C
DOI: 10.1021/acs.jafc.7b00217 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 3. Positive ion MALDI-TOF/TOF MS spectra of PC (C18:0-C18:1) (a) and MS/MS spectra of precursor with m/z at 788.6 (b) and 810.6 (c).
loss of polar headgroup; (3) [M + Na − 205]+ corresponds to loss of sodiated choline phosphate; (4) m/z values (183, 104, 147, and 86 Da) reflect the fragmentations of polar headgroup, which exist in the fragments of both the protonated precursor
further analyzed by MS2. Several main fragments were identified: (1) m/z 184 corresponds to polar headgroup [C5H14NPO4H]+, which allows other PLs to be distinguished; (2) [M + H − 183]+ and [M + Na − 183]+correspond to the D
DOI: 10.1021/acs.jafc.7b00217 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Table 1. Assignment of the m/z Values Detected in the Positive Ion MALDI-TOF/TOF MS Spectra of Four Shrimp PCs
a
m/z
P. chinesis
M. rosenbergii
M. nipponense
E. superba
732
16:0/16:1 + H+
16:0/16:1 + H+
16:0/16:1 + H+
16:0/16:1 + H+
5, 6, 10
734
16:0/16:0 + H+
nda
nd
nd
5, 6, 10
754
nd
nd
16:0/16:1 + Na+ 16:1/18:3 + H+
16:1/18:3 + H+
5, 37
756
16:0/18:3 + H+
16:0/18:3 + H+ 16:0/16:0 + Na+
16:0/18:3 + H+ 16:0/16:0+Na+
16:0/18:3 + H+
5, 22
758
16:0/18:2 + H+
16:0/18:2 + H+
16:0/18:2 + H+
nd
5, 8, 11
760
16:0/18:1 + H+
16:0/18:1 + H+
16:0/18:1 + H+
16:0/18:1 + H+
5, 6, 8, 9, 11
762
16:0/18:0 + H+
nd
nd
16:0/18:0 + H+
11, 22
778
14:0/22:6 + H+ 16:1/20:5 + H+
nd
nd
16:1/20:5 + H+
38−40
780
16:0/20:5 + H+
16:0/20:5 + H+
16:0/20:5 + H+
16:0/20:5 + H+ 16:0/18:2 + Na+ 16:1/18:1 + Na+
5, 8, 11
782
18:1/18:3 + H+ 16:0/18:1 + Na+
nd
16:0/18:1 + Na+ 16:0/20:4 + H+
16:0/20:4 + H+
5, 6, 8−11
784
16:0/18:0 + Na+
18:1/18:2 + H+
16:0/18:0 + Na+ 18:1/18:2 + H+
nd
6, 8
786
18:0/18:2 + H+
18:1/18:1 + H+
nd
nd
6, 8, 10
802
nd
nd
16:0/20:5 + Na+
nd
37
804
nd
18:2/18:2 + Na+ 16:0/20:4 + Na+
18:2/18:2 + Na+ 16:0/20:4 + Na+
nd
5, 40
806
18:1/18:2 + Na+ 16:0/20:3 + Na+
18:1/18:2 + Na+ 18:2/20:4 + H+
18:1/20:5 + H+
18:1/18:2 + Na+ 16:0/20:3 + Na+
8, 38
808
18:0/20:5 18:0/18:2 18:1/18:1 18:2/20:3
H+ Na+ Na+ H+
nd
9, 11
810
nd
nd
18:0/18:1 + Na+ 18:1/20:3 + H+
nd
9, 37
828
nd
16:0/22:6 18:1/20:5 20:4/20:5 18:2/20:4
16:0/22:6 + Na+
23, 37
830
nd
nd
18:0/20:5 + Na+ 20:4/20:4 + H+ 18:1/20:4 + Na+
nd
39−42
832
nd
18:1/20:3 + Na+ 20:4/20:3 + H+
nd
nd
42, 43
+ + + +
H+ Na+ Na+ H+
18:0/20:5 18:0/18:2 18:1/18:1 18:1/20:4
+ + + +
+ + + +
H+ Na+ Na+ H+
18:0/20:5 18:0/18:2 18:1/18:1 18:1/20:4
Na+ Na+ H+ Na+
16:0/22:6 18:1/20:5 20:4/20:5 18:2/20:4
+ + + +
+ + + +
Na+ Na+ H+ Na+
references
nd, not detected. E
DOI: 10.1021/acs.jafc.7b00217 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 2. Fatty Acid Profiles of Purified PCsa from Shrimp Lipids and Camellia Oil fatty acid
a
P. chinesis (%)b c
14:0 16:0 16:1 18:0 18:1n9 18:2n6 20:0 20:1 18:3n3 20:2 20:3n3 20:4n3 EPA (20:5n3) DHA (22:6n3)
nd 14.93 nd 5.32 51.91 24.15 0.28 0.31 0.44 nd 0.37 nd 1.44 0.85
EPA + DHA ΣSFA ΣMUFA ΣPUFA
2.29 20.53 52.22 27.25
± 0.02 ± ± ± ± ± ±
0.01 0.12 0.06 0.01 0.01 0.02
± 0.02 ± 0.01 ± 0.03
M. rosenbergii (%)b nd 18.39 0.44 6.83 36.82 28.46 0.44 0.34 0.77 0.35 0.91 0.13 3.38 2.74
± ± ± ± ± ± ± ± ± ± ± ± ±
M. nipponense (%)b nd 15.68 1.54 6.34 42.16 28.91 0.42 nd 0.55 nd 0.57 0.21 2.49 1.13
0.06 0.01 0.04 0.08 0.02 0.01 0.04 0.02 0.03 0.02 0.01 0.07 0.05
6.12 25.66 37.6 36.74
3.62 22.44 43.7 33.86
± ± ± ± ± ±
0.03 0.02 0.01 0.09 0.22 0.01
± 0.02 ± ± ± ±
0.03 0.01 0.04 0.02
E. superba (%)b 1.28 23.94 1.65 3.06 32.76 17.46 nd nd 1.28 0.84 nd nd 10.04 7.69
± ± ± ± ± ±
0.01 0.04 0.02 0.03 0.07 0.04
± 0.01 ± 0.03
± 0.02 ± 0.05
17.73 28.28 34.41 37.31
camellia oil (%)b nd 8.95 nd 2.11 78.87 9.23 nd 0.56 0.28 nd nd nd nd nd
± 0.01 ± 0.01 ± 0.03 ± 0.04 ± 0.01 ± 0.03
nd 11.06 79.43 9.51
Results are expressed as the mean ± SD (n = 3). bPercentage of the total peak area of fatty acids. cnd, not detected.
and sodiated precursor; (5) [M + H − TMA − RnCOOH]+ and [M + Na − TMA − RCOOH]+ correspond to the loss of a trimethylamine group (TMA, 59 Da) and a fatty acid; (6) [M + H − TMA]+ or [M + Na − TMA]+ corresponds to the loss of TMA; (7) two acyl groups [RCO]+ correspond to the detached fatty acyl residues; (8) [M + H − RCOOH]+ or [M + Na − RCOONa]+ results from the loss of fatty acid and sodiated fatty acid. The last two fragments provided extra information on the composition of the two fatty acyl residues in PC molecules. Obviously, this showed that not only protonated PC precursors but also the sodiated PC precursors yielded multiple fragment ions. The question as to how to identify the MS2 fragments produced from [M + H]+ or from [M + Na]+ would be an influential factor to determine molecular structures correctly. In fact, through the analysis of fracture mode, we found that [M + Na − TMA]+, [M + Na − 205]+, and [M + Na − RCOOH − TMA]+ were characteristic fragments produced by [M + Na]+ and [M + H − RCOOH − TMA]+ was a characteristic fragment of [M + H]+. Those fragments could be used as proof to confirm the precursor types. Representative MS and MS2 fragmentation spectra of molecular species are presented in Figure 3. They clearly showed that the positive ion MALDI spectra of reference (C18:0−C18:1) PC contained two precursor ions, [M + H]+ and [M + Na]+ (Figure 3a). One characteristic fragment ion at m/z 184 corresponded to polar headgroup [C5H14NPO4H]+;23,24 the less intense m/z ions at 104 choline [C5H13NOH]+, m/z 86 dehydrocholine [C5H12N]+, and m/z 147 [C2H5O4PNa]−13 existed in the fragments of both the two precursor ions (Figure 3b,c). Furthermore, the MS2 fragmentation spectra of [M + H]+ at m/z 788.6 contained multiple fragment ions that allowed for its identification as [(18:0− 18:1)PC + H]+ (Figure 3b). Two of these fragments, m/z 265 and 267, were identified as detached oleic and stearic acyl groups. Two other fragment ions at m/z 447 and 445 corresponded to the simultaneous loss of both the TMA group and either an oleic acid or a stearic acyl acid. m/z 504 and 506 corresponded to the loss of stearic fatty acid and oleic fatty acid, respectively. m/z 605 corresponded to the loss of
polar headgroup. Figure 3c shows the MS2 fragmentation spectra of [M + Na]+ at m/z 810.6. Two fragment ions at m/z 467 and 469 corresponded to loss of both the TMA group and either stearic fatty acid or oleic fatty acid. Two fragment ions at m/z 526 and 528 corresponded to the loss of stearic fatty acid and oleic fatty acid, respectively. A specific fragment ion at m/z 605 corresponded to loss of sodiated choline phosphate. The fragment ions at m/z 627 and 751 corresponded to loss of polar headgroup and TMA group, respectively. Consequently, [M + H − 18:0 − TMA]+ and [M + H − 18:1 − TMA]+ at m/z 445 and 447, respectively, were specific fragments of [M + H]+ (Figure 3b). The fragment ions, such as [M + Na − TMA]+, [M + Na − 205]+, [M + Na − 18:0 − TMA]+, and [M + Na − 18:1 − TMA]+ at m/z 751, 605, 467, and 469, respectively, were specific fragments produced by [M + Na]+ (Figure 3c). Conversely, through analysis of the fracture mode of MS2 fragments, it could be inferred that the m/z 810.6 was [18:0/ 18:1 + Na]+ instead of [18:1/20:3 + H]+ or [18:0/20:4 + H]+, because no characteristic fragments were found if the m/z 810.6 was [18:1/20:3 + H]+ or [18:0/20:4 + H]+. This method was applied to each m/z peak of shrimp PCs and enabled the identification of the constitutive PC molecular species in other food sources. Table 1 provides the structural assignments of molecular species of purified PC from the four shrimps. The four shrimp PCs showed different compositions of molecular species. In the cases of M. rosenbergii and M. nipponense, four species of ω-3 (18:3, 20:3, 20:5, and 22:6) fatty acyl residues and two species of ω-6 (18:2, 20:4) fatty acyl residues were identified. Species determination data for shrimp PCs were in agreement with their fatty acid profiles (Table 2), with high amounts of 16:0, 18:1, 18:2, and 18:0 being by far the predominant one. It was worth noting that the amounts of EPA (20:5n3) and DHA (22:6n3) were obviously higher in E. superba than in others (Table 2). Antioxidative Activity. Measurement of the disappearance of DPPH free radical is widely used for the assessment of antioxidative activity of various samples.16,25 DPPH shows a maximum absorbance at 517 nm, which disappears upon reaction with antioxidant. The DPPH free radical scavenging F
DOI: 10.1021/acs.jafc.7b00217 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry activities of various shrimp PCs were investigated, and the results are shown in Figure 4. All of the shrimp PCs exhibited
Figure 4. Scavenging effects on DPPH radical of shrimp PCs.
DPPH radical scavenging activity. For M. rosenbergii, and E. superba, the scavenging rates increased with the sample concentration in the range of 0−3 mg/mL, and then it achieved balance with no significant change (P < 0.05). E. superba PC showed the highest EC50, and the lowest value was found for P. chinesis PC. M. rosenbergii PC and M. nipponense PC showed intermediate antioxidative activity. The antioxidant efficiency of these fractions was evaluated by the lowest concentration capable of inhibiting 50% of the radical. Therefore, the descending order of antioxidative activity of shrimp PCs was as follows: P. chinesis > M. nipponense > M. rosenbergii > E. superba. A possible explanation was that P. chinesis and M. nipponense contained higher unsaturated fatty acyl compositions (MUFA + PUFA) in PC molecule than other shrimp species (Table 2). This point was in accordance with a previous study that found the unsaturation degree in fatty acyl chain of PLs was closely associated with their antioxidant capacity.17 Oxidative Stability. Figure 5 shows the oxidative stability of camellia oils with and without shrimp PCs during 240 h of storage at 60 °C. The influence of PC addition on the oxidative stability of camellia oil was investigated by measuring the peroxide value. The control sample without added PC fraction was oxidized rapidly with the increase of oxidation time. With the oxidation of 240 h, the peroxide value reached 35.5 mequiv/kg oil. However, the added PC fraction shows inhibition of the excessive growth of peroxide value during the oxidation process. Moreover, with the increase of PC content, the oxidative stability of camellia oil was enhanced (Figure 5a−d). Most notably, the effect of 0.2% addition of all the shrimp PCs studied was better than 0.02% addition of tocophenol on the oxidative stability of camellia oil. When the addition of PC was up to 0.4%, the oxidative stability of the camellia oil was not significantly enhanced (P > 0.05) (data not shown). Therefore, the appropriate additional quantity of all the shrimp PC evaluated was 0.2%, which was enough to exert good antioxidant effect in the camellia oil model system. Furthermore, the antioxidant capacity of the various shrimp PCs in camellia oil was compared. The oxidative stabilities of camellia oil enriched with various shrimp PCs was in the order P. chinesis > M. nipponense > M. rosenbergii > E. superba (Figure 5e). This indicated that shrimp PC could exhibit antioxidative activity in camellia oil in low concentration (
