A comprehensive study of lipid profiles of round scad (Decapterus maruadsi) based on lipidomic with UPLC-Q-Exactive Orbitrap-MS

Food Research International 133 (2020) 109138

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A comprehensive study of lipid profiles of round scad (Decapterus maruadsi) based on lipidomic with UPLC-Q-Exactive Orbitrap-MS Chen Hea, Zexin Suna, Xingchen Qua, Jun Caoa, , Xuanri Shena, Chuan Lia,b, ⁎

T

⁎

a

Hainan Provincial Engineering Research Centre of Aquatic Resources Efficient Utilization in the South China Sea, College of Food Science and Engineering, Hainan University, Haikou 570228, China b Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic University, Dalian 116034, China

ARTICLE INFO

ABSTRACT

Keywords: Round scad (Decapterus maruadsi) Stereospecific analysis Lipidomics Lipid profiles UPLC-Q-Exactive Orbitrap-MS EPA DHA

Round scad (Decapterus maruadsi) is rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). However, there is no comprehensive information covering its lipidomic profile. In this study, lipid profiles including fatty acid composition, distribution and detailed structure information were explored using gas chromatography-flame ionization detector (GC-FID) and ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap-MS). The results showed that triacylglycerol (TG) was the dominant class, and the EPA and DHA were concentrated as glycerophospholipid (GP). The saturated fatty acids and monounsaturated fatty acids were mainly esterified in positions 1 and 3 of TG and sn-1-position of GP, while the polyunsaturated fatty acids (PUFA) were mainly esterified in sn-2-position of TG and GP. A total of 1282 species from six classes (21 subclasses) including glycerolipids (GL), sphingolipids (SP), GP, fatty acyls (FA), saccharolipids (SL) and prenol lipids (PR) were identified. Several molecular species were characterized with PUFA, especially EPA and DHA in GP. Considering the superior fatty acids composition and distribution of round scad, it is deserved for further exploitation of its marine lipid source on account of the healthy and nutritional functions.

1. Introduction Lipids are subclassified into eight categories: glycerolipids (GL), glycerophospholipids (GP), saccharolipids (SL), sphingolipids (SP), fatty acyls (FA), sterols (ST), polyketides (PK) and prenol lipids (PR) (Fahy et al., 2005). Over the last decades, there has been a growing recognition of the effects of dietary fat on well-being, health and disease prevention (Valdés, Cifuentes, & León, 2017). Fish lipid as an essential bioactive component in marine fish is widely known for its high level of long-chain polyunsaturated fatty acids (LC PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have been evidenced with an array of health benefits in all life stages (Dyall, 2015). FAs were reported to have better utilization and oxidative stability when they are distributed in GP rather than triacylglycerols (TG) (Michalski et al., 2013). Previous researches have established that FAs

especially n-3 PUFA esterified in the sn-2 position showed a superior bioavailability to that in the sn-1,3 positions of TG (Linderborg et al., 2019; Michalski et al., 2013). Thus, it is necessary to study the distribution of FAs among different classes and esterification positions. However, the characterization of fatty acid and a certain class of lipid profile cannot comprehensively analyze the fish lipid nutrition. Lipidomic, an extensive study of all lipids in a biological system at a specific time, allows the qualitative and quantitative analysis of a large set of lipid molecular species simultaneously (Li, Vosegaard, & Guo, 2017). Recently, LC-MS-based lipidomics due to its high sensitivity and accurate quantitation, has been applied for complicated lipids analysis from aquatic products to investigate lipid composition and nutritional value. Successful applications were conducted in 15 lipid subclasses among three thermal processing methods on tilapia fillets (Shi et al., 2019) and 12 major lipid subclasses of four fish species (Wang et al., 2019).

Abbreviations: UPLC-Q-Exactive Orbitrap-MS, ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry; GL, glycerolipids; SP, sphingolipids; GP, glycerophospholipids; FA, fatty acyls; SL, saccharolipids; PR, prenol lipids; TG, triacylglycerols; DG, diacylglycerols; Cer, ceramides; SM, sphingomyelins; So, sphingoshines; PC, phosphatidylcholines; PE, phosphatidylethanolamines; CL, cardiolipins; PA, phosphatidic acids; PI, phosphatidylinositols; PS, phosphatidylserines; PG, phosphatidylglycerols; PM, phosphatidylmethanols; LPC, lysophosphatidylcholines; LPE, lysophosphatidylethanolamines; LPI, lysophosphatidylinositols; LPS, lysophosphatidylserines; LPG, lysophosphatidylglycerols; MGDG, monogalactosylmonoacylglycerols; Co, coenzyme; DHA, docosahexaenoic acids; EPA, eicosapentaenoic acids; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids ⁎ Corresponding authors at: College of Food Science and Engineering, Hainan University, No. 58, Renmin Avenue, Haikou 570228, China. E-mail addresses: [email protected] (J. Cao), [email protected] (C. Li). https://doi.org/10.1016/j.foodres.2020.109138 Received 9 October 2019; Received in revised form 28 February 2020; Accepted 29 February 2020 Available online 02 March 2020 0963-9969/ © 2020 Elsevier Ltd. All rights reserved.

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Round scad (Decapterus maruadsi), a marine coastal warm-water wild fish belonging to the family mackerel, mainly harvests the eastern Indian Ocean and Western Central Pacific. Although the total capture of Decapterus spp. and aquaculture yield exceeded 1.29 million tons in 2016 (FAO, 2018), earning its forefront place in marine fishing output, it is continuous still a low-valued dark-fleshed fish. Many reports have focused on round scad novel applications of protein such as antioxidative peptides (Hu et al., 2020), iron-binding peptide (Jiang et al., 2019) and physiochemical properties during pH-shifting and heating (Sun et al., 2019). To our best knowledge, there has been no detailed investigation of the nutritional value of the lipid in round scad. Therefore, characterizing fatty acids distribution and lipidomic analysis were performed in edible parts of round scad. In this work, comprehensive investigations of lipid profile, including lipid content, fatty acid composition among different lipid classes and the stereospecific analysis in TG and GP were conducted. In addition, the molecular species of total lipids were determined using ultra-highperformance liquid chromatography coupled with hybrid quadrupoleorbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap-MS). This study will help to improve the lipidomic coverage of fish lipids and estimate the nutritional value of bioactive lipid compounds for health care, thereby promoting the consumption of round scad.

which was first activated with 15 mL of chloroform. The TL without internal standard was dissolved in 1 mL chloroform and added to the activated cartridge. Then the neutral lipids (NL), SL, and GP were eluted 5 times with 10 mL chloroform containing 1% acetic acid, 10 mL acetone: methanol (9:1, v/v) and 10 mL methanol, respectively. The eluents were collected and dried with a stream of N2. The NL residues were further separated by a Florisil SPE cartridge (2 g, 10 mL) (Wu et al., 2015). The Florisil SPE cartridge was firstly activated using n-hexane (6 mL). The NL residues were dissolved in chloroform and separated by the activated cartridge. Subsequently, the TG, diacylglycerols (DG), monoacylglycerols (MG) and free fatty acids (FFA) were eluted 4 times with n-hexane: ethyl ether (85:15, v/v; 12 mL), n-hexane: ethyl ether (70:30, v/v; 12 mL), n-hexane: ethyl ether (25:75, v/v; 12 mL) and n-hexane: ethyl ether (95:5, v/v; 12 mL), respectively. The eluents were collected and dried with a stream of N2. The FAs in TG, DG, MG were transesterified to FAME according to a previous study (Cruz-Hernandez et al., 2004). The derivation of FFA was carried out using 1 mL of 13–15% BF3-MeOH solution for 10 min in a 90 °C bath. Then, 2 mL hexane and 2 mL deionized water were added (Bravi, Marconi, Sileoni, & Perretti, 2017). Finally, the supernatant was collected and analyzed by GC-FID system. 2.5. Stereospecific analysis of TG

2. Materials and methods

Fresh round scad (40 tails, weight: 41.75 ± 5.05 g; length: 17.26 ± 1.95 cm) were purchased from a Sanxi Road Trade Market (Haikou, China) in May 2019 and divided into three groups randomly. After the removal of viscera, the edible parts were homogenized and stored at −80 °C until analysis.

The method for TG enzymatic hydrolysis was used by Lei et al. (2013). The enzymatic hydrolysate of TG was separated with aminopropyl SPE cartridge (NH2 SPE, 500 mg, 3 mL) (Pernet, Pelletier, & Milley, 2006). The TG hydrolysate was loaded after the column was activated with 4 mL of nhexane. Then, 4 times with n-hexane: ethyl acetate (85:15, v/v; 8 mL) were performed and sn-2-MG was obtained by eluting 3 times with methylene chloride: methanol (2:1, v/v; 6 mL). Finally, the eluent (sn-2-MG) was dried with a stream of N2 and analyzed by GC-FID.

2.2. Chemicals

2.6. Stereospecific analysis of GP

Fatty acid methyl esters (FAME) standards (GLC #463) were obtained from NuChek-Prep (Elysian, MN, USA). Lysophosphatidylcholine (LPC 12:0) standard, porcine pancreatic lipase and phospholipase A2 were purchased from Sigma-Aldrich (Shanghai, China). Chromatographically grade solvents were purchased from Tedia Company (Fairfield, OH, USA; n-hexane), Merck (Darmstadt, Germany; acetonitrile, methanol, isopropanol, chloroform) and Aladdin® (Shanghai, China; methyl acetate).

The GP enzymatic hydrolysis was accorded to a reported method (Schroter, Suss, & Schiller, 2016) with slight modifications. The GP was dissolved in 2 mL ethyl ether; Then, 0.1 mL phospholipase A2 solution (50 μg/mL in tris-HCl buffer (1 mol/L, pH 8.0) containing 0.5 mM CaCl2 and 0.05% butylated hydroxytoluene) were added. After 3 h of the vigorous shaking, methanol and chloroform were added. The organic phase was evaporated under a stream of N2 and then separated into FFA and lysoglycerophospholipids (Lyso-GP) using an NH2 SPE cartridge (Pernet et al., 2006).

2.1. Sample pretreatment

2.3. Determination of total lipid and fatty acids

2.7. Lipidomics workflow by LC/MS

Total lipid (TL) extraction was carried out according to the modified method (Folch, Lees, & Stanley, 1957). Briefly, approximately 1 g fish sample was homogenized with a mixture of 9 mL methanol/chloroform (1:2, v/v) and 2.25 mL 0.88% potassium chloride solution. Methyl tridecanoate was added as the internal standard. After shaking for 2 h, the organic phase was collected and dried with a stream of N2. The lipids recovered were stored at −80 °C until use. The fatty acid methyl esterification was performed according to the alkaline methyl esterification method (Cruz-Hernandez et al., 2004) and subsequently analyzed by Agilent 7890A gas chromatographyflame ionization detector (GC-FID) equipped with a CP-Sil 88 capillary column (100 m × 0.25 mm × 0.2 μm, Agilent, USA), according to a previous study (Cao et al., 2014). The oven temperature was set at 45 °C for 4 min, then increased to 175 °C at 13 °C/min and kept for 27 min. The final temperature was to 215 °C at 4 °C/min and held for 35 min.

2.7.1. Sample preparations Lipids were extracted from 50 mg of the freeze-dried samples using the modified Folch et al. (1957) method. The final lipid extract was resuspended with isopropanol/methanol (1:1, v/v), and then the LPC 12:0 standard was added for semi-quantification. Samples were centrifuged and the supernatant was transferred for LC-MS analysis. 2.7.2. Ultra-performance liquid chromatography separation The qualitative and quantitative analysis of lipid molecules were performed on an Ultimate 3000LC system (Thermo, Bremen, Germany) equipped with an ACQUITY UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm; Waters). The sample chamber and column temperatures were maintained at 10 °C and 55 °C, respectively. The injection volume was 1 μL and the flow rate was set to 0.4 mL/min. The mobile phases were phase A: acetonitrile/water (60/40, v/v, 0.1% acetic acid, 10 mmol/L ammonium formate) and phase B: acetonitrile/isopropanol (10/90, v/ v, 0.1% acetic acid, 10 mmol/L ammonium formate). The gradient elution program was 5% B initially, followed by a linear gradient to 100% B over 17 min.

2.4. Fatty acid distribution among lipid classes The oil samples were separated by a silica solid-phase extraction cartridge (Si SPE cartridge, 2 g, 6 mL) (Rincon-Cervera et al., 2019), 2

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2.7.3. Mass spectrometric conditions Samples were performed on a Q-Exactive hybrid quadrupoleOrbitrap mass spectrometer (Thermo, Bremen, Germany) in both ESI positive and negative modes. The parameter settings were as follows: collision gas and dissociation gas, nitrogen (99.999%); capillary temperature, 320 °C; spray voltage, 3.8 kV (ESI+), 3.0 kV (ESI−); resolution: 70,000 (MS), 17,500 (MS/MS). Full scan spectra were measured range from 150 to 2000 m/z. Instrument tuning and mass calibration were performed with sodium formate and leu-enkephalin was used for accurate mass locking ([M+H]+: 556.2771 Da; [M−H]−: 554.2615 Da). The lipid molecular species were identified according to accurate mass and fragment matching using Lipidsearch Software (Thermo, Bremen, Germany) and the online LIPID MAPS database. The semi-quantification analysis was conducted by the area ratio of each species to that of the internal standard.

The fatty acid profiles of TL indicated the amounts of saturated fatty acids (SFA, 861.11 mg/100 g sample), monounsaturated fatty acids (MUFA, 452.10 mg/100 g sample) and PUFA (977.64 mg/100 g sample) (Table 1). C16:0 (324.85 mg/100 g sample) was the most abundant SFA, followed by C18:0 (123.54 mg/100 g sample). The major MUFA was oleic acid (9cC18:1, 129.45 mg/100 g sample), accounting for more than a quarter of cis MUFA. DHA (384.30 mg/100 g sample) and EPA (148.84 mg/100 g sample) were the dominant PUFA. The significantly higher absolute amount of n-3 PUFA (750.60 mg/ 100 g sample) was observed than that of n-6 PUFA (227.04 mg/100 g sample). Such tendency could attribute to the fact that small pelagic fishes consume zooplankton feeding on phytoplankton, which is characterized with richness in n-3 PUFA while a lack of n-6 PUFA (Murillo, Rao, & Durant, 2014). It is incredibly essential when evaluating the nutritional contribution of fish species to the dietary requirements to follow the recommendations of human consumption levels. Several indicators were used to evaluate the nutritional value, such as the EPA + DHA levels, PUFA/SFA radios, and n-6/n-3 PUFA radios. The Food and Agriculture Organization (FAO) has recommended the daily consumption of 250 mg EPA + DHA for adults to prevent coronary heart disease and a higher dose for those with coronary heart disease (Elmadfa & Kornsteiner, 2009). Therefore, approximately 394 g of round scad can be consumed to meet this weekly recommendation (2100 mg EPA + DHA per week). Besides, the PUFA/SFA ratio was reported to have a negative correlation with cardiovascular disease (RinconCervera et al., 2019). In the present study, the rate of PUFA/SFA (1.14) was far superior to the recommended value of FAO/WTO of 0.4–0.5. The global diets tend to take in much higher amounts of n-6 fatty acids than n-3 fatty acids because of the abundance of vegetable oils intake, which was reported to increase the risk of chronic disease (Dohrmann et al., 2019). Thus, an appropriate n-6/n-3 PUFA ratio of below 4:1 is raised by FAO/WTO and a decrease consumption in dietary n-6/n-3 PUFA is conductive to reduce the risk of cardiovascular disease and

2.8. Statistical analysis All samples were repeated in triplicate. The results are presented as means ± standard deviations (means ± SD). One-way analysis of variance (ANOVA) and Duncan's multiple comparison were used to compare the differences between mean values. All of the data were performed with SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). A level of probability at p < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Lipid content and fatty acid profiles As shown in Table 1, the TL content of round scad was 3.79 g/100 g wet sample, according to which it was categorized as a low-fat fish (2–4% of wet weight) (Prato & Biandolino, 2012). The TL content in round scad was higher than some wild marine fishes (Rincon-Cervera et al., 2019), so it could be a potential marine fish oil source.

Table 1 Main fatty acid profiles of total lipid and different lipid classes from Decapterus maruadsi by GC-FID. TL

TG

DG

MG

FFA

SL

GP

Content

(g/100 g sample) 3.79 ± 0.25

(% of total lipid) 65.99 ± 0.73

5.00 ± 0.61

9.05 ± 0.68

0.01 ± 0.00

7.36 ± 0.30

6.51 ± 0.49

Fatty acid profiles C14:0 C16:0 C17:0 C18:0 C20:0 C22:0 Total SFA 9cC16:1 9cC18:1 11cC18:1 13cC22:1 Total cis MUFA 9c12cC18:2n-6 C18:3n-3 C20:5n-3 EPA C22:4n-6 C22:5n-3 DPA C22:6n-3 DHA Total PUFA Total n-3 PUFA Total n-6 PUFA EPA + DHA n-6/n-3 PUFA/SFA

(mg/100 g raw sample) 76.89 ± 9.40 324.85 ± 16.03 83.47 ± 2.98 123.54 ± 7.39 35.54 ± 0.56 31.57 ± 1.65 861.11 ± 61.27 68.27 ± 2.80 129.45 ± 25.67 26.06 ± 1.70 86.77 ± 8.93 452.10 ± 34.10 35.85 ± 3.22 80.78 ± 3.46 148.84 ± 18.52 17.51 ± 0.58 26.23 ± 0.68 384.30 ± 17.67 977.64 ± 48.68 750.60 ± 37.06 227.04 ± 11.62 533.15 ± 23.01 0.30 1.14

(% of total fatty acids) 6.66 ± 0.09 5.88 ± 0.05 25.25 ± 0.14 b 20.38 ± 0.09 d 1.15 ± 0.03 0.88 ± 0.00 6.89 ± 0.09 e 8.16 ± 0.08 d 1.79 ± 0.04 1.43 ± 0.02 1.38 ± 0.11 1.06 ± 0.03 44.72 ± 0.32 b 39.04 ± 0.11 d 5.17 ± 0.01 5.36 ± 0.12 8.61 ± 0.09 a 8.65 ± 0.08 a 2.01 ± 0.10 1.96 ± 0.02 5.58 ± 0.07 4.37 ± 0.12 22.37 ± 0.24 a 21.10 ± 0.10 b b 1.81 ± 0.05 1.66 ± 0.02 c 4.68 ± 0.04 a 3.96 ± 0.02 b 6.90 ± 0.06 b 6.82 ± 0.09 b 0.23 ± 0.00 0.25 ± 0.01 0.96 ± 0.01b 1.39 ± 0.14 a 13.57 ± 0.08 e 20.70 ± 0.66 d 31.24 ± 0.12 d 38.06 ± 0.18 b 27.36 ± 0.11 d 34.04 ± 0.37 c 3.88 ± 0.09 d 3.85 ± 0.05 d 20.46 ± 0.14 d 27.88 ± 0.54 c 0.14 0.11 0.70 0.97

5.81 ± 2.17 32.34 ± 0.57 a 1.94 ± 0.47 14.52 ± 2.15 b 0.61 ± 0.01 0.57 ± 0.15 58.16 ± 2.44 a 4.89 ± 0.66 8.24 ± 0.13 a 3.26 ± 0.28 0.87 ± 0.22 18.00 ± 0.73 c 3.65 ± 0.78 a 0.74 ± 0.12 d 3.76 ± 0.39 d 1.10 ± 0.10 0.83 ± 0.05 b 7.83 ± 1.59 f 22.90 ± 0.66 e 13.17 ± 1.72 e 9.73 ± 0.25 a 11.59 ± 1.80 e 0.74 0.39

4.23 ± 0.26 24.55 ± 0.41 b 0.86 ± 0.10 9.07 ± 0.08 c 0.78 ± 0.05 1.00 ± 0.02 41.66 ± 0.15 cd 3.44 ± 0.01 4.82 ± 0.42 d 1.36 ± 0.29 1.18 ± 0.25 12.90 ± 0.80 d 1.30 ± 0.02 d 2.25 ± 0.39 c 11.34 ± 0.09 a 0.21 ± 0.01 0.89 ± 0.01 b 28.18 ± 0.47 b 46.68 ± 1.70 a 43.16 ± 1.25 a 3.82 ± 0.02 d 40.18 ± 1.31 a 0.09 1.12

6.45 ± 0.16 22.52 ± 0.03 c 0.98 ± 0.04 10.61 ± 0.36 b 1.01 ± 0.09 0.61 ± 0.11 44.19 ± 0.86 bc 4.62 ± 0.02 7.73 ± 0.13b 2.91 ± 0.43 2.59 ± 0.01 17.45 ± 0.36 c 1.72 ± 0.06 c 3.53 ± 0.21 b 3.14 ± 0.02 d 2.30 ± 0.41 1.06 ± 0.03 b 23.77 ± 0.58 c 36.31 ± 0.12 c 32.31 ± 0.68 c 6.50 ± 0.63 b 26.91 ± 0.60 c 0.20 0.82

0.94 ± 0.02 19.21 ± 0.80 e 1.24 ± 0.00 17.55 ± 0.53 a 0.89 ± 0.04 1.01 ± 0.17 42.92 ± 1.99 c 1.05 ± 0.01 6.46 ± 0.00c 2.02 ± 0.05 0.97 ± 0.01 11.49 ± 0.26 e 1.07 ± 0.01 e 2.04 ± 0.06 c 6.32 ± 0.34 c 0.71 ± 0.89 1.64 ± 0.00 a 30.52 ± 0.42 a 45.03 ± 2.24 a 40.73 ± 0.28 b 4.73 ± 0.17 c 36.67 ± 0.18 b 0.11 1.05

The results are presented as means ± SD (n = 3). Values in the same row with different letters are significantly different (p < 0.05). Abbreviations were: TL, total lipid; TG, triacylglycerols; DG, diacylglycerols; MG, monoacylglycerols; FFA, free fatty acids; SL, saccharolipids; GP, glycerophospholipids. 3

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A 35 a

g/100 g fatty acids

c

15 10

d hi

TG

DG

SFA

60

j

SL

MUFA

PUFA

m

cd

d

d

0

f

g

sn-2

TG

sn-1,3

c

5

h

h

sn-1

sn-2

GP

g

TG

h j

i

n l

DG

e k

j

l

MG

d

g

f lm kl

mn

SL

GP

50

n-3 PUFA

e

10

b

a

cd

f

10

f

GP

n-6 PUFA DHA

15

40

c

30

0

g

b

40

20

MG

k

D

70

50

e

j h

i

l

n-3 PUFA EPA

a

b

20

0

Percent (%)

B 20

PUFA

25

5

C

MUFA

Percent (%)

g/100 g fatty acids

30

SFA

n-6 PUFA

EPA

a

b

c

c

30

d

DHA

d

d

20

10

0

e

efg ef

sn-2

g

TG

sn-1,3

e

efg

fg fg

sn-1

fg

GP

sn-2

Fig. 1. Positional distribution of (A) total SFA, total cis MUFA and total PUFA, (B) n-3 PUFA, n-6 PUFA, EPA and DHA in triacylglycerols (TG), diacylglycerols (DG), monoacylglycerols (MG), saccharolipids (SL) and glycerophospholipids (GP) from Decapterus maruadsi (g/100 g fatty acids). Stereospecific analysis of (C) total SFA, total MUFA and total PUFA, (D) n-3 PUFA, n-6 PUFA, EPA and DHA in TG and GP from Decapterus maruadsi (% of total fatty acids). Values with different letters are significantly different (p < 0.05).

cancer (Rincon-Cervera et al., 2019). From a nutritional standpoint, round scad was highly recommended because of its pretty low ratio of n-6/n-3 (0.38). Similar results were reported in other marine species (Rincon-Cervera et al., 2019). Thus, round scad is a perfect food for human health to improve dietary structure.

The amounts of SFA, MUFA, PUFA, n-3 PUFA, n-6 PUFA, EPA and DHA (g/100 g fatty acids) were calculated and presented in Fig. 1. It was apparent that fatty acids were concentrated as TG due to its high proportion in TL. However, a few interesting observations were performed concerning the distribution and composition among different classes. The PUFA levels followed the order of TG > GP > SL > MG > DG. Quantitatively, TG and GP had considerable amounts of PUFA (20.61 g/100 g fatty acids and 2.93 g/100 g fatty acids, respectively). n-3 PUFA were predominant over the n-6 PUFA in all classes. EPA and DHA exhibit several benefits for human health. EPA + DHA was mainly concentrated in TG (68.10 g/100 g fatty acids) and GP (12.04 g/100 g fatty acids). The previous study showed that fatty acids were provided with better utilization and better antioxidant abilities when concentrated in GP rather than in TG (Michalski et al., 2013). It is a remarkable fact that the leading contributors to n-3 PUFA in all classes were DHA and EPA, especially in GP (EPA + DHA accounting for more than 90% of total n-3 PUFA) (Table 1).

3.2. Fatty acid distribution among different classes The lipids recovered from round scad were separated into TG, DG, MG, FFA, SL and GP and then determined (Table 1). TG (65.99%) was the dominant constituent of the total lipids. It was noteworthy that high levels of DG (5.00%) and MG (9.05%) were observed in lipid, which might attribute to that the TG were hydrolyzed into partial glycerides (DG and MG) and FFA under the action of active endogenous lipases in the fish (Zhou et al., 2019). Besides, SL and GP occupied 7.36% and 6.51%, respectively. In general, SFA and PUFA showed dominant relative contents over MUFA in TG (44.72%, 31.24% and 22.37%, respectively), DG (39.04%, 38.06% and 21.10%, respectively), MG (58.16%, 18.00% and 22.90%, respectively) and SL (44.19%, 36.31% and 17.45%, respectively), whereas PUFA predominated significantly over SFA and MUFA in FFA (46.68%, 41.66% and 12.90%, respectively) and GP (45.03%, 42.92% and 11.49%, respectively). C16:0 dominated the total SFA, ranging from 19.21% (in GP) to 32.34% (in MG). The major MUFA was 9cC18:1 (4.82% in FFA to 8.65% in DG). DHA and EPA predominated overwhelmingly among PUFA, accounting for over 60% of total PUFA.

3.3. Stereospecific analysis of triacylglycerols and glycerophospholipids Regarding the fatty acid esterification position in TG, the proportion of total SFA in sn-1, 3-positions was slightly higher than that in the sn-2position. The MUFA were principally esterified in the positions 1 and 3 while the total PUFA were principally distributed in the sn-2-position. Furthermore, n-6 PUFA and n-3 PUFA were preferentially occupied sn2-position. The level of DHA interesterified on sn-2 position was 4

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concentration in GP profiles with EPA (DHA) predominated greatly over those without EPA (DHA) with an exception of that in Lyso-GP (Fig. 2).

Table 2 Fatty acid composition (% of total fatty acids) in different classes of Decapterus maruadsi by UPLC-Q-Exactive Orbitrap-MS.

TG DG Cer SM So PC PE CL PA PI PS PG PM LPC LPE LPI LPS LPG FA MGDG

SFA

MUFA

PUFA

EPA

DHA

30.34 45.08 56.50 10.98 94.44 28.39 35.75 8.96 19.46 42.08 48.37 48.20 100 58.74 24.80 66.16 60.97 nd nd 13.24

22.11 4.31 42.85 81.52 5.56 13.61 8.19 36.97 2.51 9.22 2.02 41.70 nd 6.14 0.56 nd nd nd nd 1.72

47.55 50.61 0.65 7.50 nd 58.00 56.06 54.07 78.03 48.70 49.61 10.10 nd 35.11 74.64 33.84 39.03 100 100 85.05

5.23 6.78 nd nd nd 9.39 2.49 1.14 1.93 5.89 0.12 nd nd 8.43 4.18 nd nd nd 9.85 16.30

9.38 37.23 nd nd nd 21.31 40.04 26.21 38.84 33.34 43.81 3.87 nd 18.62 50.14 32.55 39.03 100 85.95 68.74

3.4.1. Profiling of glycerophospholipids Analysis of UPLC-Q-Exactive Orbitrap-MS allowed the characterization of 13 classes of GP and 723 molecular species (Fig. 2, Supplementary materials Table S3). It has been reported that dietary marine EPA/DHA-enriched GP have many nutritional benefits on antitumor activity, brain function, glucose and lipid metabolism (Zhang, Xu, Wang, & Xue, 2019). A high proportion of PUFA, especially DHA was observed in GP and distributed in sn-2 position (Table 2, Supplementary materials Table S3). The exact structure of an unknown lipid molecule could be analyzed according to its first-stage MS and MS/MS spectra. For instance, for the measured m/z 806.57 (molecular ion [M+H]+) of an unknown phosphatidylcholine (PC), PC (38:6) could be deduced tentatively grounded on the previous formula (Liu et al., 2017). Through collision-induced dissociation (CID), the product ions at m/z 568.34, 550.33, 496.34, 478.33, and 184.07 were observed in the MS/MS spectra (Fig. 3). Among these fragment ions, the fragment at m/z 184.07 was corresponding to the characteristic ion to the head group of PC. Fragments at m/z 496.34 and 568.34 were characterized as [LPC (16:0)] + and [LPC (22:6) + CH2OH] +, respectively. The deduced result of MS/MS spectra was in agreement with the PC (38:6) deduced by first-stage MS spectra. The sn-2-position of GP shows a preference for PUFA, so the PC at m/z 806.57 was identified as PC (16:0/22:6). It is now accepted that GP are the fundamental components of biomembrane, which are found in all organisms ranging from bacteria to evolved creatures (Zhang et al., 2019). The predominant PUFAs, especially the abundance of DHA and EPA in the PC, phosphatidylethanolamines (PE), phosphatidylinositols (PI) and phosphatidylserines (PS) provided the best agreement with the results obtained from GC-FID analysis, which indicated a superior nutritional value of round scad considering a higher bioavailability of fatty acids supplied as GP. PC and PE were the major components, occupying 98.08% of total GPs. A total of 243 species PC were characterized. PC (16:0/22:6) was the predominant species of PC, with relative concentration of 898.07 nmol/g as [M+H]+ at m/z 806.5694. At least 153 kinds of PE were identified. PE (18:0/22:6) (236.09 nmol/g) was major PE species, corresponding to [M+H]+ at m/z 792.5538. PC and PE as the most plentiful classes of GP play critical parts in regulating whole-body energy, lipid metabolism and lipoprotein secretion (Colin & Jaillais, 2020). Previous studies have explained the function of PC in neuroprotective and very-low-density lipoprotein (VLDL) secretion modulation (Chen et al., 2019). In addition, recent studies indicated that marine EPA/DHA-enriched PC and ethanolamine plasmalogens (PlsEtn) showed a unique bioactivity in relieving hyperlipidemia and atherosclerosis (Ding et al., 2020; Zhang et al., 2019). Regarding the other classes of GP, CL (123 species, 0.80 nmol/g), PA (23 species, 0.43 nmol/g), phosphatidylglycerols (PG, 11 species, 1.00 nmol/g), PI (52 species, 81.09 nmol/g) and PS (57 species, 60.60 nmol/g) were only found at trace concentration. Although accounting for minor parts of GP, they are extremely essential in physiological processes. CL as mitochondria-specific glycerophospholipids, interacte with many mitochondrial membrane enzymes and contribute to mitochondrial processes such as mitochondrial fusion and division (Kojima et al., 2019). PA can function in cell activities through activating or synergistically activating enzymes (Colin & Jaillais, 2020). PG perform specific functions as a signaling transduction molecule of inflammation and an acknowledged marker of pulmonary development of the fetus (Ali et al., 2019). PI are capable of regulation in the signaling transduction pathway and membrane dynamics (Ali et al., 2019). PS are not only necessary for healthy nerve cell membranes but also a key signal marker for certain diseases (Colin & Jaillais, 2020).

nd, not detected. Abbreviations were: SFA, saturated fatty acids; MUFA, monounsaturated fatty acids, PUFA, polyunsaturated fatty acids; DHA, docosahexaenoic acids; EPA, eicosapentaenoic acids; FA, fatty acyls; Cer, ceramides; SM, sphingomyelins; So, sphingoshines; PC, phosphatidylcholines; PE, phosphatidylethanolamines; CL, cardiolipins; PA, phosphatidic acids; PI, phosphatidylinositols; PS, phosphatidylserines; PG, phosphatidylglycerols; PM, phosphatidylmethanols; LPC, lysophosphatidylcholines; LPE, lysophosphatidylethanolamines; LPI, lysophosphatidylinositols; LPS, lysophosphatidylserines; LPG, lysophosphatidylglycerols; MGDG, monogalactosylmonoacylglycerols.

significantly higher than that on sn-1, 3-positions, whereas EPA mainly distributed in the external positions. For positional fatty acid locations in GP, the total SFA and total MUFA exhibited a preference for sn-1position, whereas total PUFA preferred to esterify on sn-2-position. The level of DHA esterified on sn-2 position was significantly higher than that on sn-1-position, whereas EPA principally esterified in the sn-1position (Fig. 1, Supplementary materials Table S1). Studies have revealed that fatty acids, especially n-3 LC-PUFA, were better utilized when located preferentially on the sn-2 position of TG, and the bioavailability of medium-chain fatty acids on external positions was more outstanding (Linderborg et al., 2019; Michalski et al., 2013). Besides, in consideration of the better oxidative stability of fatty acids distributed in the sn-2-position (Michalski et al., 2013), these specific characteristics were favorable for the efficient absorption and utilization of lipids. 3.4. Lipidomic profiles of round scad The different classes of lipid species were separated within 20 min as exhibited in Supplementary materials Fig. S1. At least 878 and 404 molecular species from six classes (21 subclasses) were characterized in positive and negative modes, respectively (Supplementary materials Table S2–S6). TG, DG, ceramides (Cer), sphingoshines (So) and coenzyme (Co) were determined only in positive mode, while cardiolipins (CL), phosphatidic acids (PA), lysophosphatidylglycerols (LPG), FA and monogalactosylmonoacylglycerols (MGDG) were detected in negative mode. Considerable amounts of PUFA were observed in all classes except for Cer, sphingomyelins (SM) and So (Table 2). GP and GL were the predominant classes, accounting for 62.59% and 36.55% of total lipid profiles, respectively. SP, FA, SL and PR were only determined in trace proportion (0.86%). EPA + DHA were concentrated in GP (72.55%), followed by GL (27.29% for TG and 0.16% for DG). The relative 5

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Fig. 2. Molecular species numbers (A) and relative concentration (B) in total lipid profiles (1), lipid profiles with EPA (DHA) (2) of Decapterus maruadsi. (C) Comparisons of relative concentration composition in main lipid classes of Decapterus maruadsi.

Additionally, five classes of Lyso-GP were determined at minor species and relative concentrations, with a total concentration of 36.43 nmol/g including 40 species of LPC, 14 species of lysophosphatidylethanolamines (LPE), 3 species of lysophosphatidylinositols (LPI), 2 species of lysophosphatidylserines (LPS) and 1species of LPG. Lyso-GP are derived from their corresponding GPs, which lack one of the fatty acyls due to the hydrolysis of phospholipase. According to Wang et al. (2019), lyso-GP were possible to be the hydrolysate of their corresponding GP under the action of phospholipase in fishes (not alive), which revealed the lipid profiles were in dynamic change during storage time.

3.4.2. Profiling of glycerolipids In the present study, at least 444 and 27 molecular species were identified from two classes of GL (TG and DG; Fig. 3, Supplementary materials Table S4). Both of them were detected in positive mode as [M +NH4]+, [M+H]+, and [M+Na]+ ions. TG (16:0/16:0/18:1) (157.16 nmol/g) and TG (18:3/18:2/18:2) (154.77 nmol/g) were dominant species. In accordance with informed literature (Duffin, Henion, & Shieh, 1991), the major productions that formed from the TG [M+NH4]+ were neutral loss of one fatty acid ([M–RCOO] +) and the acyl fatty acids chain ([RC = O]+) in MS/MS spectra. As shown in Fig. 4, the fragments at m/z 577.52 and 551.50 were formed resulted 6

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Relative intensity

100

184.07

RT: 14.81 AV: 1 NL:9.46E7 FTMS + p ESI d Full ms2 806.5694

75 50 25 0 150

496.34 550.33 568.34 478.33

250

350

450

m/z

550

806.57

650

750

850

Fig. 3. MS/MS spectrum and fragmentation pathways of PC (16:0/22:6) as [M+H]+ at m/z 806.5694.

Fig. 4. MS/MS spectrum and fragmentation pathways of TG (16:0/16:0/18:1) as [M+NH4]+ at m/z 850.7858.

from neutral loss of [C15H31COO]– and [C17H33COO]–, corresponding to the fatty acids C16:0 and C18:1, respectively. Besides, the corresponding ions [C15H31C]O]+ (m/z 239.24) and [C17H33C]O]+ (m/z 265.25) were also presented in the MS/MS spectra. Therefore, the TG with m/z 850.7858 could be identified as TG (16:0/16:0/18:1). The digestion, absorption properties of TGs are closely associated with their fatty acid esterification position, as mentioned above. The lipidomic results further indicated round scad was favorable sources of fatty acids. DHA (EPA)-TG, as one of the mainstream DHA (EPA) products in market, has been reported to provide superior bioavailability

when compared to DHA (EPA)-ethyl ester (Ding et al., 2013). There were considerable amounts and numbers of TG found containing functional LC-PUFAs, indicating benefits for human nutrition and massive potential as sources of DHA (EPA) products. 3.4.3. Profiling of other lipids Supplementary materials Table S5 shows the SP species and concentration in round scad. Fifty-one kinds of SM, 22 kinds of Cer and 6 kinds of So were identified. SP have been typically found in eukaryotic membranes, comprising a small but essential constituent (10–20%). It 7

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was reported that the SP composition of marine crab Dromia dehanni hemolymph might be essential in the anti-cancer activity (Rethnapriya, Ravichandran, Gobinath, Tilvi, & Devi, 2019). Five SL, three FA and one PR were also detected in round scad (Supplementary materials Table S6). In spite of the low amounts of these classes, they are well-documented for their health functions. SL have attracted researchers' interest due to its natural PUFA source and recognized bioactivities such as anti-inflammatory and antiproliferative activities (Fernando, Nah, & Jeon, 2016). The five SLs identified in the present study were regarded as bioactive species according to the presence of C16 and C18 SFAs and LC-PUFAs (Rey et al., 2019). Moreover, Co (Q10) was detected in round scad, which is known to perform excellent anti-oxidation and anti-inflammatory functions. Oral administration of Co (Q10) was considered as a potential therapeutic approach of hypertension, cardiovascular disease and metabolic syndrome (Ghaffari & Roshanravan, 2020). In recent years, many marine natural products have revealed a series of biological activities (Liang, Luo, & Luesch, 2019). Round scad as a low-valued marine fish, is mainly featured by significant amounts of EPA (DHA)-TG and EPA (DHA)-GP, high PUFA/SFA and low n-6/n-3 PUFA ratios, which indicated the potential applications in serving as dietary supplements for infants (Wang et al., 2019) and the improvement of some physiological functions such as lipid metabolism and brain function (Wang, Xue, Zhang, & Wang, 2018; Zhang et al., 2019). Besides, various functional lipid classes showed multiple possible applications in therapeutic agents and a considerable utilization prospect in lipid nutrition.

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodres.2020.109138. References Ali, A. H., Zou, X., Abed, S. M., Korma, S. A., Jin, Q., & Wang, X. (2019). Natural phospholipids: Occurrence, biosynthesis, separation, identification, and beneficial health aspects. Critical Reviews in Food Science and Nutrition, 59(2), 253–275. https:// doi.org/10.1080/10408398.2017.1363714. Bravi, E., Marconi, O., Sileoni, V., & Perretti, G. (2017). Determination of free fatty acids in beer. Food Chemistry, 215, 341–346. https://doi.org/10.1016/j.foodchem.2016.07. 153. Cao, J., Deng, L., Zhu, X., Fan, Y., Hu, J., Li, J., & Deng, Z. (2014). Novel approach to evaluate the oxidation state of vegetable oils using characteristic oxidation indicators. Journal of Agricultural and Food Chemistry, 62(52), 12545. https://doi.org/ 10.1021/jf5047656. Chen, J., Lin, S., Sun, N., Bao, Z., Shen, J., & Lu, X. (2019). Egg yolk phosphatidylcholine: Extraction, purification and its potential neuroprotective effect on PC12 cells. Journal of Functional Foods, 56, 372–383. https://doi.org/10.1016/j.jff.2019.03.037. Colin, L. A., & Jaillais, Y. (2020). Phospholipids across scales: Lipid patterns and plant development. Current Opinion in Plant Biology, 53, 1–9. https://doi.org/10.1016/j. pbi.2019.08.007. Cruz-Hernandez, C., Deng, Z., Zhou, J., Hill, A. R., Yurawecz, M. P., Delmonte, P., ... Kramer, J. K. (2004). Methods for analysis of conjugated linoleic acids and trans-18: 1 isomers in dairy fats by using a combination of gas chromatography, silver-ion thinlayer chromatography/gas chromatography, and silver-ion liquid chromatography. Journal of AOAC International, 87(2), 545–562. Ding, L., Zhang, L., Shi, H., Xue, C., Yanagita, T., Zhang, T., & Wang, Y. (2020). EPAenriched ethanolamine plasmalogen alleviates atherosclerosis via mediating bile acids metabolism. Journal of Functional Foods, 66. https://doi.org/10.1016/j.jff.2020. 103824. Ding, N., Xue, Y., Tang, X., Sun, Z.-M., Yanagita, T., Xue, C.-H., & Wang, Y.-M. (2013). Short-term effects of different fish oil formulations on tissue absorption of docosahexaenoic acid in mice fed high-and low-fat diets. Journal of Oleo Science, 62(11), 883–891. https://doi.org/10.5650/jos.62.883. Dohrmann, D. D., Putnik, P., Bursac Kovacevic, D., Simal-Gandara, J., Lorenzo, J. M., & Barba, F. J. (2019). Japanese, Mediterranean and Argentinean diets and their potential roles in neurodegenerative diseases. Food Research International, 120, 464–477. https://doi.org/10.1016/j.foodres.2018.10.090. Duffin, K. L., Henion, J. D., & Shieh, J. (1991). Electrospray and tandem mass spectrometric characterization of acylglycerol mixtures that are dissolved in nonpolar solvents. Analytical Chemistry, 63(17), 1781–1788. https://doi.org/10.1021/ ac00017a023. Dyall, S. C. (2015). Long-chain omega-3 fatty acids and the brain: A review of the independent and shared effects of EPA, DPA and DHA. Frontiers in Aging Neuroscience, 7, 52. https://doi.org/10.3389/fnagi.2015.00052. Elmadfa, I., & Kornsteiner, M. (2009). Fats and fatty acid requirements for adults. Annals of Nutrition and Metabolism, 55(1–3), 56–75. https://doi.org/10.1159/000228996. Fahy, E., Subramaniam, S., Brown, H. A., Glass, C. K., Merrill, A. H., Jr., Murphy, R. C., ... Dennis, E. A. (2005). A comprehensive classification system for lipids. European Journal of Lipid Science and Technology, 46(5), 839–861. https://doi.org/10.1194/jlr. E400004-JLR200. FAO (2018). The State of World Fisheries and Aquaculture 2018-Meeting the sustainable development goals. Rome, (Part 1). Licence: CC BY-NC-SA 3.0 IGO. Fernando, I. S., Nah, J. W., & Jeon, Y. J. (2016). Potential anti-inflammatory natural products from marine algae. Environmental Toxicology and Pharmacology, 48, 22–30. https://doi.org/10.1016/j.etap.2016.09.023. Folch, J., Lees, M., & Stanley, G. S. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226(1), 497–509. Ghaffari, S., & Roshanravan, N. (2020). The role of nutraceuticals in prevention and treatment of hypertension: An updated review of the literature. Food Research International, 128, 108749. https://doi.org/10.1016/j.foodres.2019.108749. Hu, X., Yang, X., Wang, T., Li, L., Wu, Y., Zhou, Y., & You, L. (2020). Purification and identification of antioxidant peptides from round scad (Decapterus maruadsi) hydrolysates by consecutive chromatography and electrospray ionization-mass spectrometry. Food and Chemical Toxicology, 135, 110882. https://doi.org/10.1016/j.fct. 2019.110882. Jiang, H., Zhang, W., Chen, F., Zou, J., Chen, W., & Huang, G. (2019). 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4. Conclusion Round scad was shown to contain large amounts of PUFA, especially DHA and EPA, and have a proper nutritional contribution. The lipids consisted of TG, MG, DG, FFA, SL and GP, among which, TG were dominant and the EPA + DHA were concentrated as GP. The stereospecific analysis of TG and GP indicated these PUFA were prone to be utilized and less likely to be oxidized. The lipid classes, fatty acid compositions, and relative concentration of each lipid profile were acquired by UPLC-Q-Exactive Orbitrap-MS. DHA and EPA were further confirmed as the dominating fatty acids. A high proportion of PUFA was observed in GP and distributed in the sn-2 position. Hence, considering the large production while inadequate application of round scad, it is deserved for further exploitation of its marine lipid source on account of the healthy and nutritional functions. CRediT authorship contribution statement Chen He: Methodology, Investigation, Writing - original draft. Zexin Sun: Investigation, Formal analysis. Xingchen Qu: Investigation, Formal analysis, Data curation. Jun Cao: Supervision, Funding acquisition, Writing - review & editing. Xuanri Shen: Resources, Funding acquisition. Chuan Li: Project administration, Funding acquisition, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Key Research and Development Program of China (2018YFD0901103), Hainan Provincial Natural Science Foundation of China (319QN158) and Scientific Research Foundation of Hainan University (KYQD1608). 8

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