Changes in lipid profiles of dried clams (Mactra chinensis Philippi and

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Food and Beverage Chemistry/Biochemistry

Changes in lipid profiles of dried clams (Mactra chinensis Philippi and Ruditapes philippinarum) during accelerated storage and prediction of shelf life Hongkai Xie, Dayong Zhou, Xiaopei Hu, Zhongyuan Liu, Liang Song, and Bei-Wei Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03047 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 9, 2018

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Journal of Agricultural and Food Chemistry

Changes in lipid profiles of dried clams (Mactra chinensis Philippi and Ruditapes philippinarum) during accelerated storage and prediction of shelf life

Hongkai Xie†,₸, Dayong Zhou₸,§*, Xiaopei Hu†,₸, Zhongyuan Liu₸,§, Liang Song₸,§, Beiwei Zhu†,₸,§,¶*

† Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China ₸ National Engineering Research Center of Seafood, Dalian 116034, People’s Republic of China § School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, People’s Republic of China ¶ Tianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center, Tianjin 300457, People’s Republic of China Corresponding Authors: Dayong Zhou₸,§. E-mail: [email protected]; Tel: +86-411 86323453

Beiwei Zhu†,₸,§,¶. E-mail: [email protected]; Tel: +86-411 86323262

ACS Paragon Plus Environment


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Abstract: To predict the shelf life through Arrhenius model and evaluate the changes in lipid profiles, two dried clams were stored at 50 °C and 65 °C and collected periodically for analysis. The predicted shelf life of two dried clams were 530 ± 14 and 487 ± 24 hours (24 °C), and the relative errors between the actual and predicted values were 5.7 and 6.8%, respectively. During accelerated storage, the peroxide value, p-anisidine value, thiobarbituric acid-reactive substances value, total oxidation value, acid value and free fatty acid content all increased, while the levels of triacylglycerol,

phosphatidylcholine,

phosphatidylethanolamine,

major

glycerophospholipid molecular species and polyunsaturated fatty acid (PUFA) decreased. Moreover, content of phospholipid containing PUFA decreased significantly than that of triacylglycerol containing PUFA. Results indicated that the Arrhenius model was suitable for the shelf life prediction of dried clams and accelerated storage caused loss in quality of dried clams in terms of lipids.

Keywords: dried clam, lipid profile, oxidation, hydrolysis, accelerated storage, shelf life prediction


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INTRODUCTION Clam, one of the most abundant marine bivalves, is cultivated and harvested worldwide. Its global production was about 5.2 million tons in 2015, which ranks first in production among shellfish species.1 Clam is regarded as a “healthy” food and appreciated by consumers because it is rich in various components beneficial to human health, including long-chain polyunsaturated fatty acids (LC-PUFA), peptides, minerals, vitamins, essential amino acids and other nutrients.2-4 However, as a representative shellfish, clam is a highly perishable food product due to the spoilage brought about rapidly by microbiological growth and lipid oxidation.5 Drying is an important processing method for clams as it can reduce the water activity of the fresh aquatic products matrix and inhibit microbial growth.6 The dehydrated clams can be easily preserved. Moreover, they are rich in nutrition, unique in flavor, easy to transport, and popular with consumers.7 Shelf life is the duration for which a product can be preserved under a given storage condition and can still meet a certain quality level.8 Real-time and accelerated storage tests are the common methods used to evaluate the shelf life of food products, but the real-time tests take a long period.9 Thus, to shorten the experimental time, accelerated storage coupled with the commonly used Arrhenius Equation Modeling is widely used to assess the shelf life of oils or oil products such as biscuits and bread with limited stability.10-13 For the aforementioned oils and oil products, peroxide value (POV) is normally taken as the representative index of quality reduction during storage.10-13 According to GB 10136-2015, POV is also used as an indicator to evaluate the shelf



life of dried aquatic products. However, to the best of our knowledge, no study regarding the shelf life prediction of aquatic products through accelerated storage coupled with Arrhenius Equation Modeling has been reported. Furthermore, previous research related to the changes in n-3 LC-PUFA enriched lipid during accelerated storage has mainly focused on marine fish oils.14,15 The various marine sources of n-3 LC-PUFA differ in terms of their lipid classes. Omega-3 LC-PUFA is present primarily as triacylglycerol (TAG) in fish. However, in shellfish, a substantial proportion of n-3 LC-PUFA is bound in phospholipids (PL).16-17 However, little information is available about the impact of accelerated storage conditions on the lipids present in shellfish species, especially clams. Thus, the objective of the present study is to investigate the changes in lipid components in two clams (Mactra chinensis Philippi (MP) and Ruditapes philippinarum (RP)) during accelerated storage. Furthermore, this study will offer researchers in food industry a reference for designing accelerated tests and for predicting the shelf life of dried aquatic products. To fulfill this goal, POV, thiobarbituric acid-reactive substances value (TBARS), p-anisidine value (AnV), acid value (AV), total oxidation value (TOTOX), lipid content, lipid classes, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents, fatty acids (FAs) composition, EPA and DHA contents, glycerophospholipid (GP) molecular species, as well as lipolytic enzyme and lipoxygenase (LOX) activities in the dried clams during accelerated storage were determined.


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MATERIALS AND METHODS Materials Fresh clams, MP and RP, were obtained from a local market in Dalian, Liaoning, China. Deuterated chloroform, deuterated methanol, triethyl phosphate, cesium carbonate, D2O, 4-methylumbelliferone, linoleic acid, TAG (20:0, 20:0, 20:0), docosahexaenoic acid (DHA, C22:6 n-3) and eicosapentaenoic acid (EPA, C20:5 n-3) were

purchased

from

Aladdin

Glycerylphosphatidylcholine

Reagent

(GPCho

Co.,

Ltd.

12:0/12:0,

(Shanghai,

China).

16:0/18:1)

and

glycerylphosphatidylethanolamine (GPEtn 12:0/12:0, 16:0/18:1) were provided by Avanti Polar Lipids, Inc. (Alabaster, AL, USA). HPLC grade n-hexane, isopropanol, chloroform and methanol were purchased from Spectrum Chemical Mfg. Corp. (Gardena, CA, USA). All other reagents were analytical grade and obtained from Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Sample Preparation and Accelerated Storage Treatment Fresh clams were boiled for ten minutes and the entire boiled meat was dried in a lightproof DGH-9030A hot-air oven (Yiheng Science and Technology Co., Ltd., Shanghai, China) at 70 °C for 21 hours to obtain dried clams. The dried clams were divided into three parts, which were stored at 24 °C (room temperature), 50 °C and 65 °C, respectively. To predict the shelf life of clams, the samples used to determine the POV were collected at regular intervals of 40 hours until day-15, and were collected for the final time on day-20. For the determination of TBARS, AnV and AV, the sample collection



was performed at regular intervals of 5 days. Samples used for the determination of lipid classes, phospholipid classes, fatty acids and phospholipid molecular species were collected only at the start and end of the storage period. Further, samples stored at 65 °C were used for the determination of EPA and DHA when the POV limit was reached. All samples were stored at -30 °C immediately after collection. The sample labels 0D-MP, 50 °C-20D-MP and 65 °C-20D-MP represent dried MP before storage, dried MP collected on day-20 at 50 °C and 65 °C, respectively. The same naming scheme was used for the RP clams: 0D-RP, 50 °C-20D-RP and 65 °C-20D-RP. The sample 65 °C-280 h-MP refers to dried MP collected after 280 h at 65 °C and the sample 65 °C-200 h-RP refers to dried RP collected after 200 h at 65 °C. Lipid Extraction A modified MTBE (methyl tert-butyl ether) method was employed for the total lipid extraction from dried clams according to our previous study.18 The extracted lipids were stored at -30 °C for further analysis within 2 weeks. Peroxide Value (POV) The POV was measured according to the American Oil Chemists’ Society (AOCS) official method AOCS Cd 8-53.19 Thiobarbituric Acid Reactive Substances Assay (TBARS) The TBARS was determined as described by Khan20 with a slight modification. Briefly, 0.5 g of clam powder was mixed with 2 mL of 10% (w/v) trichloroacetic acid (TCA) solution and 2 mL of distilled water. The mixture was vortexed for 2 min and centrifuged at 8000 g for 5 min. Then, 1 mL of supernatant was mixed with 1 mL of


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0.01 M 2-thiobarbituric acid (TBA) solution, and then heated in a boiling water bath for 25 min. The absorbance of the solution was measured at 532 nm using a TECAN Infinite M200 microplate reader (Tecan, Durham, USA). The TBARS value, expressed as mg malondialdehyde (MDA)/kg clam powder, was calculated from a standard curve prepared with 1,1,3,3-tetramethoxypropane. p-Anisidine Value (AnV) The AnV was determined as described by Okpala19 with a slight modification. Briefly, 0.5 g clam powder was mixed with 5 mL n-hexane. The mixture was sonicated for 10 min, and centrifuged at 8000 g for 5 min. 2.5 mL supernatant was mixed with 0.5 mL of 0.5% (w/v) p-anisidine solution, and then the mixture reacted for 10 min at 25 °C. The AnV was recorded at 350 nm by a spectrophotometer and calculated using the equation: AnV = [25(1.2A2 – A1)] / m

(Eq. 1)

where A1 and A2 are the absorbance recorded before and after adding p-anisidine and m is the mass of sample (g). Total Oxidation Value (TOTOX) The TOTOX was calculated based on the POV and TBARS as described by Wanasundara & Shahidi22 using the following equation: TOTOX = 2POV + TBARS

(Eq. 2)

Acid Value (AV) The AV was determined by alkaline titration according to the AOCS official method Cd 3d-63.19



Lipid Classes Analysis Lipid class compositions were determined using an Iatroscan MK-6S thin layer chromatography-flame ionization detection (TLC-FID) Analyzer (Iatron Inc., Tokyo, Japan), according to the method reported by Yin.23 TAG and FFA were quantitatively determined and expressed as mg/g dried clam (on a dry basis), and were calculated by standard curve method with purified TAG and DHA as standards. Phospholipid Classes Analysis The determination of PL class composition was conducted using an Avance III 400 MHz NMR spectrometer (Bruker, Karlsruhe, Germany) (9.4 T), according to the method reported by Liu.24 The quantitative analyses of PC and PE were performed by a Shimadzu LC-20AD Prominence HPLC system (Shimadzu Corp., Kyoto, Japan) combined with an Alltech ELSD 6000 detector (Alltech, Deerfield, IL, USA).25 The gas flow rate was 2 L/min and ELSD tube temperature was 70 °C. PC and PE were separated on an Agilent Zorbax RX-SIL (4.6 × 250 mm, 5 µm) column at a flow rate of 1.0 mL/min, and the mobile phase was (A) methyl alcohol: water: glacial acetic acid: triethylamine (85:15:0.45:0.05, v/v) and (B) n-hexane: isopropanol: A (20:48:32, v/v). The elution gradient was as follows: 90-70% B, 0-20 min; 70-5% B, 21-35 min; 90% B, 36-41 min. The lipid sample concentration was 2 mg/mL, and the injection volume was 10 µL. The PC and PE contents were calculated by standard curve method with GPCho (C16:0/18:1) and GPEtn (C16:0/18:1) as standards. Fatty Acid Analysis


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The determination of FA composition was performed using an Agilent 7890A Gas Chromatography/5977A Mass Spectrometer (GC-MS) system (Palo Alto, CA, USA) equipped with an Agilent HP-5-MS capillary column (30 m × 0.25 mm, 0.25 µm).23 The EPA and DHA contents were calculated by standard curve method with EPA (C20:5 n-3) and DHA (C22:6 n-3) as standards. Furthermore, the FA composition of TAG and PL were also determined in this study. TAG and PL were fractionated by the method reported by Rouser.26 Briefly, 0.5 g of clam oil was applied to a silicic acid column (2.5 cm i.d. × 30 cm h; 200-300 mesh silicic acid powder (20 g), Qingdao Haiyang Chemical Co., Ltd, Tsingtao). The column was first eluted with n-hexane/diethyl ether (9:1, v/v, 150 mL), and then the TAG fraction of the oil was eluted with n-hexane/diethyl ether (9:2, v/v, 165 mL). Then, the column was successively eluted with diethyl ether (150 mL) and ethyl acetate (150 mL). Finally, methanol (300 mL) was used to elute the PL fractions. A rotary evaporator was used to remove the solvents under vacuum at 40 °C. The purity of TAG and PL was determined by TLC-FID. Phospholipid Molecular Species Analysis GP molecular species were characterized by direct infusion mass spectrometry in this study according to the method reported by Gang.27 Semi quantitative analyses of major molecular species of GPCho and GPEtn in MP and RP during accelerated storage were carried out by using triple quadrupole mass spectrometry in MS/MS mode with precursor-ion scanning (PIS) and neutral loss scanning (NLS) modes according to our preliminary study.16



Enzyme Extraction and Activities Assay Crude LOX was extracted and determined by using the method of Gata.28 The lipase activities were determined spectrophotometrically at 234 nm with a linoleic acid substrate solution. LOX catalyzes the formation of conjugated diene hydroperoxide from linoleic acid and the conjugated double bonds have characteristic absorption at 234 nm. One unit (U) of LOX activity was defined as an increase absorbance of 1 per minute at 234 nm. Lipase activities were determined by a kit method according to the method reported by Zheng.29 Crude lipases were extracted and the lipase activities were determined according to the manufacturer’s instructions (Jiancheng Technology Co., Nanjing, China). Crude phospholipase was extracted and the phospholipase activity was determined according to the method of Motilva.30 4-Methylumbelliferyl oleate was used as the substrate to measure the aforementioned lipase activities. This substrate liberated fluorescent 4-methylumbelliferone after lipase hydrolysis, which was determined at the excitation wavelength of 320 nm and emission wavelength of 420 nm by using an Infinite F200 Pro microplate reader (Tecan Group Ltd., Männedorf, Switzerland). One unit (U) of activity was defined as 1 nmol of released 4-methylumbelliferone per minute at 37 oC. Shelf Life Prediction The POV of MP and RP at 50 °C and 65 °C were obtained to fit the kinetic model and the observed POV was used to predict their POVs after storage at 24 °C. The


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following equations were adopted for the oxidation kinetic model analysis and shelf life prediction: Zero-order model: POV = k0 t + POV0

(Eq. 3)

First-order model: ln (POV) = k t + ln (POV0)

(Eq. 4)

Arrhenius equation: ln (k) = -EA / RT + lnk0

(Eq. 5)

Shelf life prediction: SL = [ln (POVlim) – ln (POV0)] / [k0 e (−EA / RT)]

(Eq. 6)

where k0 and k are the reaction rate constants, POV and POV0 are the POV at storage time t (h) and the initial value, k0 is a preexponential factor, EA is the activation energy (J mol−1), T is the absolute temperature, and R is the molar gas constant (8.3144 J K−2 mol−1). POVlim is 47.28 meq/kg lipid according to GB 10136-2015 for dried aquatic products. Statistical Analysis The experiments were carried out in triplicate and the results were expressed as mean ± SD. The statistical analysis was performed by SPSS analytical software version 20.0 (SPSS Inc., Chicago, IL, USA), including One-way ANOVA analysis and independent-samples T test (p < 0.05). Significant differences between the means of parameters were determined by the Duncan test (p < 0.05). RESULTS



Change in Moisture Content during Accelerated Storage The moisture contents of the two clam tissues during accelerated storage are shown in Table 2S. Results indicated that the moisture contents of 0D-MP and 0D-RP were 6.89 ± 0.29% and 7.99 ± 0.27%, respectively. The moisture contents of MP and RP decreased throughout the 20 days of accelerated storage. After accelerated storage for 20 days, the MP tissue retained 2.26 ± 0.11% of moisture at 50 °C and 2.09 ± 0.13% of moisture at 65 °C, respectively, while the corresponding values for 0D-RP were 2.17 ± 0.09% of moisture at 50 °C and 1.79 ± 0.05% of moisture at 65 °C. Changes in POV, TBARS, AnV, TOTOX and AV during Accelerated Storage The POV (Figure 1A) and TOTOX (Figure 1D) showed a time-dependent increase during the initial 15 days of storage and then decreased. However, the TBARS (Figure 1C), AnV (Figure 1B) and AV (Figure 1E) continuously increased throughout the 20 days of accelerated storage. By contrast, the initial values of POV, TOTOX, TBARS and AnV of RP were higher than the corresponding values of MP. After accelerated storage at 50 °C and 65 °C for 20 days, the differences in all the aforementioned oxidative indices between MP and RP were enlarged. Moreover, for the two clam species, the oxidative indices including POV, TOTOX, TBARS and AnV during accelerated storage all showed a similar tendency of higher values at higher storage temperatures. Changes in Lipid Class TLC-FID chromatogram of the lipids is shown in Figure 2A. The lipid contents determined by the MTBE method for 0D-MP, 50 °C-20D-MP, 65 °C-20D-MP, 0D-RP,


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50 °C-20D-RP, and 65 °C-20D-RP were 6.01 ± 0.19, 5.98 ± 0.17, 6.24 ± 0.12, 9.02 ± 0.26, 8.67 ± 0.14, and 8.72 ± 0.11% on a dry basis, respectively. TLC-FID analysis suggested that the lipids from 0D-MP contained 20.06 ± 1.09% of TAG, 0.17 ± 0.02% of monoacylglycerol (MAG), 68.22 ± 1.36% of polar lipids (PoL), 2.30 ± 0.17% of FFA and 9.25 ± 0.48% of cholesterol (CHO), respectively, while the corresponding values for 0D-RP were 24.11 ± 0.37, 0.15 ± 0.03, 69.05 ± 0.75, 1.88 ± 0.46 and 4.97 ± 0.08%. The accelerated storage caused an increase in the percentage of FFA and a decrease in that of PoL. Moreover, the percentages of TAG, CHO and MAG hardly changed during the accelerated storage (Table 1). Absolute quantification analysis indicated that the 0D-MP and 0D-RP samples contained 15.1 ± 0.3 and 21.5 ± 0.2 mg/g TAG on a dry basis, respectively (Figure 3A). The TAG contents in 0D-MP and 0D-RP significantly decreased during storage, which dropped to 13.6 ± 0.2 and 20.7 ± 0.1 mg/g, respectively, at 50 °C after 20 days, and dropped to 11.8 ± 0.8 and 18.9 ± 0.5 mg/g, respectively, at 65 °C after 20 days. Obviously, the higher storage temperatures caused a larger reduction in TAG contents. The 0D-MP and 0D-RP samples contained 4.7 ± 0.2 and 6.7 ± 0.4 mg/g FFA on a dry basis, respectively. After 20 days of accelerated storage, the corresponding values increased to 7.3 ± 0.3 and 9.2 ± 0.3 mg/g, respectively, at 50 °C, and increased to 5.8 ± 0.3 and 7.4 ± 0.1 mg/g, respectively, at 65 °C. Interestingly, for FFA contents, the higher storage temperature caused a smaller increase. Changes in Phospholipid Class 31

P NMR chromatogram of the phospholipids is shown in Figure 2B. The results



indicated that PL in 0D-MP consisted of 43.46 ± 0.42 mol% of PC, 41.91 ± 0.10 mol% of PE/ phosphatidylserine (PS)/ phosphatidylinositol (PI), 5.80 ± 0.38 mol% of phosphatidylglycerol (PG), 5.30 ± 0.05 mol% of lysophosphatidylcholine (LPC) and 3.53 ± 0.10 mol% of phosphatidic acid (PA). The corresponding values for 0D-RP were 42.73 ± 0.21, 39.40 ± 1.84, 7.63 ± 2.16, 6.27 ± 0.21 and 3.98 ± 0.33 mol% (Table 2). The accelerated storage caused an increase in the molar percentage of LPC and a decrease in that of PC. HPLC chromatogram of the PC and PE is shown in Figure 2C. HPLC-ELSD analysis indicated that 0D-MP and 0D-RP contained 875.03 ± 4.05 and 966.31 ± 41.50 mg/100 g PC on a dry basis, respectively. After 20 days of accelerated storage, the corresponding values decreased to 829.30 ± 20.75 and 860.21 ± 23.25 mg/100 g, respectively, at 50 °C, and decreased to 775.30 ± 11.05 and 811.68 ± 9.70 mg/100 g, respectively, at 65 °C (Figure 3B). The 0D-MP and 0D-RP contained 266.01 ± 0.15 and 346.32 ± 5.25 mg/100 g PE on a dry basis, respectively. The contents of PE in 0D-MP and 0D-RP decreased along with the storage time. The PE contents dropped to 260.91 ± 3.60 and 297.01 ± 7.35 mg/100 g, respectively, at 50 °C after 20 days, and dropped to 238.36 ± 15.55 and 261.74 ± 4.2 mg/100 g, respectively, at 65 °C after 20 days. Obviously, the higher storage temperature caused a larger reduction in the contents of PE and PC. Changes in Fatty Acids Highly pure (> 95%) TAG and PL were recovered from column chromatography (Figure 2D). The FA compositions of TAG and PL recovered from 0D-MP and


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0D-RP are shown in Table 3. TAG and PL from the two clam species had similar FA profiles. Specifically, they were abundant in palmitic acid (C16:0), EPA (C20:5 n-3) and DHA (C22:6 n-3). It is noteworthy that PUFA (50.62-56.35% of total FAs), especially EPA (14.45-36.35% of total FAs) and DHA (11.03-27.22% of total FAs), constituted the majority of the total FAs for the two clam species. For both MP and RP, the accelerated storage caused a decrease in the relative percentages of PUFA, EPA and DHA in PL and TAG. For PL and TAG containing PUFA, EPA and DHA, the higher storage temperature caused a larger reduction. Moreover, at the same storage temperature, the PUFA in PL showed a greater reduction than that in TAG for the two clam species. In detail, for PL containing PUFA in RP and MP, 20 days of storage at 65 °C resulted in 18.0% and 23.3% of reduction in PUFA content, respectively. For TAG containing PUFA in MP and RP, the 20 days storage at 65 °C caused 15.5% and 21.0% of reduction in PUFA content, respectively. The results of absolute quantification analysis of EPA and DHA are presented in Figure 4. It was found that 0D-MP and 0D-RP contained 627.28 ± 2.71 and 714.20 ± 10.03 µg/g EPA on a dry basis, respectively. After 20 days of accelerated storage, the corresponding values decreased to 371.63 ± 4.41 and 427.10 ± 8.82 µg/g, respectively, at 50 °C, and decreased to 367.44 ± 1.68 and 426.16 ± 2.97 µg/g, respectively, at 65 °C. The 0D-MP and 0D-RP samples contained 1336.77 ± 20.95 and 1544.90 ± 34.56 µg/g DHA on a dry basis, respectively. After 20 days of accelerated storage, the corresponding values decreased to 756.03 ± 12.09 and 894.81 ± 9.80 µg/g,



respectively, at 50 °C, and decreased to 717.22 ± 6.67 and 844.61 ± 10.21 µg/g respectively, at 65 °C. According to the results from shelf life prediction, the POV limit is reached for MP and RP stored at 65 °C at the storage times of 280 h and 200 h, respectively. At this point, the EPA content in MP and RP stored at 65 °C decreased by 20.43% and 16.49%, respectively. Similarly, the DHA content in MP and RP stored at 65 °C reduced by 25.76% and 17.14%, respectively. Changes in Phospholipid Molecular Species First-stage MS spectra of GPCho and GPEtn are shown in Figure 2E and 2F, respectively. In this study, GPCho and GPEtn were selectively detected in the positive ion mode by using PIS for m/z 184 and NLS for m/z 141, respectively. The measured m/z values were consistent with the molecular ion ([M]+) of GPCho and the quasi-molecular ion ([M+H]+) of GPEtn, respectively. Then, the values of x and y (which represent the number of carbons and double bonds of the fatty acid in GP, respectively) for the major GPCho and GPEtn were determined based on their measured molecular mass according to the formulas reported in our previous work. 14,16

Moreover, the MS/MS data of the unknown GPCho ([M–H+HCOO]–) and GPEtn

([M–H]–) were acquired in the negative ion mode by using EPI scanning. The product ions including characteristic fatty acid anions ([RCOO]–) of the unknown GP were present in the MS/MS data. Then, the molecular species of unknown GPCho or GPEtn were characterized according to the first-stage MS and MS/MS information. For example, based on the measured m/z 832 (molecular ion ([M]+)) of the


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unknown GPCho, phosphatidyl GPCho 40:7 could be tentatively deduced according to the equation reported previously. 16,18 Through EPI scanning, the product ions with m/z 876, 816, 552, 506, 327 and 281 were observed in the MS/MS spectrum (Figure 1S). Among these product ions, fragments at m/z 281 and 327 were identified as the FA anions ([RCOO]–) of 18:1 and 22:6, respectively (Figure 2S). The sn-2 of GP is usually the preferred position for PUFAs.31 Therefore, the unknown GPCho with measured m/z 832 was identified as phosphatidyl GPCho 18:1/22:6. In addition, the fragment with m/z 506 was identified as [LGPCho 18:1–CH3]–, m/z 552 was identified as [LGPCho 22:6–CH3]–, m/z 816 was identified as [M–CH3]– and m/z 876 was identified as [M–H+HCOO] – (Figure 2S). GPCho and GPEtn from the two clams had similar molecular species profiles (Table 1S). Among them, 18:0/20:5, 16:0/22:5, 16:0/20:4, 18:0/18:4 and 18:1/18:3 were the predominant species of the phosphatidyl subclass, 22:4/16:0 and 22:5/18:0 were the predominant species of the plasmenyl subclass, while 16:0/22:5, 22:5/16:0, 18:0/22:6 and 22:6/18:0 were the predominant species of the plasmanyl subclass. Obviously, most of the predominant glycerophospholipid molecular species in clam contained PUFA, mainly EPA (C20:5 n-3) and DHA (C22:6 n-3). Moreover, the storage for 20 days caused a significant decrease in the total intensity of GPEtn and GPCho molecular species, especially EPA (C20:5 n-3) and DHA (C22:6 n-3), in lipids from the two clam species (Figure 5). Changes in Lipolytic Enzymes and LOX Activities The activities of LOX and lipolytic enzymes in the two clam tissues during



processing are presented in Figure 6. In fresh clam MP, the enzymatic activities for LOX, lipase and phospholipase were 2.22 ± 0.06, 1.41 ± 0.05 and 1.49 ± 0.04 U/g dry tissue, respectively, while the corresponding values for fresh RP were 2.61 ± 0.11, 1.30 ± 0.12 and 1.60± 0.05 U/g dry tissue. The enzymatic activities for LOX and lipolytic enzymes in MP and RP showed significant decrease during the boiling process. After 10 min of boiling, the MP tissue retained 23.18 ± 0.03% of LOX, 14.09 ± 0.02% of lipase and 18.65 ± 0.02% of phospholipase, respectively, while the corresponding values for RP tissue were 22.14 ± 0.02%, 21.92 ± 0.03% and 18.28 ± 0.04%, respectively. The drying process further decreased the activities of all three enzymes. After drying process, the MP tissue retained 3.74 ± 0.02% of LOX, 3.80 ± 0.03% of lipase and 2.60 ± 0.03% of phospholipase, respectively, while the corresponding values for RP tissue were 3.17 ± 0.01%, 2.61 ± 0.03% and 3.74 ± 0.04%, respectively. Shelf Life Prediction For both MP and RP, the first-order model fitted the changes in POV better than the zero-order model, based on the value of R2 (Table 4). Arrhenius equations for MP and RP were ln (k) = −1328.2/T – 1.8813 and ln (k) = −2158.3/T + 0.8358, respectively. EA values of RP and MP were 11.04 kJ mol−1 and 17.94 kJ mol−1, respectively. The predicted shelf life for MP and RP stored at 24 °C were 530 ± 14 h and 487 ± 24 h, respectively, according to Eq. 6, while the observed values for the dried MP and RP stored at 24 °C were 560 ± 40 h and 520 ± 0 h, respectively. The relative error (RE) of shelf life at 24 °C between the actual values and the predicted values was 5.7% for


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MP and 6.8% for RP. Results indicated that the higher the initial value of POV, the shorter the shelf life. DISCUSSION Dried aquatic products are common products in the market, as they possess a longer shelf life than fresh aquatic products. However, lipid oxidation still occurs in dried aquatic products during the storage period, which leads to a decline in the quality. Generally, a long experimental period is needed for the study of lipid changes in dried aquatic products under normal storage conditions. Accelerated storage can speed up the lipid oxidation and shorten the experimental period. Thus, accelerated storage conditions were used in this study to investigate the changes in lipid profiles of dried clams and to predict their shelf life.32 POV is widely used to reflect the degree of lipid oxidation in the primary period. In this study, the increase in POV of clam during the initial 15 days of accelerated storage reflected the formation and accumulation of lipid hydroperoxides.33 The subsequent sharp decrease in POV may be due to the decomposition of lipid hydroperoxides.34,35 Similar variation of POV was observed in sunflower oil during accelerated storage, where the POV increased during the initial 20 days of storage and then decreased.36 MDA and aldehydes are the main representatives of secondary oxidation products, and they are commonly measured by TBARS and AnV assays, respectively. Therefore, the increase in TBARS and AnV in clams during accelerated storage indicated the decomposition of hydroperoxides into secondary oxidation products. Lu37 and



Topuz14 also observed the increase in TBARS and AnV values of dried shrimp (Acetes chinensis) and anchovy fish oil during accelerated storage, respectively. The TOTOX value reflects the initial and later stages of the lipid oxidation and provides a better assessment of the progress of lipid oxidation.38 Moreover, lower TOTOX indicates better stability of the lipid. Therefore, the increase in TOTOX of clams during accelerated storage further confirmed the occurrence of lipid oxidation and indicated the instability of lipid. Topuz14 also observed the increase in TOTOX of anchovy fish oil during accelerated storage. Overall, the RP had significantly higher values in all oxidation indices including POV, TBARS, AnV and TOTOX than those of MP, indicating the relatively higher oxidation degree of RP compared to MP. After accelerated storage for 20 days, the differences in all the aforementioned oxidative indices between MP and RP were enlarged, indicating that the oxidation rate was directly proportional to the initial oxidation degree of sample. Higher oxidative degree indicated more unstable hydroperoxides as well as more highly reactive radicals decomposed from hydroperoxides, which greatly promote the oxidation process.39 This could be supported indirectly by the study of Hrncirik,40 where the difference in POV of virgin olive oils extracted from three olive species was enlarged during accelerated storage. The lipid deterioration rates at higher temperatures (50 °C and 65 °C) were obtained by the accelerated test, which were extrapolated to the deterioration rates at lower temperatures (24 °C) through the Arrhenius equation.41 In this study, the predicted shelf life of MP was longer than that of RP, which may be due to the


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differences in initial degree of oxidation. Lee42 reported that the predicted shelf life of semi-dried Pacific saury was 60 days (1440 hours) at 5 °C. This reported shelf life is longer than the results in the study, which may be caused by the lower storage temperature. In the present study, the accelerated storage in a lightproof hot-air oven caused a significant decrease in the relative percentages of PUFA (especially EPA and DHA) in both PL and TAG of the two clams in a temperature-dependent manner. Moreover, according to our results, 22-24% of lipoxygenase was retained in clam tissue after the boiling process. D'souza43 also reported that the lipoxygenase in mackerels (Somberus sombrus) contributed to the oxidation during boiling treatment. However, most of the lipoxygenase was inactivated and probably only 3-4% remained after the hot-air treatment. Hence, the lipoxygenase may play a limited role in the lipid oxidation during accelerated storage. Thus, the reduction in PUFA content indicated the occurrence of autoxidation because this spontaneous oxidation process involves unsaturated lipids and oxygen without light and catalyst.44 Similarly, Chakraborty reported that the relative percentages of DHA, EPA and PUFA in refined fish oil were also significantly decreased after 12 days of accelerated storage.15 Our results indicated that PL containing PUFA decreased more rapidly than TAG containing PUFA, which may be due to the preferential oxidation of PL and the prolonged induction period before oxidation of TAG.44,45 Moreover, the contents of GPCho and GPEtn containing EPA and DHA in clams were also decreased, which further confirmed the oxidation of



PUFA. The accelerated storage also reduced the contents of TAG, PC and PE in dried clams in a temperature-dependent manner. Moreover, the amounts of the major molecular species of GPCho and GPEtn in dried clams also decreased. As described above, the TAG and PL containing PUFA were significantly degraded due to the occurrence of autoxidation, which may be responsible for the decrease in TAG, PC and PE as well as their molecular species. Moreover, according to our results, 22-24% and 2.6-3.8% of lipolytic enzymes were retained in clam tissues after boiling treatment and hot-air treatment, respectively. Kaneniwa46 and Toyes47 also suggested that lipolytic enzymes played an important role in lipid hydrolysis upon boiling treatment of silver carp muscle and drying treatment of viscera from scallop, respectively. Hence, the increase in FFA content and AV in dried clams during accelerated storage indicated the slight hydrolysis of esterified lipids by the surviving enzymes, which also contributed to the decline in contents of TAG, PC and PE. Hoehne-Reitan has reported that neutral lipase and phospholipase were abundant in bivalve shellfish.48 Previous studies also suggested that lipase and phospholipase played a part in the hydrolysis of lipids including PL and TAG during accelerated storage of milk and sunflower oil.49 Furthermore, clams are considered as a rich source of PL and TAG containing n-3 LC-PUFA, especially EPA and DHA.16,24 Recently, much attention has been given to n-3 LC-PUFA in the PL and TAG forms due to their high bioavailability, high tissue-delivery capacity and good health promoting effects compared to those in ethyl


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ester.50 However, our results indicated that PL and TAG contents, as well as their EPA and DHA components, were significantly decreased during storage due to hydrolysis and oxidation, indicating a decrease in the nutritional value in terms of lipids. In conclusion, lipid oxidation and degradation occurred in dried MP and RP during accelerated storage, which were reflected by the increase in all oxidation indices including POV, TBARS, AnV and TOTOX and the decrease in contents of TAG, PC, PE and major GP molecular species as well as PUFA percentage. Moreover, the predicted shelf life for MP and RP at 24 °C were 530 ± 14 and 487 ± 24 hours, respectively, according to Arrhenius model, while the observed values for the dried MP and RP stored at 24 °C were 560 ± 40 h and 520 ± 0 h, respectively. By calculation, the REs of shelf life at 24 °C between the actual values and the predicted values were 5.7% and 6.8%, respectively. Therefore, the accelerated storage test coupled with Arrhenius Equation Modeling is suitable for the shelf life prediction of dried clams. In general, all indices suggested that the storage caused deterioration in the quality and nutrition value of dried clams.



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Abbreviations Used Mactra chinensis Philippi (MP); Ruditapes philippinarum (RP); Peroxide value (POV); p-Anisidine value (AnV); Thiobarbituric acid-reactive substances (TBARS); Acid value (AV); Free fatty acid (FFA); Phospholipid (PL); Triglyceride (TAG); Phosphatidylcholine

(PC);

Phosphatidylethanolamine

(PE);

Glycerylphosphatidylcholine (GPCho); Glycerylphosphatidylethanolamine (GPEtn); Polyunsaturated fatty acids (PUFA); Precursor-ion scanning (PIS); Neutral loss scanning (NLS); Malonaldehyde (MDA); Long chain polyunsaturated fatty acids (LC-PUFA);

Glycerophospholipid

(GP);

Trichloroacetic

acid

(TCA);

Docosahexaenoic acid (DHA, C22:6 n-3); eicosapentaenoic acid (EPA, C20:5 n-3); MP before storage (0D-MP); MP storage for 20 days at 50 °C (50 °C-20D-MP); MP storage for 20 days at 65 °C (65 °C-20D-MP); RP before storage (0D-RP); RP storage for 20 days at 50 °C (50 °C-20D-RP); RP storage for 20 days at 65 °C (65 °C-20D-RP); 65 °C-280 h-MP were dried MP collected on 280 h; 65 °C-200 h-RP were dried RP collected on 200 h at 65 °C; Thin layer chromatography-flame ionization detection (TLC-FID); Mass spectrometer (GC-MS); Monoacylglycerol (MAG);

Polar

lipids

(PoL);

Cholesterol

(CHO);

Phosphatidylserine

(PS);

Phosphatidylinositol (PI); Phosphatidylglycerol (PG); Lysophosphatidylcholine (LPC); Phosphatidic acid (PA); Fatty acids (FAs); Relative error (RE). nd, not detected.


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Acknowledgment This work was financially supported by “Public Science and Technology Research Funds Projects of Ocean (201505029)”, “Project of Distinguish ed Professor of Liaoning Province (2015-153)”, “Program for Liaoning Excellent Talents in University (LR2015006), “Liaoning Provincial Natural Science Foundation of China (2015020781)”, and Supported by Program for “Dalian High-Level Innovative Talent (2015R0007)”

Supporting Information Table 1S. Changes in GP molecular species (%) of MP and RP during accelerated storage. Table 2S. Changes in moisture content (%) of MP and RP during accelerated storage. Figure 1S. MS/MS spectra for precursor ions of m/z 832. Figure 2S. Fragmentation pathways of phosphatidyl glycerophosphocholine 18:1/22:6 in MS/MS. This material is available free of charge via the Internet at http://pubs.acs.org.



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Figure captions Figure 1. Changes in POV (A), AnV (B), TBARS (C), TOTOX (D), and AV (E) of MP and RP during accelerated storage. Different letters and numbers in the same line indicate significant difference (P < 0.05). Bars represent standard deviations (n = 3).

Figure 2. TLC-FID chromatogram of lipid class (A),

31

P NMR chromatogram of

phospholipid class (B), HPLC chromatogram of PC and PE in dried clams (C), the purity analysis of TAG and PL separated by column chromatography (D), First-stage MS spectra of GPCho (molecular ion [M]+) and GPEtn (quasi-molecular ion ([M+H]+)) (E, F).

Figure 3. Changes in contents of TAG, FFA, PE and PC in MP and RP during accelerated storage (on a dry basis). Values of different groups with different lower case letters (a-e) and upper case letters (A-E) are significantly different at P < 0.05.

Figure 4. Changes in contents of EPA and DHA in MP and RP during accelerated storage. Values of different groups with different lower case letters (a-f) and upper case letters (A-E) are significantly different at P < 0.05.

Figure 5. (A) Total intensity of GPCho molecular species and total intensity of GPCho molecular species containing EPA and DHA; (B) Total intensity of GPEtn molecular species and total intensity of GPEtn molecular species containing EPA and



DHA. Values of different groups with different lower case letters (a-e) and upper case letters (A-E) are significantly different at P < 0.05.

Figure 6. Changes in enzyme activities of RP (A) and MP (B) (on a dry basis) upon different treatments. LOX, LPS and LPLS represent lipoxygenase, lipase and phospholipase, respectively. Values of different groups with different lower case letters (a-c), upper case letters (A-C) and numbers (1-3) are significantly different at P

Changes in lipid profiles of dried clams (Mactra chinensis Philippi and

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