Effects of high hydrostatic pressure processing on purine, taurine, cholesterol, antioxidant micronutrients and antioxidant activity of squid (Todarodes pacificus) muscles

Food Control 60 (2016) 189e195

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Effects of high hydrostatic pressure processing on purine, taurine, cholesterol, antioxidant micronutrients and antioxidant activity of squid (Todarodes pacificus) muscles Yifeng Zhang a, Gang Wang a, Yafang Jin a, Yun Deng a, *, Yanyun Zhao b a

Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, SJTU-Bor Luh Food Safety Center, Department of Food Science and Technology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China Department of Food Science and Technology, 100 Wiegand Hall, Oregon State University, Corvallis, OR, USA

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 March 2015 Received in revised form 28 July 2015 Accepted 29 July 2015 Available online 31 July 2015

This study investigated the effect of high hydrostatic pressure processing (HHP, at 0.1 [control], 200, 400 or 600 MPa) on purine, taurine, cholesterol, antioxidant micronutrients (Mn, Se, Fe, Zn, Cu, vitamin B2, A and E), DPPH, and reducing power of squids during 10-day storage at 4
C. Compared with the control, pressurization did not change the contents of purine, vitamin A and E, Mn and Fe in the squid samples on day 0. After 10-days of storage, HHP at 600 MPa caused maximum decreases in cholesterol, hypoxanthine, adenine and Fe, and produced small reductions in guanine, vitamin B2, DPPH, reducing power, and TBARS. No significant differences were found in cholesterol, reducing power, vitamin B2 or A between 200 MPa and 400 MPa treated samples. Both pressurization and storage time did not affect the levels of taurine, DHA, EPA, Mn and Cu. This study provided a strategy to decrease the cholesterol and purine contents with minimal antioxidant activity loss in seafood using HHP. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Squid High hydrostatic pressure Purine Cholesterol Antioxidant micronutrients Antioxidant property

1. Introduction Currently, nutrition, health, safety, convenience and diversity are the major factors driving the global food industry. Squid products have attracted considerable attention as a source of high amounts of good quality protein and healthy u3 series polyunsaturated fatty acids (Deng et al., 2011). Moreover, squids are also potential sources of antioxidant micronutrients (such as vitamins and minerals) (Li, 2003; Yang, Wang, Li, Huang, & Chi, 2013). These nutrients are essential for humans, and help to improve imbalanced dietary habits and prevent lifestyle-related diseases (Evans & Halliwell, 2001). Fox example, vitamin E is not only an essential nutrient for humans, but also acts as a chain-breaking antioxidant that specifically inhibits the oxidation of polyunsaturated fatty
-Puigmartí, acids (PUFAs) (Evans & Halliwell, 2001; Molto
pez-Sabater, 2011). High intake of Permanyer, Castellote, & Lo vitamin E contributes to reduce the risk of disorders associated with free radicals, such as atherosclerosis, cancer, cataracts, and cell

* Corresponding author. E-mail address: [email protected] (Y. Deng). http://dx.doi.org/10.1016/j.foodcont.2015.07.044 0956-7135/© 2015 Elsevier Ltd. All rights reserved.

damage connected to ischemia and reperfusion (Barba, Esteve, & Frigola, 2012a, b). Vitamin B2 also acts as an antioxidant that can help rid the body of damaging free radicals and its intake has been associated with decreased plasma homocysteine levels. Sufficient vitamin B2 could be obtained from natural food sources because of its water-solubility (Yang et al., 2013). The antioxidant activity of vitamin A (retinol) is conferred by the hydrophobic chain of polyene units that can quench singlet oxygen, neutralize thiyl radicals and combine with, and stabilize, peroxyl radicals (Ahmed, Islam, Khan, Huque, & Ahsan, 2004). Some microelements (such as manganese, selenium, iron, zinc and copper) are commonly referred to as antioxidant minerals and are required for the activity of certain antioxidant enzymes (Ahmed et al., 2004; Evans & Halliwell, 2001). Squids are currently regarded as a rich source of taurine, purine and cholesterol (Deng, Luo, Wang, & Zhao, 2015; Li, 2003). Taurine (2-aminoethanesulfonic acid) is a free amino acid found ubiquitously in the animal body that has important roles in several essential biological processes, such as calcium modulation, bile acid conjugation, antioxidation, membrane stabilization and immunity (Chen, 2006). Taurine synthetic activity in humans is weak, and supplemental taurine could be obtained from seafood (Chen, 2006;

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Li, 2003). Furthermore, human studies have revealed that the administration of taurine with n3 PUFAs has hypolipidemic and antiatherogenic effects, compared with n3 PUFAs supplementation alone (Chen, 2006; Yang et al., 2013). Like other seafood, squids are rich in purines (such as adenine, guanine, hypoxanthine and xanthine) and are thus classified as purine-rich foods. Purines play crucial roles in the storage of genetic information and in the processes of making and breaking nucleotides. Hypoxanthine and xanthine have been linked to off-flavors in food and are important markers for determining the freshness of seafood (Wang, Ling, Sun, Chu, & Zhou, 2008). Consumption of high-purine food raises uric acid and is associated with hyperuricemia and an increased risk of incident gout, which is a common and excruciatingly painful inflammatory arthritis (Lou, 1998). Therefore, low dietary purine intake is suggested for sufferers of gout to reduce the serum uric acid concentration (Wang et al., 2008). In addition, squids also have relatively high cholesterol, mostly the high-density lipoprotein cholesterol. Cholesterol is the major sterol (95% of total sterols), acting as a critical component of cell membranes, and is the precursor of all steroid hormones and vitamin D (Clariana & García
novas, Regueiro, 2011; Medina-Meza, Barnaba, & Barbosa-Ca 2014). On the other hand, consumption of cholesterol has been related to the incidence and severity of cardiovascular incidents (Chen, 2006). Squids have been designated as major contributors to dietary cholesterol; therefore, it is important to determine the level of cholesterol in food, including seafood. Increasing appreciation of the nutritional and functional properties of squid encourages studies on the effects of processing on product attributes to minimize quality degradation. High hydrostatic pressure processing (HHP) is considered a valuable nonthermal processing technology, allowing the extension of the shell-life of food products (Kim & Ahn, 2013). It inactivates and inhibits microorganisms, and can activate or inactivate enzymes, as well as producing safe and minimally processed foods with satis
s, Møller, factory nutritional and organoleptic qualities (Andre Adamsen, & Skibsted, 2004; Chevalier, Le Bail, & Ghoul, 2001;
, Comaposada, Arnau, & Garriga, 2014; VegaStollewerk, Jofre
lvez et al., 2011; Zhang, Jiao, Lian, Deng, & Zhao, 2015). When Ga HHP technology is employed in food processing, complete evaluation of the effects of process variables on essential nutrients and functional components is vital to define treatment conditions that avoid the loss of important food properties of foods and to obtain seafood with high health benefits for the consumer. Recently, studies have investigated the effect of HHP on microorganism inactivation, protein denaturation, enzyme activation and inactivation, and modification of physicochemical properties (e.g. proximate composition, color, texture and acidity) in squids and other seafood (Gou, Choi, He, & Ahn, 2010; Gou, Xu, Choi, Lee, & Ahn, 2010; Hu et al., 2013; Zhang et al., 2015). However, little information is available on the effects of HHP treatment on the purine, cholesterol, taurine, antioxidant micronutrients and their antioxidant activities in squids. Processing and storage may maintain, decrease or increase the levels of bioactive substances, and then modify bioavailability of these compounds in food. Therefore, to ensure that processed foods supply consumers with all nutrients in their most available form, comply with their requirements and maintain their organoleptic proprieties, it is fundamental to monitor and compare the influences of the main operative variable and storage conditions on nutritional components present in foods. The objectives of this work were 1) to investigate the influences of different pressure levels on the content and activity level of cholesterol, purines, taurine, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), thiobarbituric acid reactive substances (TBARS), and antioxidant micronutrients of squids; 2) to evaluate the relationships between

these determined compounds and their antioxidant activities; and 3) to assess the stability of these components and antioxidant activities in squids during refrigerated storage. 2. Materials and methods 2.1. Sampling, packaging and pressure treatment Fresh squids (Todarodes pacificus) were purchased from a local fishery market (Shanghai, China), and transported to the laboratory in iced water. Squids (310 ± 25 g, with a mantle length of 21.5 ± 2.8 cm) were cleaned and scrubbed thoroughly in iced water. After dissection, internal organs, arms and tentacles were removed to obtain the mantle. The integrated mantle muscle was retained as the experimental sample, with an average length of 210 ± 25 mm (widest part of 165 ± 15 mm, and a thickness of 3.0 ± 0.5 mm). Subsequently, mantle muscle was gently blotted with tissue paper to remove excess water on its surface. Squid samples were individually packaged in polyamide/chlorinated polypropylene (PA/CPP) complex film bags (Yinuo Packaging Materials Co., Ltd., Shanghai, China), heat-sealed under a vacuum (Shengsong Machinery Manufacturing Co., Ltd., Shanghai, China). Sample pairs were divided into four groups: one batch was assigned as the control and was treated at 0.1 MPa; the second batch was allocated to pressurization at 200 MPa, the third batch was allocated to pressurization at 400 MPa, and the fourth batch was allocated to HHP at 600 MPa. HHP treatments were done at 20
C for 10 min in a HHP-750 unit (Kefa High Pressure Food Processing Inc., Baotou, China) with a 2.5 L of cylindrical pressure vessel and a pressure range of 0e700 MPa. The pressure increase rate was 8.3 MPa/s, and the depressurization time was less than 4 s. After HHP treatments, all the samples were stored at 4
C, and sampled at 0 and 10 d for analysis. 2.2. Determination of total cholesterol contents Cholesterol analysis was conducted using the methodology described GB/T 22220-2008 (2008). About 10 mL of 60% KOH and 30 mL ethanol were added to squid samples (0.5 g) for saponification reaction at 100
C for 1 h. The unsaponifiable portion was extracted with 40 mL petroleum ether/ether (1:1, v:v) three times. The organic layer was retained and further washed with deionized water until neutral pH, and dried over sodium sulfate. The sample was reduced to near dryness using a vacuum rotary evaporator, redissolved in 5 mL of ethanol and filtered through Millex 0.45 mm nylon membrane syringe filters for HPLC. Quantification by HPLC was carried out using an Alliance 2695 system (Waters Corp., Medford, MA, USA) liquid chromatograph system equipped with a UVeVIS detector at 206 nm. The chromatographic separation was carried out on a reversed-phase C18 column (5m, 3.2  250 mm, Waters Corp., Medford, MA, USA.) and the mobile phase was methanol with a flow rate at 1.0 mL/min. The column temperature was set at 35
C and the sample injection volume was 10 mL. 2.3. Determination of purine contents The purine contents were determined by high performance liquid chromatography (Waters 2695, Medford, MA, USA) according to the method of Lou (1998), with some modifications. Briefly, the powdered samples (about 0.2 g) were digested with an 11 mL mixture of trichloroacetic acid/methanoic acid/H2O (5/5/1: v/v) at 90
C for 12 min. The resultant hydrolysates were transferred into a 250-mL flask and dried by a rotary vacuum evaporator at 55
C. The solution was filtered via a 0.2 mm membrane filter. HPLC conditions were as follows: column, thermo scientific syncronis C18 (5 m,

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191

4.6  250 mm); mobile phase, potassium dihydrogen phosphatemethanol (2:98, v/v, pH ¼ 4); flow rate, 0.8 mL/min; column temperature, 25
C; and detector wavelength, 254 nm.

1300 W; coolant flow, 15 L/min; auxiliary flow, 0.8 L/min; nebulizer flow, 0.2 L/min; read time, 0.21 s; sample aspiration rate, 0.2 mL/ min.

2.4. Determination of taurine content

2.9. Antioxidant activity analysis

Amino acid analysis was performed using a Hitachi L-8900 highspeed amino acid analyzer (Tokyo, Japan), according to the method by Deng et al. (2015). Each analysis was done in triplicate.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and total reducing power (TRP) were used to evaluate antioxidant activity in the squids. The sample was prepared using the method described by Deng et al. (2013), with some modifications. Briefly, 5 g of minced squid samples were homogenized with 50 mL of phosphate buffer solution (PBS, pH 7.0) at 5000 rpm for 2 min. The homogenates were placed at 4
C for 24 h. After filtration, the filtrate was stored at 4
C before subsequent antioxidant activity experiments. The DPPH and TRP were determined according to the procedure of Deng et al. (2014). The DPPH radical scavenging activity was calculated from the standard curve obtained by the reaction of ascorbic acid with DPPH solution, and expressed in mg of ascorbic acid equivalents per mL of squid extract. For TRP, The absorbance was measured at 700 nm. The assays were done in triplicate.

2.5. Vitamin B2 analyses Vitamin B2 contents were determined according to GB/T 9695.28-2008 (2008) and high performance liquid chromatography (Waters 2695, Medford, MA, USA) methods. The HPLC conditions were: reversed-phase C18 column: 5 m  3.2  250 mm; flow rate: 0.8 mL/min; injection volume: 20 L; mobile phase: methanol-water (30:70, v/v, pH ¼ 4); running time: 22 min; wavelength: 462 and 522 nm for excitation and emission, respectively. 2.6. Vitamin E and A analyses

2.10. Lipid oxidation assessment Vitamin E (a-tocopherol) and A (retinol) were analyzed HPLC as described by GB/T 5009.82-2003 (2003), with some modification. About 10 g of samples were saponificated with 25 mL KOH (250 g/ 200 mL, w/v) at 80
C for 40 min, and ascorbic acid in ethanol (15 g/ L, 50 mL) was added as antioxidant. The extraction was carried out using 50 mL petroleum ether three times. After evaporating the pooled extract to dryness, 5 mL ethanol was used to resuspend the samples. The separation was carried out on a reversed-phase C18 column (5 m, 3.2  250 mm) with methanolewater (98:2, v/v) as the mobile phase. The flow rate was 1.0 mL/min and the column temperature was 35
C. UV detector was set at 325 nm for retinol and 292 nm for a-tocopherol. The quantitative data were analyzed with the Waters 2695 separation module HPLC system. All tested solutions were filtered through Millex 0.45 mm nylon membrane syringe filters before use. All measurements were done in triplicate. Vitamin E content was expressed as mg/100 g d.m.

Lipid oxidation, measured as thiobarbituric acid reactive substances (TBARS) values, was determined according to the method of Chevalier et al. (2001), with some modifications. About 5 g of squid samples were minced and homogenized with 30 mL of 7.5% trichloroacetic acid solution (containing 0.1% propylgallate and 0.1% EDTA) for 2 min. The homogenates were centrifuged at 1500  g for 5 min. Then, 5 mL of the supernatant was mixed with 5 mL of 0.01 mol/L thiobarbituric acid (TBA) reagent, and heated in boiling water for 30 min. After heating, the samples were cooled under running tap water. The absorbance of the TBA color reaction was then determined by a UV-1800 spectrophotometer (Shimadzu Co, Japan) at 530 nm. TBA was calculated from the standard curve obtained by the reaction of 1,1,3,3 tetraethoxypropane with the TBA reagent. The TBA content in the squids was expressed in mg of malonaldehyde per kg of squid (mg MDA/kg).

2.7. EPA and DHA analyses

2.11. Statistical analysis

Fat extraction and fatty acid compositions were determined as described by Deng et al. (2011). Briefly, lipid extracts were converted into fatty acid methyl esters (FAME) and then analyzed with an Agilent 6890 Gas Chromatograph (GC) with a 5973 MSD system (Palo Alto, CA, USA) (7697A þ 7890B). Operational conditions were as follows: the injection volume was 1 mL; the injector and detector temperatures were 200
C and 250
C, respectively; nitrogen was used as the carrier gas and the flow rate was 1 mL/min. The fatty acid peaks in the squid samples were identified by comparing the retention times with standard mixtures (Sigma, St. Louis, MO). The amounts of each fatty acid and its isomers present were expressed as percentages of the total fatty acid content. All measurements were made in triplicate.

Two-way analysis of variance (ANOVA) of the data was carried out using Statistical Packages for the Social Sciences (SPSS 19.0) software (SPSS Inc., USA). Differences among the means were determined using Duncan's multiple range test with P < 0.05.

2.8. Determination of mineral content Mineral contents were measured using an inductively coupled plasma optical emission spectrometer (OPTIMA-7000DV, PerkinElmer, Waltham, MA, USA) after microwave digestion. Approximately 0.3 g of dried sample was weighed and digested with a reagent mixture of 6 mL of HNO3 and 2 mL of H2O2. After microwave digestion, the contents were diluted to 20 mL with deionized water. Instrument operational conditions were as follows: power,

3. Results and discussion 3.1. Cholesterol, taurine and purine Table 1 shows the changes in cholesterol, taurine, and purine contents in all squid samples. These values were within the general range of 6.15e12.15 mg/g for cholesterol, 1.96e19.29 mg/g for taurine, and 11.49e117.51 mg/100 g for purine contents for untreated squid samples from the literatures (Rosa, Pereira, & Nunes, 2005; Shirai et al., 1997; Venugopal & Shahidi, 1996; Yang et al., 2013). The variations might associate with species, age, sex, reproductive status, tissue location and assay methods (Yang et al., 2013). The cholesterol contents of squids treated at 200 and 600 MPa were higher than those of control and treated at 0.1 (control) and 400 MPa samples, but no significant variations were found between 200 and 600 MPa or between 0.1 (control) and 400 MPa on day 0. The cholesterol values were higher in HHPtreated squids on day 0 than on day 10. Moreover, there were no

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Table 1 Taurine, purine, and cholesterol contents of squid samples treated under different HHP conditions. Pressure (MPa) 0.1 200 400 600

Cholestrol (mg/g d.m.)

Taurine (mg/g d.m.)

Guanine (mg/100 g d.m.)

Hypoxanthine (mg/100 g d.m.)

Adenine (mg/100 g d.m.)

0d

0d

0d

0d

0d

8.42 8.92 8.41 8.86

10 d ± ± ± ±

Ab

0.20 0.01Aa 0.08Ab 0.03Aa

8.31 6.51 6.69 4.61

± ± ± ±

Aa

0.02 0.06Bab 0.08Bab 0.52Bb

10.9 11.8 10.9 11.0

10 d ± ± ± ±

Aa

0.6 1.4Aa 2.2Aa 1.0Aa

10.6 12.6 11.9 10.4

± ± ± ±

Aa

0.6 1.4Aa 0.9Aa 1.1Aa

38.7 35.9 38.2 39.9

10 d ± ± ± ±

aA

3.2 0.8aA 1.5 aA 3.0aA

12.4 11.5 30.3 48.7

± ± ± ±

cB

0.5 0.8cB 0.5bA 0.8aA

335.0 313.2 328.2 329.3

10 d ± ± ± ±

aB

4.2 5.0aB 20.5aB 17.1aB

470.8 446.1 403.2 356.0

± ± ± ±

aA

1.4 8.7bA 2.9cA 3.1dA

107.4 88.4 98.8 90.6

10 d ± ± ± ±

aA

1.1 2.2aA 0.6aA 1.4aA

68.2 67.2 38.8 23.5

± ± ± ±

0.2aB 0.6aB 0.2bB 0.1cB

d: day. Values were expressed as average ± standard error (n ¼ 3). Different lowercase letters within the same column row denote significant differences between means at P < 0.05. Different capital letters within the same row for the same index denote significant differences between means at P < 0.05.

significant differences in the cholesterol levels between 200 and 400 MPa, and they were the lowest at 600 MPa on day 10. Cholesterol is susceptible to oxidation to form cholesterol oxidation products (COPs) or oxysterols by processing technologies. For example, the possible membrane damage or protein denaturation induced by HHP could cause oxidation of cholesterol and finally change the cholesterol contents, depending on pressure levels, food matrix, treatment time and temperature as well as storage conditions (Clariana & García-Regueiro, 2011; Medina-Meza et al., 2014).
l, Me
sza
ros, and Farkas (2003) found observed Tuboly, Lebovics, Gaa increases in COPs levels of deboned turkey meat treated at 200 and 400 MPa for 20 min during storage of 4 and 8 months at 20
C, showing that oxidation processes occurred after treatment because of free radical oxidation. Pressurization of dry-cured ham at 600 MPa did not show changes in the contents of COPs (Clariana & García-Regueiro, 2011). Moreover, HHP (above 800 MPa) can lead to
s formation of free radicals, promoting lipid peroxidation (Andre et al., 2004), which could induce cholesterol oxidation (MedinaMeza et al., 2014). The mechanism by which HHP induces cholesterol oxidation is not clear and requires further investigation. There was no significant (P > 0.05) difference among samples for taurine contents, regardless of pressure levels and storage time (Table 1). The initial guanine, hypoxanthine and adenine values of untreated squids were 38.7, 335.0 and 107.4 mg/100 g dm, respectively (Table 1). Moreover, xanthine was not detected in the squid samples. Adenine and hypoxanthine were found to be more uricogenic than guanine and xanthine (Wang et al., 2008). The changes in purine content of squids were followed during storage at different pressure levels (Table 1). Compared with the control, HHP did not affect the contents of guanine and hypoxanthine, whereas adenine decreased by 15.6e17.7% after HHP. There were no differences (P > 0.05) in guanine, adenine and hypoxanthine in squids after different HHP treatments on day 0. After 10-days of storage, the levels of guanine (except for the 600 MPa sample) and adenine decreased markedly; however, the hypoxanthine content increased significantly. Currently, we could not explain why the guanine content of squids treated at 600 MPa was higher on day 10 than on day 0. Previous studies stated that pressurization of dry-cured ham does not modify purines and pyrimidines contents; however, treatment at 600 and 900 MPa caused a decrease in guanosine and
s, Garcíaan increase in adenosine, respectively (Clariana, Horto Regueiro & Castellari, 2011). Lou (1998) also found that in the grass shrimps, the adenine content decreased steadily, while hypoxanthine content increased during 120 h of storage at 5
C; and guanine content also increased slightly, reached a peak at 72 h, and then decreased slightly. At day 10, the guanine increased with increasing in pressure level, whereas adenine and hypoxanthine decreased with increasing pressure. This may indicate that the pressure was sufficiently high to lower the adenine and hypoxanthine content. The decreasing adenine levels suggested an enzymatic effect of AMP deaminase or adenosine deaminase: the

degradation of ATP to IMP was caused by endogenous enzymes in the kuruma prawn muscle (Matsumoto & Yamanaka, 1991). Adenine deaminates to form the purine base hypoxanthine, which is soluble and is released quickly from foods during processing (Lou, 1998). Our results showed that different HHP levels could modify protein structure and change enzyme activities; consequently having a major impact on the patterns of purine compounds in stored squid. We also concluded that suitable HHP treatments could reduce the amount of purines present in squid during storage. 3.2. Vitamin B2, A and E The initial contents of Vitamin B2, A and E in untreated squid (control) were 0.79 ± 0.06, 0.67 ± 0.02 and 77.2 ± 0.2 mg/g d.m., respectively (Table 2). These values agreed with the results of 0.11e10.00 mg/g for Vitamin B2, 0.03e4.51 mg/g for Vitamin A, and 0.02e2.70 mg/g for Vitamin E from Venugopal and Shahidi (1996) and Yang et al. (2013). Compared with the control, a significant increase (1.1e38.0%) in vitamin B2 activity was observed when HHP at 100e600 MPa was applied (P < 0.05), whereas no significant differences (P > 0.05) were obtained between 400 MPa and 600 MPa on day 0 or between 200 MPa and 400 MPa on day 10. There were no significant differences in the contents of vitamin A or E among all samples on day 0. During storage, the levels of vitamin A and E in HHP treated samples were lower on day 10 than on day 0 (P < 0.05); however, the vitamin B2 level in all samples remained
-Puigmartí et al. (2011) stable during the period of the study. Molto also observed no significant changes in a-, g-, and d-tocopherols when they applied HHP (400e600 MPa/22e27
C/5 min) in mature human milk. However, other studies formed contrary opinions to
lvez et al. (2011) stated that HHP at our results. Vega-Ga 300e800 MPa for 3 min resulted in significant losses of vitamin E in Aloe vera gel after 35 days of storage. Barba et al. (2012a, b) observed a significant increase in vitamin E activity (7e28%) of orange juiceemilk after the application of HHP (100e400 MPa for 9 min). This discrepancy could be related to the fact that both the processing conditions and the food matrix can influence the degradation of lipidic constituents via oxidation, as well as the release of vitamin E linked to proteins or phospholipids, thereby
lvez et al., 2011). directly affecting the vitamin E content (Vega-Ga Our results do not agree with those reported by de Ancos, Gonzalez, and Cano (2000), who noted that the vitamin A content of
e increased by 45% as a result of application of persimmon pure 350 MPa for 5 min. In addition, Sancho et al. (1999) also found that in strawberry ‘coulis’, vitamin B2 retention was not significantly affected by HHP (400e600 MPa/25
C/30 min). A determination of increased content of a particular nutrient may result from either moisture loss and thereby a ‘concentration’ of the nutrient, or the process itself may free the nutrient from the cellular matrix, such that the analytical determination is higher (Barba et al., 2012a, b).

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Table 2 Vitamin B2, vitamin A, and vitamin E contents of squid samples treated under different HHP conditions. Vitamin B2 (mg/g d.m.)

Pressure (MPa)

0d 0.1 200 400 600

0.79 0.92 1.07 1.09

Vitamin A (mg/g d.m.) 10 d

± ± ± ±

Ab

0.06 0.01Aab 0.03Aa 0.02Aa

0.84 0.95 0.93 1.04

0d ± ± ± ±

Ac

0.02 0.03Ab 0.02Ab 0.03Aa

0.67 0.61 0.67 0.62

Vitamin E (mg/g d.m.) 10 d

± ± ± ±

Aa

0.02 0.03Aa 0.03Aa 0.03Aa

0.65 0.34 0.30 0.32

0d ± ± ± ±

Aa

0.01 0.02Bb 0.01Bb 0.02Bb

77.2 69.7 74.7 80.6

10 d ± ± ± ±

Aa

0.2 6.1Aa 6.2Aa 1.1Aa

50.6 ± 1.1Ba 41.6 ± 1.1Bb 34.3Bc 38.6 ± 3.3Bbc

d: day. Values were expressed as average ± standard error (n ¼ 3). Different lowercase letters within the same column row denote significant differences between means at P < 0.05. Different capital letters within the same row for the same index denote significant differences between means at P < 0.05.

3.3. Antioxidant minerals Table 3 shows the antioxidant mineral contents of untreated and treated samples of squids at different pressure levels after 10 days of storage. The predominant antioxidant mineral elements in the raw squid samples (zero day storage) were Mn, Se, Fe, Zn and Cu. Zn was found to be the most abundant antioxidant mineral with a value of 6.3 mg/100 g d.m., while Mn was the least abundant with a value of 0.15 mg/100 g d.m. These values were comparable with the observations from other studies of 0.03e0.55 mg/100 g for Mn, 0.38e3.21 mg/100 g for Se, 0.90e10.46 mg/100 g for Fe, 2.01e12.19 mg/100 g for Zn, and 0.45e1.22 mg/100 g Cu contents (Anna & Kamila, 2013; Chen, 2006; Yang et al., 2013). The untreated squids stored for 10 days showed a significant decrease (P < 0.05) in mineral content of approximately 3.2e50% compared with the initial value of the untreated raw squids (zero day storage), except for Cu. On day 0, the concentrations of Se and Fe (except for 600 MPa) in the samples treated at 200e600 MPa were lower than those in the untreated samples. At day 10, the contents of Zn in the samples treated at 400 or 600 MPa were lower those in other two samples, however, the levels of Fe showed an opposite trend. Overall, after HHP treatments, the levels of the mineral contents (except for Fe) were either decreased or unchanged in squids between day 0 and day 10. Similar results were reported by Vega
lvez et al. (2011) that significant decrease in mineral contents Ga (Ca, Na, K) of Aloe vera gels treated with 300 and 500 MPa for 3 min was observed. However, various studies showed that HHP treatment (200e400 MPa) increases the concentration of ionic calcium in milk as well as the level of total calcium and phosphate in the serum phase of milk, with a maximum effect at 300 MPa (Barba et al., 2012a, b). With regard to the mineral profiles, the mineral contents may vary widely, depending on the food matrix, the distribution of minerals, the level of ionized minerals, tissue structure and type, pressure intensity, treatment time and temperature. 3.4. Antioxidant activity, EPA, DHA and TBARS The antioxidant capacity of squid muscles treated by HHP was

measured using DPPH and TRP assays. DPPH assays relate to radical and electron scavenging, whereas the TRP assay focuses on reducing oxidizing ability. The antioxidant capacity of squid muscles is mainly attributed to proteins, peptides or amino acids in the tissue (Deng et al., 2013) The DPPH and TRP values obtained ranged from 2.28 to 10.59 mg/ml AAE, and 0.061 and 0.1 in all samples, respectively (Table 4). The DPPH values increased with increased pressure, and were lower on day 0 than on day 10. In the TRP assays, the highest values were found for 600 MPa treated samples, with no significant changes among the other samples. Patras, Brunton, Da Pieve, and Butler (2009) demonstrated that total antioxidant
es was increased by 67.89% after 15 mon activity of blackberry pure of 600 MPa HHP treatment, while total antioxidant activity in squid samples increased by 257% after 10 min of 600 MPa HHP treatment in this study. Storage time did not affect the TRP values. A similar
zquez-Gutie
rrez et al. (2013), who behavior was observed by Va found that higher antioxidant activity (DPPH and TRP values) was observed in onions when the pressure applied increased at pres
lvez et al. (2011) reported that sures of 300 or 600 MPa. Vega-Ga DPPH free radical-scavenging activity increased after 35 days of storage in all 300e500 MPa pressurized samples (P < 0.05),
rez-Jacobo reaching its highest activity at 300 MPa. However, Sua et al. (2011) found that high pressure (100e300 MPa) homogenization processing did not change apple juice TRP and DPPH values. The no changes or increased antioxidant activity might be related to changes in the tissue matrix induced by high hydrostatic pressures (e.g. tissue disruption), which might lead to a better extractability of antioxidant components (McInerney, Seccafien,
zquez-Gutie
rrez et al., 2013; VegaStewart, & Bird, 2007; Va G
alvez et al., 2011). Lipid oxidation is a free-radical chain reaction involving initiation, propagation and termination stages (Medina-Meza et al., 2014). As can be seen in Table 4, a clear trend towards an increase in squid TBARS values were observed when applying pressures from 0.1 to 600 MPa, reaching the TBARS value at 600 MPa. The TBARS values in HHP-treated samples were lower on day 0 than on day 10. A significant increase in TBARS value was observed at the end of storage for all HHP-treated samples. Previous studies

Table 3 Mineral contents (mg/100 g d.m) of squid samples treated under different HHP conditions. Pressure (MPa)

Mn

Se

Fe

0d

10 d

0d

10 d

0d

0.1 200 400 600

0.15 ± 0.07aA 0.1aA 0.1aA 0.15 ± 0.07aA

0.14 ± 0.01aA 0.14 ± 0.03aA 0.14aA 0.15 ± 0.01aA

0.45 ± 0.07aA 0.4abA 0.3bA 0.3bA

0.3 ± 0.14aA 0.16 ± 0.06aA 0.1aA 0.1aA

3.75 1.65 1.75 3.7

Zn 10 d ± ± ± ±

0.34aA 0.21bA 0.64bA 0.84aA

3.3 1.55 0.15 0.6

Cu

0d ± ± ± ±

0.14aA 0.07bA 0.07dB 0.14cB

6.3 6.85 6.25 6.55

± ± ± ±

0.00abA 0.21aA 0.35bA 0.07abA

d: day. Values were expressed as average ± standard error (n ¼ 3). Different lowercase letters within the same column row denote significant differences between means at P < 0.05. Different capital letters within the same row for the same index denote significant differences between means at P < 0.05.

10 d

0d

10 d

6.05 ± 0.07cA 6.2 ± 0.14bcA 6.4abA 6.5aA

0.7aA 0.7aA 0.55 ± 0.07bA 0.7aA

0.7 ± 0.02aA 0.8aA 0.6aA 0.7 ± 0.01aA

194

Y. Zhang et al. / Food Control 60 (2016) 189e195

Table 4 DPPH, total reducing power, TBARS, EPA and DHA values of squid samples treated under different HHP conditions. Pressure (MPa) 0.1 200 400 600

DPPH (mg/ml AAE)

TRP (OD700)

0d

0d

2.28 4.78 5.64 8.15

10 d ± ± ± ±

Bd

3.32 0.03 0.64Bc 6.54 Bb 0.45 8.17 Ba 0.45 10.59

± ± ± ±

Ad

0.12 0.24Ac 0.20Ab 0.26 Aa

0.061 0.065 0.064 0.092

10 d ± ± ± ±

Ab

0.002 0.004Ab 0.006Ab 0.007Aa

0.067 0.072 0.074 0.100

± ± ± ±

Ab

0.004 0.004Ab 0.017Ab 0.011Aa

TBARS (mg MDA/kg w.m.)

EPA (mg/g d.m.)

0d

0d

0.455 0.498 0.571 0.647

10 d ± ± ± ±

Ac

0.04 0.02Bbc 0.06Bab 0.06Ba

0.540 0.648 0.794 1.204

± ± ± ±

Ad

0.04 0.01Ac 0.08Ab 0.02Aa

3.73 3.67 3.78 3.87

DHA (mg/g d.m.)

10 d ± ± ± ±

Aa

0.03 0.07Aa 0.30Aa 0.18Aa

3.47 3.43 3.55 3.65

0d ± ± ± ±

Aa

0.07 0.01Aa 0.06Aa 0.17Aa

15.8 16.3 16.3 15.8

10 d ± ± ± ±

Aa

0.1 0.4Aa 2.2Aa 1.4Aa

15.8 15.8 16.4 16.5

± ± ± ±

0.3Aa 0.1Aa 0.3Aa 0.5Aa

d: day. DPPH (1,1-diphenyl-2-picrylhydrazyl) in mg ascorbic acid equivalents/of squid extract; C: TRP (total reducing power) in absorbance values at 700 nm; d: day. TBARS: thiobarbituric acid reactive substances; DHA: docosahexaenoic acid (22:6 n3); EPA: eicosapentaenoic acid (20:5 n3). Values were expressed as average ± standard error (n ¼ 3). Different lowercase letters within the same column row denote significant differences between means at P < 0.05. Different capital letters within the same row for the same index denote significant differences between means at P < 0.05.

showed that washed muscle fibers and minced pork treated with HHP in a range of 300e800 MPa for 20 min showed a linear relationship between pressure and TBA number (Medina-Meza et al., 2014). A similar phenomenon was reported by Pereda, Ferragut, Quevedo, Guamis, and Trujillo (2008), who confirmed that 300 MPa treatments resulted in higher TBARS values compared
lez, Carrasco, and with 200 MPa treatments. Cava, Ladero, Gonza Ramírez (2009) found that in dry-cured ham and loins, the TBA values significantly increased, even at lower pressure (200 and 300 MPa), regardless of the time of treatment. Proteins, peptides or amino acids in the squid tissues possess significant antioxidant activity (Deng et al., 2013), which may be related to their hydrophobic nature and orientation of potential antioxidant side chains at the lipid interface; however, they can also bind prooxidant metals to phosphoseryl residues (Pereda et al., 2008). As shown in Table 3, HHP treatments resulted in more or less release of iron ions, which could promote free radical formation via Fenton's mechanism, which supported the results of a previous study (Medina-Meza et al., 2014). Orlien, Hansen, and Skibsted (2000) also found that, at the same time, pressure contributes to the denaturation of myoglobin species, causing greater exposure of the catalytical heme group. In this study, no statistically significant differences in either the EPA or DHA contents were detected between pressurized and untreated samples (Table 4). The EPA and DHA contents were in accordance with previous research results of 2.4e15.5 and 12.7e34.7 mg/g, respectively (Liang & Hwang, 2000; Venugopal & Shahidi, 1996; Yang et al., 2013). Our results agreed with a study by Cruz-Romero, Kerry, and Kelly (2008), in which there was no alteration in the percentage of fatty acids in oysters after HHP at 260, 500 and 800 MPa. According to the correlation analysis results among HHP pressure, storage time and quality indices in squid samples (data not shown), the relationships between pressure and DPPH, vitaminB2 were both significant (P < 0.001), and pressure also had a significant (P < 0.01) correlation with reducing power and TBARS. Notably, storage time was highly correlated with cholesterol, vitamin E, vitamin A, hypoxanthine, adenine, and Se in squid samples (P < 0.001). There was a negative correlation between TBARS and cholesterol (R ¼ 0.88, P < 0.001) and adenine (R ¼ 0.88, P < 0.001); however, cholesterol was positively correlated with vitamin E (R ¼ 0.76, P < 0.001) and vitamin A (R ¼ 0.86, P < 0.001) in squid samples. Meanwhile, the data indicated that the correlation of DPPH between reducing power and TBARS was very high (jRj  0.80, P < 0.001). These results revealed that HHP pressure significantly affected the antioxidant properties, and storage time significantly reduced the content of cholesterol, vitamin E, vitamin A and hypoxanthine in squid samples.

4. Conclusions The changes in purine, taurine, cholesterol, antioxidant micronutrients, antioxidant activity, and lipid oxidation of HHP processed squid muscles during refrigerated storage were observed. HHP possesses potential advantages as a cold-pasteurization technique by reducing the content of cholesterol and purine (hypoxanthine, adenine), delaying the losses of vitamin B2 and antioxidant activity, as well as maintaining the contents of taurine, DHP, EPA, Mn and Cu. However, HHP also promoted lipid oxidation and Fe loss during storage. Collectively, the maximum lipid oxidation and reductions in purine and cholesterol in squids occurred at 600 MPa, where the antioxidant activity and vitamin B2 changes of squid were minimized after storage for 10 days. More studies are under the way in our laboratory to investigate the oxidation mechanism of cholesterol and proteins in seafood induced by HHP. Acknowledgments This research was supported by the National Natural Science Foundation of China (Nos. 31271955, 31301586) and the AgriþX Program of Shanghai Jiao Tong University (Nos. Agri-X2014006). Special thanks to SJTU Instrumental Analysis Center for expert assistance with the analysis of the vatmine contents. References Ahmed, L., Islam, S. N., Khan, M., Huque, S., & Ahsan, M. (2004). Antioxidant micronutrient profile (vitamin E, C, A, copper, zinc, iron) of colostrum: association with maternal characteristics. Journal of Tropical Pediatrics, 50(6), 357e358. de Ancos, B., Gonzalez, E., & Cano, M. P. (2000). Effect of high-pressure treatment on the carotenoid composition and the radical scavenging activity of persimmon fruit purees. Journal of Agricultural and Food Chemistry, 48(8), 3542e3548.
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