WANG Qi1), 2), IKEGAME Keita3), TAKAHASHI Koretaro3), XUE Changhu4), ZHANG Weinong1), 2), WANG Hongxun1), HOU Wenfu1), and WANG Yuming4), *

?

Comparison of Lipids in Organs of the StarfishAssociated with Different Treatments

WANG Qi, IKEGAME Keita, TAKAHASHI Koretaro, XUE Changhu, ZHANG Weinong, WANG Hongxun, HOU Wenfu, and WANG Yuming

1),,430023,2),,430023,3),,041-8611,4),,266003,

Lipids were extracted from organs of the starfishassociated with different treatments (raw-control, boiling and heating), and then analyzed for lipid content, lipid oxidation index, lipid classes and fatty acid composition. Results showed that boiling softened the hard starfish shells, thus facilitating the collection of starfish organs. As compared with raw organs, the boiled organs had lower water content and higher lipid content, possibly due to the loss of water-holding capacity caused by protein denaturation. Both boiling and heating increased the peroxide value (PV), thiobarbituric acid (TBA) value and carbon value (CV) of lipids. Despite slight increases in the content of complex lipids, associated lipid composition had no substantial variations upon boiling and heating. For simple lipids, the content of 1, 2-diglyceride decreased in boiled and heated organs, with free fatty acids observed on thin layer chromatography (TLC). However, neither boiling nor heating significantly changed the fatty acid compositions of simple or complex lipids in starfish organs, suggesting that these two treatments had no significant effects on complex lipids in starfish organs. Together, our results indicated that boiling of starfish soon after capture facilitated the handling and extraction of useful complex lipids consisting of abundant glucosylceramide and eicosapentaenoic acid (EPA)-bounded phospholipids.

; organs; lipids; boiling; heating

1 Introduction

The starfishis widely distributed in the North Pacific and its outbreak can cause severe damage to fishing and aquacultural grounds for benthic shellfish such as scallops (Sloan and Aldrige, 1981; Dare, 1982). In the Nemuro Bay (Hokkaido) of northern Japan, physical removal of starfish is routinely practiced in scallop culture grounds. As a result, a large number of starfish are collected and accumulated as wastes. The amount of starfish wastes has been estimated to be approx. 15000 tons per year in Hokkaido (Ito, 1991). Currently, the treatment of these starfish wastes remains problematic.

Despite being a nuisance to Japanese fishermen, starfish have been boiled to make snacks and utilized as a traditional Chinese medicine in some inshore areas in China. Several studies have attempted to utilize starfish waste as fertilizer and growth regulator for plants (Line, 1994; Ishii, 2007). Recent studies have shown that starfish organs are a good source of functional lipids such as glycoylceramide and phospholipids rich in eicosapen- tanoic acids (EPA) (Hossain., 2006; Shah., 2008). Hence, it is thought that starfish can be developed to be a good source of functional food.

As the organs of starfish rot fast, immediate boiling becomes an inevitable and economical way to prevent their rotting upon capture. In this study, lipids were extracted from the organs of starfish associated with different treatments (raw, boiling and heating), and then analyzed for changes in lipid content, lipid oxidation index, lipid classes and fatty acid composition, which potentially affect their utilization.

2 Materials and Methods

2.1 Materials

The starfishwas collected on the coast of Nemuro, Hokkaido, Japan and transported to the laboratory on ice immediately after collection. The starfish bodies were cut into two halves using scissors. One half was boiled in water for 30min and the organs were separated and defined as the boiled group. The organs of untreated starfish were separated, homogenized and pooled into two groups: one stored on ice until use (referred to as the raw group), and the other sealed in plastic bags and kept in boiling water for 30min (referred to as the heated group).

All the chemical regents and solvents were of analytical grade and purchased from Japan.

2.2 Total Lipid Extraction

Lipids were extracted from starfish organs with a solvent combination of chloroform, methanol and distilled water according to Bligh and Dyer (1959) with slight modifications (reduced amount of material and reagent in proportion). Lipid extract was redissolved in chloroform and stored under argon gas in dark at ?30℃ till further analysis. Water content was determined according to the Association of Analytical Communities (AOAC) method (Cunniff, 1995).

2.3 Lipid Oxidation Analysis

2.3.1Determination of peroxide value

Approx. 0.5g of lipid sample was mixed in a conical flask containing 30mL of glacial acetic acid and chloroform (v:v=3:2), to which 0.5mL of saturated potassium iodide was added. The mixture was kept in dark for 10min, and then 30mL of distilled water and 0.5mL of freshly prepared 1% starch solution were added. After shaking, the mixture was titrated with 0.01N sodium thiosulfate. The peroxide value (PV) was expressed in unit of milliequivalents (meq) per kg of lipid (Cunniff, 1995).

2.3.2 Determination of thiobarbituric acid value

Thiobarbituric acid (TBA, as malonaldehyde) was determined colorimetrically. A portion (50–200mg) of tissue sample was weighed into a 25 mL volumetric ?ask, to which 1mL of 1-butanol solvent was added. The mixture wasmade to 25mL with 1-butanol and then mixed. Five mL of the mixture was pipetted into a dry stoppered test tube, to which 5mLof TBA reagent was added. The TBA reagent was prepared by dissolving 200mg of 2-TBA in 100mL of 1-butanol, ?ltered and stored at 4℃ forless than 7d. The test tubewas stoppered, vortexed, and then placed in a 95℃ water bath for 120min. The mixture was cooled and absorbance was measured at 532nm with a JASCO V-630 spectrophotometer (Tokyo, Japan). A reagentblank was measured for each treatment. TBA value (mg of malonaldehydekgof tissue) was calculated from a standard calibration curve generated with known amounts of 2-TBA (Natseba, 2005; Wrolstad, 2005; Shah, 2010).

2.3.3 Determination of carbon value

2,4-Dinitrophenylhydrazine (2,4-DNPH) solution was prepared by dissolving 50mg of 2,4-DNPH in 100mL of 1-butanol containing 3.5mL of concentrated HCl. Lipid sample (20–200mg) was weighed and solved in a 10mL volumetric flask containing 1-butanol to the volume. One mL of lipid solution was transferred into a 15mL test tube and mixed with 1mL of 2,4-DNPH solution. The test tube was stoppered and incubated at 40℃ for 20min. After cooling, 8mL of 8% KOH in 1-butanol was added to the test tube, and then centrifuged at 2000×for 5min. The absorbance of the upper phase was measured at 420nm with a JASCO V-630 spectrophotometer (Tokyo, Japan). The concentration of carbonyl compound in the lipid (carbon value, CV) was calculated from a standard calibration curve generated with known amounts of-octylaldehyde and expressed as μmolLlipid (Shah, 2010).

2.4 Fractionation of Total Lipids

Simple lipid (non-polar lipid) and complex lipid (polar lipid) were separated from the total lipid using Sep-Pak Vac 12cc silica cartridges (Waters Corporation, Milford, MA, USA). Two hundred mg of lipid samples were loaded onto the top of the cartridges. Then, simple and complex lipids were respectively eluted with chloroform and methanol in sequential order (Shah, 2008).

2.5 Lipid Class Composition Analysis

The lipid class composition of simple lipid was determined via a commercial silica gel 60F254 (Merck KGaA, Darmstadt, Germany) thin-layer chromatograph (TLC) plate with a single step development system consisting of-hexane: diethyl ether: acetic acid (v:v:v=80:20:1,). The plate was sprayed with 3% copper (Ⅱ) acetate ?8% phosphoric acid reagent and heated at 120℃ for 15min. Each spot was identified by authentic lipid standards and the lipid composition was scanned and then analyzed using Scion Image (Scion Corporation, Frederick, Maryland, USA). Lipid composition of complex lipid was determined by TLC with the solvent system of chloroform: methanol: water (v:v:v=65:25:4,) and 3% copper (Ⅱ) acetate ?8% phosphoric acid reagent as detection reagent (Prevot and Mordret, 1976).

2.6 Fatty Acid Composition Analysis

Fatty acid methyl esters were derived from the lipid samples. Briefly, dried lipid samples were dissolved in 1mL of-hexane, and then 0.2mL of methanolic 2N-NaOH solution was added. The mixture was shaken and kept at 50℃ for 20sec. Then, 0.2mL of methanolic 2N-HCl solution was added. The-hexane layer was collected, concentrated and analyzed using a Hitachi 163 Gas Chromatograph (Tokyo, Japan) connected to a PEG-20M liquid phase-coated G-300 column (1.2mmi.d.×40m, 0.5mm; Chemicals Evaluation and Research Institute, Saitama, Japan) with flame ionization detection. The temperature of the column, detector and injector were 170, 250 and 240℃, respectively. The fatty acids were identified by comparing the peak retention times with authentic standards (GL Sciences Inc. Tokyo, Japan) based on the linear relationship between the carbon number unit or double bond number of fatty acid and the logarithm of corresponding retention times (Shah, 2008).

2.7 Statistical Analysis

Analysis of Variance (ANOVA) was used to minimize the error, and student’s t-test used to determine significant differences among lipid properties of different treatments. Differences were considered statistically significant at<0.05. All statistics were performed using SPSS16.0.

3 Results and Discussion

3.1 Total Lipid Contents

As shown in Table 1, wet-based total lipid content in-creased and water content decreased in boiled organs compared with those of the raw group. It was likely due to the loss of water-holding capacity caused by protein denaturation during boiling. For the heated group, wet- based total lipid content remained at the same level and water content barely decreased. We speculated that protein denaturation occurred during heating, but the released water sealed in the plastic bag likely mixed with the organs after cooling. Dry-based lipid contents were also calculated, showing no substantial changes upon boiling or heating.

Table 1 Lipid and water contents of organs of starfish (A. amurensis) associated with different treatments

Notes: mean±SD;=3; different letters in the same row indicate statistically significant difference (<0.05).

3.2 Lipid Oxidation Index

Lipid oxidation is a complex process, in which unsaturated fatty acids react with molecular oxygen via a free radical chain mechanism. This forms fatty acyl hydroperoxides, which is generally called peroxides or primary products of the oxidation (Gray, 1978). The primary auto-oxidation is followed by a series of secondary reactions, leading to the degradation of lipid and the development of oxidative rancidity. Lipid oxidation in muscle foods can be examined by measuring primary or secondary changes with a variety of methods. The suitability of each of these methods depends on the type of product and the way it is processed and stored (Coxon, 1987), as well as the degree of correlation of the method with sensory analysis (Igene and Pearson, 1979). Methods that measure primary changes such as oxygen uptake, loss of polyunsaturated fatty acids and formation of hydroperoxides (measured as PV), are generally suitable to measure low levels of oxidation in uncooked products stored at low temperatures (Coxon, 1987). By comparison, the TBA value analysis is more widely used (Melton, 1983), which has frequently been found to produce useful correlation with sensory scores in studies regarding the development of warmed-over flavor (WOF) in cooked meats (Poste., 1986). Methods (CV test) that involve measurement of other secondary changes such as formation of carbonyls hydrocarbons and fluorescent products have also been used to study lipid oxidation (Melton, 1983; Kamarei and Karel, 1984).

For a full-scale study on lipid oxidation in organs of starfish, the oxidation indices were expressed by PV, TBA value and CV (Table 2). The PV increased slightly after boiling and had no significant change after heating. The TBA value and CV also increased slightly after boil-ing and heating. Compared with the case of raw materials, only moderate lipid oxidation occurred during boiling and heating even after starfish organs were kept in boiling water for 30min. Further study is needed to test whether boiling or heating for a shorter time can minimize the increases in oxidative indices.

Table 2 Lipid oxidation indices of organs of starfish (A. amurensis) associated with different treatments

Notes: mean±SD;=3; different letters in the same row indicate statistically significant difference (<0.05).

3.3 Lipid Class Composition

The complex lipid content in total lipids of raw organs (30.30%) relatively increased (<0.05) after boiling (34.88%) and heating (35.52%). We speculated that the non-polar simple lipid was more easily released into hot water and decomposed more thoroughly than complex lipid, as the latter’s polarity, special structure and combination pattern in the organ prevent its removal and de-composition.

Table 3 Lipid class composition of simple lipids (% of total simple lipids) in organs of starfish (A. amurensis) associated with different treatments

Notes: mean±SD;=3; different letters in the same row indicate statistically significant difference (<0.05);not detected.

As shown in Table 3, free fatty acid was not detected in lipids of raw organs, but only occurred after boiling and heating. In contrast, the content of 1,2-diglyceride decreased in starfish organs after boiling and heating. For the remaining components, no obvious changes were observed in their content. We concluded that the simple lipids slightly decomposed upon boiling and heating.

As shown in Table 4, there was no obvious change in lipid class composition of starfish organs caused by boiling and heating. In particular, the contents of main functional lipids such as phosphatidylcholine, phosphatidyle- thanolamine, ceramide dihexoside and ceramide monohe- xoside were not affected by boiling and heating. From the results, we concluded that complex lipids were stabilized in relevant tissues.

Table 4 Lipid class composition of complex lipids (% of total complex lipids) in organs of starfish (A. amurensis) associated with different treatments

Notes: mean±SD;=3; different letters in the same row indicate statistically significant difference (<0.05).

3.4 Fatty Acid Composition

As shown in Table 5, the proportion of polyunsaturated fatty acids (PUFA) of simple lipids decreased while that of monounsaturated fatty acids increased upon boiling and heating. For saturated fatty acids, the proportion decreased slightly in the boiled group but not in the heated group. These could be attributed to the decomposition and oxidation of simple lipids during boiling and heating. Some of the separated simple lipids could rise to the boiling water surface and react with fresh air, thus oxidized and decomposed to some extent. The changes in fatty acids composition were likely caused by several reasons such as the fast increase in the PUFA autoxidation rate (Frankel, 1998) and easy attack of isolated and lipids-incorporated PUFA by free radicals, resulting in lipid peroxides. Both monounsaturated and saturated fatty acids are more resistant to free-radical attack (Halliwell and Chirico, 1993).

As shown in Table 6, the PUFA proportion relatively increased while that of saturated fatty acids decreased in boiled and heated groups compared with raw organs. This could be attributed to the elevated content of complex lipids rich in PUFA. There was no obvious change in the proportion of monounsaturated fatty acids. Results showed that PUFA were affected much less in complex lipids than in simple lipids. As for the most important characteristic fatty acid, EPA (eicosapentaenoic acid, C20:5n-3), which is considered to form functional phospholipids (Hossain., 2006), no obvious change was detected in its proportion after boiling and heating.

Table 5 Fatty acid composition (% of total fatty acids) of simple lipids in organs of starfish (A. amurensis) associated with different treatments

Notes: mean±SD;=3; different letters in the same row indicate statistically significant difference (<0.05).

Table 6 Fatty acid composition (% of total fatty acids) of complex lipids in organs of starfish (A. amurensis) associated with different treatments

Notes: mean±SD;=3; different letters in the same row indicate statistically significant difference (<0.05).

4 Conclusions

This study showed that neither boiling nor heating treatments had substantial effects on the quality of complex lipids in organs of the starfish, particularly for functional lipids such as glucosylceramide and EPA-bounded phospholipids. However, the boiling treat- ment indeed softened the hard shell of starfish, thus facilitating the collection of the organs. Our work demonstrated that simple boiling is a useful method to treat large amounts of starfish in order to prevent their deterioration and obtain large-scale functional complex lipids.

Acknowledgements

This work was supported by the International Science and Technology Cooperation Program of China (Grant No.2010DFA31330), and partially by the Sakura Program of Japan Society for Promotion of Science.

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報(bào)告指出，亞洲資本市場(chǎng)呈現(xiàn)機(jī)構(gòu)化趨勢(shì)，中國(guó)在股票和企業(yè)債券方面占全球的權(quán)重將顯著提升。在零售投資文化日益風(fēng)靡的環(huán)境下，隨著存款儲(chǔ)蓄機(jī)構(gòu)追求更高的回報(bào)率，亞洲資本市場(chǎng)將不斷增長(zhǎng)，相關(guān)股本和固定收益證券將更多地在國(guó)內(nèi)消化。瑞信預(yù)期這些資產(chǎn)將呈現(xiàn)機(jī)構(gòu)化趨勢(shì)，流向養(yǎng)老金、保險(xiǎn)資金及共同基金。

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(Edited by Wei Liuzhi)

10.1007/s11802-013-1956-8

ISSN 1672-5182, 2013 12(3): 413-417

. Tel: 0086-532-82032597 E-mail: wangyuming@ouc.edu.cn

(March 11, 2012; revised May 4, 2012; accepted June 25, 2012)

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2013