Received Date: March 18, 2014; Accepted: May 27, 2014; Published Date: June 19, 2014
Citation: Osawa A, Ito K, Fukuo N, Maoka T, Tsuruoka H et al. (2014) Changes of Carotenoids in Atlantic Salmon by Heat Cooking and the Singlet Oxygen Quenching Activities of the Artificially Produced Carotenoids. J Food Process Technol 5:332. doi:10.4172/2157-7110.1000332
Copyright: © 2014 Osawa A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Carotenoids are widely distributed in food such as vegetables, fruits, fish and crustacean animals, and are thought to play an important role in human health. Although the above materials are often heated for cooking, few studies have reported the change of dietary carotenoids by these processes. In this study, we analyzed the carotenoids in heat cooked (steamed, grilled, fried, and microwaved) Atlantic salmon fed mixtures of astaxanthin, adnirubin, and canthaxanthin, (6 : 3 : 1, all trans) (salmons ingested feed containing 80 mg Panaferd AX/kg) for two years, using a silica gel HPLC column, and compared with carotenoids contained in raw salmon for the first time. As a result, the cis-carotenods (9-cis astaxanthin, 13-cis astaxanthin, 13-cis canthaxanthin, 13-cis adonirubin) derived from salmon fed carotenoids were clearly increased in heat cooked salmon. The rates of cis-isomers/total (trans + cis-isomers) were microwave heating (21-32%), steaming and grilling (17-24%), and frying (14-21%), respectively. We also examined the singlet oxygen quenching activities of the isolated natural and cis-isomer carotenoids (trans- or ciscanthaxanthin, adonirubin, astaxanthin and adonixanthin), and concluded that there were no significant differences between trans and cis-isomers (IC50 2.4-7.4 μM).
Carotenoids; Atlantic salmon; Singlet oxygen quenching activity; Heat cooking
Carotenoids are C40 isoprenoid pigments (tetraterpenes) with long conjugated double bonds that possess colors ranging from yellow through to orange and red . These pigments are widely distributed in food such as vegetables, fruits, fish and crustacean animals, and are thought to play important roles in human health, such as the prevention of cancer , metabolic syndrome , and eye disease , due to their antioxidative activity.
The Red color in wild Atlantic salmon (salmo salar) muscle derives from astaxanthin along with some minor carotenoids such as canthaxanthin, adonirbin, adonixanthin, zeaxanthin and antheraxanthin. These carotenoids originate from crustacean planktons, which are food for salmon, and salmon can store them in muscle and on the body surface. Also, salmon can reduce them on their body surface .
The structures of carotenoids change rapidly by oxidation or heat treatment. Since foods containing caroteonoids are often heat cooked for eating, studies on the change of carotenoids in vegetables (broccoli, spinach, green beans, cabbage, carrots, tomatoes and potatoes) [6,7] and fruits (orange, peach, mango and papaya) by heat cooking processes (microwaving, boiling and steaming) were reported previously [8-11]. On the other hand, only a few studies on carotenoid changes by heat treatment exist for seafood (edible fish, shell and crustaceans) have been reported [the change of the total carotneoid amount by heat treatment was reported for farmed (astaxanthin or canthaxanthin fed) rainbow trout in some studies [12,13].
In this study, we analyzed carotenoids in several heat cooked (steaming, grilling, frying and microwaving) Atlantic salmon, fed astaxanthin, canthaxanthin and adonirubin, using a silica gel HPLC column and compared to those contained in raw salmon. This is the first report comparing the changes of carotenoids by exhaustive heat treatments.
We also report for the first time the antioxidative activities [singlet oxygen (1O2) quenching activities] of isolated carotenoids from raw and cooked salmon [4 carotenoid from raw salmon (all-trans canthaxanthin, adonirubin, astaxanthin and adonixanthin) and 4 cisisomer carotenoids from cooked salmon (13-cis-canthaxanthin, 13-cisadonirubin, 9-cis-astaxanthin, and 13-cis-astaxanthin)].
Preparation of atlantic salmon
Four male farmed atlantic salmon (1.335 kg, 1.367 kg, 1.478 kg, 1.644 kg) were fed Panaferd AX [astaxathin : adonirubin : canthaxanthin (6 : 3 : 1, all trans)] for 2 years (salmons ingested feed containing 80 mg Panaferd AX/kg) were used in this study. Two of them (1.367 kg and 1.478 kg) were used for carotenoids analysis in raw and cooked fillets, and the others (1.335 kg and 1.644 kg) were used to isolate carotenoids for 1O2 quenching activity. These were gifts from JX Nippon Oil and Energy Corporation (Japan, Tokyo). The muscle of each salmon were cut into 12 fillets (approximately 80 g/ fillet) and stored in a -80°C freezer.
Preparation of raw and cooked salmon
After defrosting for 12 hours at <5°C, the stored fillets were divided in to 5 groups (Group A-E, 4 fillets in each group). Each group was cooked as follows [Group A: uncooked, Group B: steamed over boiling water for 12 min, Group C: heated on a medium open grill for 15 min, Group D: fried in oil at 180-200°C for 2 min, and Group E: microwaved for 4 min at full power (500 W maximum output)]. The heat condition in each group was decided according to general cook books [14-16] to achieve over 75°C at the core.
HPLC analysis of carotenoids in cooked and raw salmon
The cooked fillets of each group (Group A-E) were cut into 15- 20 cubes (2.0 cm×2.0 cm×2.0 cm, approximately), and extracted by stirring in 200 ml acetone for 1 hour at room temperature (×3 times). The extracts (600 ml) were added to hexane (600 ml) and H2O (600 ml), and partitioned in a 2 l separating funnel. The upper layer (red) was collected and dehydrated with sodium sulfate anhydrate, and the amount of total keto-carotenoids (astaxanthin, adonixanthin, adonirubin, and canthxanthin) in the upper layer was assessed using an Optical Density (OD) of 470 nm [extinction coefficient (absorbance of 1% concentration) of 2,100 was adopted for quantification] .
Each extract was concentrated to dryness in vacuo to give red oil (3.8-4.5 g/80 g fillet). To analyses the carotenoids contained in each group, the red oil was dissolved in 5 ml hexane : acetone (82:18) solution and 10 μl of the solution was subjected to silica gel High Performance Liquid Chromatography (HPLC) (Cosmosil 5SL-II, 4.6 x 250 mm, Nacalai Tesque Inc., Kyoto, Japan) developed with hexane : acetone (82: 18) at a flow rate of 1 ml/min [using this condition, the standard carotenoids, trans-canthaxanthin (1), 13cis-canthaxanthin (2), trans-adonirubin (3), 13cis-adonirubin (4), trans-astaxanthin (5), 9cis-astaxanthin (6), 13cis-astaxanthin (7), and trans-adonixanthin (8) were eluted at Retention Time (Rt) 11.1 min, 11.8 min, 16.4 min, 18.9 min, 26.0 min, 32.0 min, 33.16 min and 34.7 min, respectively (Figure 1)]. The peaks of 1, 3, 5, 7 and 8 (Group A), and 1, 2, 3, 4, 5, 6, 7 and 8 (Group B-E) were observed in each experiment, and the amount of each carotenoid peak was calculated from the calibration curve of the standard (the average of 4 experiments is shown as the amount of carotenoid in Table 1).
|Total Carotenoid (µg/g)||4.6±0.6||5.5±1.0||4.8±1.0||5.3±1.7||4.7±1.1|
|Compositon (% of total)|
|cis-astaxanthin (6 +7)||4.9±0.4||10.7±1.5**||1.1±2.1**||9.0±0.6**||13.0±1.3**|
Table 1: Carotenoid content and composition in raw and cooked salmon muscle. Values are the means ± SD, n = 4 n.d.: not detected (Group A: raw, B: steaming, C: grilling, D: frying, E: microwaving). Significant difference compared with the group A: *p < 0.000, ** p < 0.05.
HPLC was carried out using a Hitachi L-7100 intelligent pump and L-7400 DAD detector. The maximum absorbance was measured in the range of 200-600 nm.
Data were analyzed using SPSS Statistics 22 software (IBM co.). Means of carotenoid value in each group (A-E) were compared by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (significance level was set at p<0.05). The percentage values shown in Table 1 were transformed using the arcsine transformation.
1H Nuclear Magnetic Resonance (NMR) spectra were measured with a Bruker AVANCE400. Chemical shifts were referenced to tetramethylsilane. Data processing was performed using Top Spin- NMR software (version 3.0) (Bruker BioSpin). High Resolution Electro Spray Ionization Mass Spectrometry (HRESI-MS) was recorded with a JEOL JMS-T100LP mass spectrometer. UV-VIS spectra were recorded with a Hitachi U-3200.
Isolation of carotenoids in cooked salmon for 1O2 quenching activity
To examine 1O2 quenching activities, carotenoids [1-8] were isolated from the fillets treated with microwave heating (group E treatment) which include cis-carotenoids most abundantly. Two salmons was cut into 24 fillets (approximately 80 g/ fillet), and the fillets were microwaved for 4 min, cut to small cubes, and extracted with acetone (2 l). The extracts were concentrated to a small volume (800 ml), and partitioned between hexane-acetone (1:1)/H2O. The upper layer was concentrated to dryness (101.3 g), and applied to silica gel column chromatography [Silica Gel 60 (Kanto Chemicals) i.d. 30×200 mm, solvent: hexane]. The silica gel column was developed with hexane (500 ml), hexane-diethyl ether 1:1 (800 ml), diethyl ether (300 ml), and diethyl ether -acetone 1:1 (300 ml), stepwisely. The diethyl ether (fraction I) and diethyl ether -acetone (1:1) (fraction II) elutes that contained carotenoids were collected separately and concentrated to give red oils (I : 91.4 mg, II: 25.3 mg), respectively.
Fraction I was applied to anion exchange column chromatography (DEAE Toyopeal 650 M, Co. Ltd), [20×200 mm, solvent: hexaneacetone (1:1)] and developed with hexane-acetone (1:1). The eluted red fractions were collected (100 ml) and H2O (100 ml) was added to partition between hexane-acetone/H2O. The upper layer was concentrated to dryness (18.8 mg), then applied to silica gel column chromatography [Silica Gel 60 (Kanto Chemicals) i.d. 30×100 mm, solvent: CH2Cl2-acetone (100:1)] and developed with CH2Cl2-acetone (100:1). The red fraction was collected and concentrated to dryness (10.1 mg), and then subjected to preparative silica gel HPLC using a Cosmosil 5SL-II column (4.6×250 mm) (Nacalai Tesque Co. Ltd.) under the following conditions [flow rate: 1 ml/min, solvent: hexaneacetone (9:1), the maximum absorbance being monitored by DAD detector: 200-600 nm]. The red peaks at Rt 8.3 min (1), Rt 9.0 min (2), Rt 14.9 min (3) and Rt 17.8 min (4) were collected separately and concentrated to dryness to give pure 1 (3.2 mg), 2 (0.4 mg), 3 (0.7 mg), and 4 (0.2 mg), respectively.
Fraction II was subjected to preparative silica gel HPLC using a Cosmosil 5SL-II column (10×250 mm) (Nacalai Tesque Co. Ltd.) under the following conditions [flow rate: 3 mL/min, solvent: hexane-acetone (85:15), the maximum absorbance monitored by DAD detector: 200- 600 nm]. The red peaks at Rt 19.9 min (5), Rt 23.8 min (6), Rt 24.7 min (7) and Rt 25.7 min (8) were collected separately and concentrated to dryness to give pure 5 (5.1 mg), 6 (0.5 mg), 7 (0.2 mg), and 8 (0.1 mg), respectively.
The identities and purities of the isolated compounds 1-8 were checked by HRESI-MS and 1H NMR analysis.
1O2 quenching act
1O2 quenching activity was examined by measuring methylene blue-sensitized photooxidation of linoleic acid . To evaluate the crude carotenoid extracts (the extracts prepared for HPLC analysis) of raw and cooked salmon, 80 μl of 0.025 mM methylene blue in hexaneethanol (1:1), 280 μl of 100 mM linoleic acid in hexane- ethanol (1:1) and 200 μl hexane-ethanol (1:1) with or without 80 μl extract solution [total carotenoids 8 μM, 40 μM, and 80 μM in hexane (final total carotenoids concentration 1 μM, 5 μM and 10 μM)] were added to micro glass vials (5.0 ml). To evaluate the isolated carotenoids, 80 μl of 0.025 mM methylene blue in ethanol and 280 μl of 100 mM linoleic acid in ethanol with or without 80 μl carotenoid [10 μM, 100 μM, and 100 0 μM in ethanol (final concenarion 1 μM, 10 μM and 100 μM)] were added to micro glass vials (5.0 ml). The vials were tightly closed with a screw cap and a septum, and the mixtures were illuminated at 7,000 lux at 22°C for 3 hours in corrugated cardboard. Then, 100 μl of the reaction mixture was removed and diluted to 3.0 ml with ethanol, and absorbance at 235 nm was measured to estimate the formation of conjugates dienes . The value in the absence of carotenoid was determined and 1O2 quenching activity was calculated relative to this reference value. Activity is indicated as the IC50 (μM) representing the concentration at which 50% inhibition was observed. The IC50 value was calculated by averaging the data from triplicate experiments.
Carotenoid composition in raw and cooked salmon
The results of carotenoids analysis (total amount and composition of each carotenoid) of raw and 4 cooked (steamed, grilled, fryied and microwaved) salmon are listed in Table 1. Although no significant differences were observed in the total carotenoids among Group A-E, cis-carotenoids (2, 4, 6+7) in salmon were significantly increased by heat cooking (Group B-E) (p<0.05). This observation indicates the conversion of the trans-carotenoid to the corresponding cis-isomers. The cis-isomer of 8 was inferred to have been produced, but it was not detected due to low productivity. Although the production of cis- isomers from trans-carotenoids on cooking of vegetables (tomato and sweet potato leaves) has been reported in some previous reports [20,21], this is the first report on the detailed changes of keto carotenoids (astaxanthin, adonirubin, adonixanthin and canthaxanthin) contained in fish and shellfish (Figure 1).
In this study, the heating time for group B-E was determined based on cookbooks [14-16] to examine the composition of carotenoids contained in cooked Atlantic salmon. The core temperature transition in salmon fillets by each heating method is shown in Figure 2, and the ratio of cis-isomers/total carotenoid (trans+cis) in each cooking treatment is shown as a bar graph in Figure 3. The ratio of each cis-isomer (2, 4, 6 and 7) was Group D (19-21%)<Group B and C (21-25%)<Group E (28-32%). The ratios of all cis-isomers in group E was significantly higher than some other groups (p<0.05) (Figure 3). Since the core temperatures by microwave cooking (88-94°C) were higher than those by steaming, open fire cooking, and frying (78-82°C) (Figure 2), these results are roughly explainable from their temperatures. Although the core temperatures of Group B, C and D were almost the same, the amount of cis-carotenoids was less in Group D than in Group B and C (Figure 2) (There were no significant differences (p>0.05) between these groups). This observation may be explainable by the shorter heating time in Group D.
The ratios of cis-isomers in each heat cooking treatment were astaxanthin (14-21%) <adonirubin (19-27%) <canthaxanthin (21- 32%) (Figure 3). Since it has been reported that the β-ionone ring of carotenoids in salmon muscle was combined with hydrophobic pockets of protein (such as actomyosin) and the presence of carbonyl (C=O) and hydroxyl (OH) groups enhance this combination , astaxanthin may be more stable than canthaxanthin in salmon.
In this study, the total carotenoid amounts (cis + trans) in Group B–E was the same as in Group A, indicating no chemical reactions except the isomerization from trans to cis-isomers. Since the decrease of total carotenoids (cis + tans) by excessive heating was observed in previous studies on vegetables [20,21], we suppose that the same decrease may also occur in Atlantic salmon if the heating time is increased.
Singlet oxygen quenching activity of carotenoids in raw and cooked salmon muscle
First, we examined the 1O2 quenching activity on crude carotenoid extracts (organic layer) of raw and cooked salmon. Their IC50 values were calculated for 6.1 μM (Group A), 4.9 μM (Group B), 3.2 μM (Group C), 3.6 μM (Group D) and 2.6 μM (Group E). These observations indicated that cooking hardly influenced the 1O2 quenching activity of carotenoids when the total carotenoid amounts were not decreased.
We also examined the 1O2 quenching activities of the purified carotenoids 1-8 (purified from Group E extract). The IC50 values were 2.7 μM (1), 7.4 μM (2), 3.2 μM (3), 2.4 μM (4), 5.4 μM (5), 3.5 μM (6), 2.4 μM (7) and 2.5 μM (8), respectively. As far as we know, this is the first report concerning 1O2 quenching activities of cis-isomers 2, 4, 6, and 7. The trans carotenoid and the corresponding cis-isomer (1 vs. 2, 3 vs. 4, and. 5 vs. 6 and 7) showed almost the same 1O2 quenching activities. These results support the equal 1O2 quenching activity between raw and cooked salmon extracts. Shimidzu et al. reported that increasing the number of conjugated double bonds (C=C) and the presence of carbonyl (C=O) or hydroxyl (OH) groups enhanced the 1O2 quenching activity , but cis-isomerazation might not have affected 1O2 quenching activity in our study.
Liu and Osawa reported that cis-astaxanthin shows more potent antioxidative activity than all-trans isomer in a DPPH radical scavenging assay and a lipid peroxidation assay using rat microsomes . Clinton et al. described that more cis-carotenoids (such as lycopene) are taken up by the animal body than trans-isomer . Further biological evaluations of the carotenoids in cooked Atlantic salmon considering these points are in progress.
We thank Ms. Yui Kaseya and Ms. Nao Koue for their assistance in experiments. This work was supported by a Grant-in-Aid for Young Scientists (B) No. 23700888 from MEXT Japan.