Food Science and Technology Research, 24 (6), 991_997, 2018Copyright © 2018, Japanese Society for Food Science and Technologydoi: 10.3136/fstr.24.991

http://www.jsfst.or.jp

Original paper

Effects of Different Preparation Conditions on Fish Oil Oxidation in Heat-induced Emulsified Surimi Gels

Yuanpei Gao1, Hideto Fukushima

2, Kazuya shirota3, Yusuke kakizaki

3, Shanggui DenG4, Naho nakazawa

1, Kazufumi osako

1 and Emiko okazaki1*

1Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan

2Department of Marine Science and Resources, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan

3Maruha Nichiro Corporation, 16-2 Wadai, Tsukuba 300-4295, Japan4Department of Food Science and Technology, Zhanjiang Ocean University, 316022, Zhoushan, China

Received May 2, 2018 ; Accepted August 26, 2018

In this study, the effects of different preparation conditions on the oxidative stability of fish oil in surimi gels were investigated. Emulsified surimi gels were prepared under different conditions (mixing speed, protein concentration, and environmental conditions) to measure the oxidative degree of fish oil during storage. The oxidation of fish oil in each type of surimi gels gradually increased within the storage period. On one hand, oxidation was suppressed when the oil particle size became smaller; lipid oxidation was also suppressed under vacuum + air as well as under vacuum. On the other hand, oil particle size decreased in the surimi gels as protein concentration increased, and oxidative stability was significantly enhanced. These results indicate that the levels of emulsification affect the oxidation level of oil in surimi gel and that complete emulsification protects fish oil from oxidation.

Keywords: fish oil, surimi gel, particle size, oxidation, storage

*To whom correspondence should be addressed. E-mail: eokazaki@kaiyodai.ac.jp

IntroductionRecently, there has been increasing interest in the

incorporation of polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in foods because of their many benefits in promoting health. The benefits of fish oils can be largely attributed to the high PUFA content (Kris Etherton, 2002; Nair et al., 1997). Absorption of DHA and EPA reduces the risk of hypertension and Alzheimer’s disease (Tully et al., 2003), and prevents certain cardiac arrhythmias (Garg et al., 2006). Moreover, a previous study confirmed the benefits of EPA and DHA supplementation during pregnancy for fetal brain and retina development (Ramakrishnan et al., 2010). As noted above, EPA and DHA are among the few functional ingredients that have been clinically confirmed to be effective for human

health. To enhance the functionality and economic value of foods, researchers have successfully fortified many types of foods, such as bread, ice cream, and milk, with fish oil (Kolanowski and Berger, 1999; Newton and Snyder, 1997). Moreover, some of these functional foods have recently been marketed, and further applications of fish oil in the food industry are expected.

Surimi is an intermediate product used in the preparation of a variety of ready-to-eat seafood, which is a mechanically deboned, washed (bleached) and stabilized fish meat (Okazaki and Kimura, 2013) containing myofibrillar (Mf) protein as the main protein. Because of the spread of Japanese restaurants and culinary traditions in North America, Europe and elsewhere, surimi-based foods are widely accepted and enjoyed worldwide. Recently, surimi-based foods enriched with EPA

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and DHA have demonstrated a potentially wide applicability.Because of the high degree of unsaturated fatty acids, fish

oil is unstable and is rapidly oxidized during processing and storage to form toxic products, which limit its palatability and functional quality (Guillén and Cabo, 2002). Preventing oil oxidation in the food is thus an important task to produce better products. In previous studies, Miyashita et al. (1995) clarified that the emulsified fish oil has a higher oxidative stability than the bulk oil and Nakaya et al. (2005) reported that the oxidative degree is affected by oil particle size in an O/W emulsion. In the emulsified surimi gel, surimi protein (myofibrillar protein) contributes not only gelation properties, but also emulsification properties. Myofibrillar protein attaches to the surface of oil particles to form a membrane to stabilize the oil in the emulsified surimi gel (Gordon et al., 1992; Xiong 2007). To prevent oxidation of lipids in foods such as sausage, an effective method is isolation of oxygen or air. However, air is usually added to fish sausage during processing to obtain a better texture, despite decreasing shelf life. Several manuscripts have reported a relationship between oil size or protein concentration and the texture of emulsified surimi gel (Okazaki et al., 2006; Okazaki et al., 2002). However, lipid oxidation in emulsified surimi gels prepared by the above conditions has not been clarified. Although, DHA-enriched fish sausage was recently made available in the market (Park, 2013), the oxidative stability of omega-3 fatty acids in surimi gel has not been fully clarified. The objective of this study was to clarify the effects of different preparation conditions related to several factors, such as oil particle size, protein concentration, and environmental condition, on oil oxidation in the emulsified surimi gel.

Materials and Methods(1) Materials Frozen surimi: The Alaska pollock

(Theragra chalcogramma) surimi (FA grade) used in this study was produced by Pacific Seafoods Corp. (Portland, OR). The surimi was cut into small blocks (about 500 g), vacuum packed, and stored at ‒30 ℃ until use. Surimi contained 75.5 % moisture, 17.0 % crude protein, 0.4 % crude lipid and 7.1 % other constituents.

Fish oil: Refined fish oil, DHA-22K, was prepared from tuna and bonito by Maruha Nichiro Corp. (Tokyo, Japan) and was stored at ‒30 ℃ with nitrogen gas until use. The fish oil contained more than 99 % triglyceride, and 0.6 % tocopherol as an antioxidant. DHA and EPA contents were 26.7 % and 6.7 %,

respectively. PV and TBARS values of fish oil are 2 meq/kg oil and 30 mg MDA/kg oil, respectively.

(2) Gels preparation Formulation of the emulsified surimi paste is shown in Table 1. Frozen surimi was thawed and chopped in a universal food processor (Model UMC 5E; Stephan Machinery, Hameln, Germany), which was connected with a vacuum pump (160VP; Hitachi Ltd., Tokyo, Japan) and circulation cooling machine (TBG120SB; Toyo Seisakusho Kaisha Ltd., Tokyo, Japan), at 1,500 rpm for 1 min. Surimi paste was obtained after mixing with cold IEW and 2 % NaCl (w/w) under vacuum and 8 % (w/w) fish oil was added into surimi paste. The different mixing and protein conditions used were as follows:

(a) Different mixing speeds: High and low mixing speeds were used for surimi paste with 12 % protein concentration. High-speed mixing was performed using a stepwise increasing speed from 300 rpm to 3,000 rpm (300 rpm for 60 s; 600 rpm, 900 rpm, 1,200 rpm, 1,500 rpm for 30 s; 1,800 rpm, 2,100 rpm for 15 s; and 2,400 rpm, 2,700 rpm and 3,000 rpm for 10 s) for twice repeats (Okazaki, et al., 2002). Low-speed mixing was performed at a constant speed of 300 rpm for 4 min for twice repeats.

(b) Different protein concentrations: Different amounts of IEW were added to surimi to adjust the protein concentration to 10 %, 12 % and 14 %, NaCl was added, and then fish oil was added, followed by mixing twice at high-speed.

Emulsification of fish oil into salted surimi paste with different mixing speeds and protein concentrations was performed in a vacuum (below 50 KPa) (repeated twice), mixing with air (twice), and mixing in vacuum (once) followed with air (once) (vacuum+air).

All steps were performed at temperatures below 10 ℃. Emulsified surimi paste was packed into polyvinylidene chloride casing tubes with a diameter of 23 mm, followed by heating in a retort machine (120 ℃, 7 min). Final products were stored at 25 ℃, and oxidative parameters were measured every 10 days.

(3) Microscopic observation and oil particle size analysis The paraffin embedding method was used to perform microscopic observation of the surimi gel with fish oil. Pieces ( thickness, 2 mm) of surimi gels were f ixed in 4 % paraformaldehyde phosphate buffer solution overnight, and then fixed in 2 % osmium tetroxide (OsO4) for more than 24 h (Lillie, 1947). Samples were embedded in paraffin for 24 h and 2-µm slices were prepared, and then the slices were observed

Table 1 . Formulation of the emulsified surimi paste (g).

Protein Concentration Surimi IEW NaCl Fish oil Total

10% 580 320 20 80 1,00012% 700 200 20 80 1,00014% 820 80 20 80 1,000

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using a microscope (BZ-9000 Generation-2; Keyence Corporation, Osaka, Japan) after dewaxing and staining. Oil particle size was calculated using the software WinRoof (Mitani Corporation, Japan).

(4) Lipid extraction A single solvent extraction method described by Folch et al. (1957) was used for lipid extraction. Approximately 15 g of surimi gel samples was placed into a homogenizer cup; 50 mL of a chloroform-methanol mixture (2:1) was added, followed by homogenization at 10,000 rpm for 1 min (PT10-35GT; Kinematica Ltd., Tokyo, Japan). The mixture was filtered using a 300-mL separating funnel, and 20 mL of saturated NaCl solution was added to separate the filtrate into 2 layers. The chloroform layer was collected and dried with drops of sodium sulfate overnight. Chloroform was allowed to evaporate in a water bath at 35 ℃.

(5) Determination of peroxide value The peroxide value of lipids was determined according to the method of Takagi et al. (1978). First, 5‒50 mg of lipid was mixed with 5 mL of chloroform and 10 mL of acetic acid, followed by the addition of 1 mL of 50 % (w/v) potassium iodide solution. The reaction mixture was allowed to stand in the dark for 5 min after being isolated from the air with nitrogen, 9 mL of 2 % (w/v) cadmium acetate solution was added to stop the reaction, and the mixture was placed in a dark place until the 2 phases were clearly separated. Absorbance of the supernatant was measured at 410 nm using a spectrophotometer (Model UV-1800; Shimadzu Corp., Japan). PV was calculated using the following equation:

PV (meq/kg) =((A-B) ×60.14+0.69) /(8w)×1000 ······Eq. 1

where, A and B represent the absorbance values of the sample and blank, respectively, and w represents the sample weight (mg).

(6) Determination of thiobarbituric acid reactive substances (TBARS) Thiobarbituric acid reactive substances (TBARS) values of fish oil were determined using the method of Weng et al. (2009). A stock solution of 15 % TCA - 0.375 % TBA - 0.1 M HCl was slowly heated to 75 ℃ in a water bath to facilitate the dissolution of thiobarbituric acid (TBA). Then, 5 mL of the prepared solution mixed with 5–50 mg fish oil sample was heated for 10 min in a boiling water bath to obtain a pink color. After cooling with tap water and centrifugation at 6,000 × g for 10 min, absorbance of the supernatant was determined at 532 nm. 1,1,3,3-Tetramethoxypropane was used to prepare the MDA standard curve.

(7) Fatty acid composition Fatty acid analysis was performed according to the method of Osako et al. (2003). Crude lipids were converted into methyl esters directly with methanol containing a catalyst amount of concentrated HCl under reflux for 3 h at 130 ℃. The obtained methyl esters were separated from other accessory substances by column c h r o m a t o g r a p h y u s i n g s i l i c a g e l ( S i l i c a g e l 6 0 , 0.063‒0.200 mm; Merck, Darmstadt, Germany) and elution with dichloromethane. Fatty acids methyl esters were analyzed

on a gas chromatograph (GC-2014; Shimadzu Seisakusho Corporation, Kyoto, Japan) equipped with a capillary column (Omegawax-250, 30 m×0.25 mm i.d., film thickness, 0.25 µm; split ratio, 10:1; Supelco Japan Co., Ltd., Tokyo, Japan). Temperatures of the injector, column, and detector were held at 250 ℃, 205 ℃, and 260 ℃, respectively. Helium was used as the carrier gas and inlet rate was kept constant at 7.3 mL/min. Acquired data were quantified using the GC-Solution software (Shimadzu Seisakusho Corporation, Kyoto, Japan).

(8) Statistical analysis Data are expressed as means ± standard deviation (4 replications). Differences between variables were evaluated using Tukey-HSD test. Analysis was performed using SPSS Statistics software (SPSS Inc., Chicago, IL).

Results and Discussion(1) Microscopic observation Figure 1 shows the

microscopic observations of emulsified surimi gels under different conditions, where the black area represents oil particles. Oil particles are clearly observed in the figure and had a regular shape. However, the particle size was different in the gels under different preparation conditions. Also, the changes in oil particles in the emulsified surimi paste under different conditions were similar to those in the heat-induced gels. Okazaki et al. (2006) also reported that oil particles kept the same shape in both surimi paste and heat-induced gel. The effects of mixing speed and protein concentration on fish oil particle size are shown in Fig. 1. When the mixing speed was increased from 300 rpm to 3,000 rpm, the oil particle size decreased from 4.27 ± 3.08 µm (Fig. 1 B) to 2.22 ± 1.68 µm (Fig. 1 A), as a higher mixing speed resulted in a larger shear force. This result was similar to that reported by Okazaki, who noted that oil particle size decreased in surimi gel with an increase in mixing speed and time (Okazaki, et al., 2002). Increases in the protein concentration of the surimi paste increased the viscosity, which also leads to a larger shear force. Therefore, the diameter of oil particles in the surimi gel with protein concentrations of 10 %, 12 %, and 14 % was 3.39 ± 1.29 µm (Fig. 1 C), 2.22 ± 1.68 µm (Fig. 1 A), and 0.99 ± 0.42 µm (Fig. 1 D), respectively. After 30 days of storage, oil particle size increased in all samples with different protein concentrations, and the percent increase in oil particle size in the samples with protein concentrations of 10 %, 12 %, and 14 % was 14.12 %, 10.43 %, and 8.14 %, respectively. This suggests that the more compact the network formed in the surimi gels with higher protein concentrations and lower moisture contents, the more stable emulsion system was established to inhibit the combination of fish oils during storage. On the other hand, the changes in viscosity of the surimi with protein content are also relevant to this phenomenon.

(2) Oxidative degree of fish oil in surimi gels Most available methods for evaluating oil oxidation can be classified

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based on measurement of primary and secondary changes of oxidation. The primary oxidative products can be monitored using PV (Gray, 1978; Warner, 1995). The secondary changes are generally measured by TBARS test (Frankel, 1993; Warner, 1995).

In the present study, the oxidative degree of fish oils in the emulsified surimi gels prepared under several different conditions during storage was investigated. Figure 2 (A) shows the changes in PV of the oil in the surimi gels prepared by different mixing conditions during 30 days of storage. With increasing storage period, the PV of oil in each type of surimi gel increased gradually.

In the case of air condition, the PV of oil with high-speed mixing (small oil particle) was slightly higher than that with low-speed mixing (large oil particle) at 0-day storage because the oxygen or air content in the gel and the surface area of oil particles with high-speed mixing was higher and larger than those with low-speed mixing, respectively. These two factors accelerated oil oxidation during processing. With increasing storage period, the PV of oil with small oil particle size decreased when compared with large oil particle size in the surimi gels.

Even though the PV of oil under vacuum conditions was significantly lower than that under air conditions, the change in PV under vacuum conditions was similar to that under air conditions. At the beginning of the storage period, the PV of oil with high speed mixing was slightly but not significantly higher than that under low speed mixing. It is possible that oil oxidation was accelerated by small amounts of oxygen in surimi or water, which could not be fully removed under

vacuum conditions. In the present study, the PV of oil in surimi gels with small

oil particle size was lower than that with large oil particle size under both vacuum and air conditions. Some studies have reported that oxygen and oil are able to react efficiently when the oil size was small or the oil sample has a high ratio of surface to volume (Tan et al., 2002). However, Gohtani et al. (1999) and Nakaya et al. (2005) revealed that oils with a smaller particle size had a higher oxidative stability in the oil-in-water emulsion system prepared with the purified triglyceride, as the emulsifier molecules located at the interface between the oil and water phases may influence the mobility of the oil molecules existing in the oil phase and may result in the improvement of the oxidative stability of oil. In summary, we hypothesized that the mechanism of inhibited effects of oil oxidation with small oil particle in the emulsified surimi gel was similar to the above studies using the o/w emulsion.

Figure 2 (B) shows the changes in TBARS in the fish oil emulsified in the surimi gels. From the figure, the TBARS value of oil under air conditions was significantly higher than that under vacuum conditions; however, significant changes were not observed between high and low speed mixing. During storage, PV, which indicates the primary changes in oxidation, is more sensitive than TBARS, which indicates the secondary changes in oxidation. Therefore, it was suspected that the changes in the TBARS were different from PV with different oil particle size.

Figure 3 shows the oxidative degree of fish oil in the surimi gels with different protein concentrations (10, 12, and 14 %). Although PV and TBARS in the fish oil increased during the storage period in the 3 types of surimi gel, the oxidation of fish oil was significantly inhibited in surimi gels with high protein concentration when compared with those with low protein concentration. The fish oil particle size (0.99 ± 0.42 µm) in the gels with 14 % protein concentration, which was smaller than those (3.39±1.29 µm) in the gels with 10 % protein concentration, had more oxidative stability. On the other hand, increases in protein concentration in the surimi gel would increase the gel strength (Okazaki, et al., 2006), and Tolasa et al. (2010) also suggested that lipids could maintain oxidative stability in surimi gel when the protein matrix was stable and tight. Gao et al. (2018) reported that myofibrillar protein will attach to oil particles in order to stabilize fish oil in the surimi gel. Therefore, it was suspected that oil in the surimi gels with higher concentrations of proteins could form both smaller oil particle sizes and stronger emulsion structures with the protein network, which closely integrates with the oil particle surface to prevent oxidation.

In Fig. 3, the oil oxidation in the surimi gel prepared under different environmental conditions is shown. As noted previously, food companies usually produce surimi sausage under air conditions rather than in a vacuum in order to improve the product texture, as air bubbles in the product

Fig. 1. Microscopic observation of the emulsified surimi gel. A: Surimi gel prepared using high-speed mixing (12 % protein concentration); B: Surimi gel prepared using low-speed mixing (12 % protein concentration); C: Surimi gel prepared with 10 % protein concentration (high-speed mixing); D: surimi gel prepared with 14 % protein concentration (High-speed mixing). Numbers in the figure are oil particle size.

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contribute the soft texture. Based on the present results, however, the air may accelerate oil oxidation in the emulsified surimi gel. Hence, other types of emulsified surimi gel were prepared under “vacuum + air” (mixed in vacuum followed by air) to determine the oxidative stability of fish oil. The changes in PV in the emulsified surimi gels achieved under “vacuum + air” were significantly lower than those achieved under “air” alone during the storage period, and the oxidative stability was similar to that under vacuum conditions. At the beginning of storage, the changes in TBARS of oil under different conditions

were smal ler than PV. In the case of 14 % protein concentration, TBARS values under each condition had no significant changes. The possible reason was the slow generation of MDA, as previously noted. The results suggested that oil mixed with surimi under “vacuum” conditions could form a stable emulsion, which protects it from oxidation.

(3) Fatty acid composition Table 2 and Table 3 show the changes in fatty acid composition of the fish oil emulsified in surimi gels prepared under different mixing speeds and protein concentrations, respectively. In fish oil, the main poly-

Fig. 2. Oxidative degree of fish oil in surimi gels that prepared using different mixing speeds. A and B show the changes in PV and TBARS of fish oil in the surimi gel during storage, respectively. H and L represent the high-speed mixing and low-speed mixing, respectively. V and A represent the vacuum condition and air condition, respectively. Different letters indicate significant differences (P < 0.05).

Fig. 3. Oxidative degree of fish oil in surimi gels using prepared by different protein concentrations. A, B, and C represent the changes in peroxide value of fish oil emulsified in surimi gel with protein concentration of 10 %, 12 %, and 14 %, respectively. D, E and F represent the changes in TBARS of fish oil emulsified in surimi gel with protein concentration of 10 %, 12 %, and 14 %, respectively. Different letters indicate significant differences (P < 0.05).

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unsaturated fatty acids are EPA and DHA. Based on Table 2, the content of EPA in each sample decreased significantly after 30 days of storage. However, no significant ( p > 0.05) changes were observed among the samples prepared under vacuum and vacuum + air, and the content of EPA under air conditions was significantly ( p < 0.05) lower than under the other conditions after 30 days of storage. DHA content decreased significantly after storage only in gels under low-speed mixing with air. Based on Table 3, the gels prepared under vacuum and vacuum+air showed similar changes in EPA and DHA. After storage, EPA and DHA were significantly lower in gels with lower protein concentrations.

Miyashita et al. (1993) reported that DHA and EPA would have adopted a more tightly packed conformation in an aqueous solution, and it might have been difficult for free radicals and/or oxygen to attack the substrates in emulsions. In this study, fish oil was emulsified in the surimi, and the emulsion state was formed with surimi protein under the conditions of vacuum mixing and higher protein concentration, and then oxidation was inhibited. The results showed that changes in saturated fatty acids and mono-unsaturated fatty acids were lower than those in unsaturated fatty acids after 30 days of storage. However, the total content of unsaturated fatty acids was decreased as oxidation progressed during storage.

ConclusionsAddition of n-3 PUFA-rich oils is required for the

development of functionality enhanced surimi-based seafood products. Lipid oxidation in surimi gels under different preparation conditions was demonstrated in the present study. On the one hand, oils with smaller particle size in surimi gel, which were prepared under high-speed mixing or high protein concentration, had better oxidative stability; on the other hand, oxidation of oils could be efficiently prevented when oil was emulsified with surimi under vacuum followed by mixing under air. This approach allows the addition of functional fish oil in surimi with high oxidative stability to enhance the commercial value and produce better surimi-based products containing high amounts of oil.

Acknowledgements This study was supported by the project “A Scheme to Revitalize Agriculture and Fisheries in Disaster Area through Deploying Highly Advanced Technology” of the Ministry of Agriculture, Forestry and Fisheries, Japan.

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Table 3 . Changes in the fatty acid composition of fish oil emulsified in surimi gels prepared with different mixing speed after 30 days of storage (%).

Before High-Speed Low-Speed

Storage Vacuum Air Vacuum+Air Vacuum Air Vacuum+Air

EPA 6 .7±0 .1 6 .3±0 .0 6 .1±0 .1 6 .3±0 .1 5 .7±0 .0 5 .6±0 .2 5 .9±0 .3DHA 26 .7±1 .3 26 .9±0 .4 26 .6±0 .8 26 .5±1 .1 26 .3±0 .3 25 .9±0 .1 26 .4±0 .0SFA 34 .7±0 .5 34 .3±0 .8 36 .1±1 .3 35 .6±1 .1 36 .2±1 .3 36 .5±0 .6 36 .0±0 .5

MUFA 24 .4±0 .7 25 .0±1 .0 24 .0±0 .9 23 .5±0 .9 24 .3±0 .5 24 .8±0 .6 23 .9±0 .7PUFA 40 .9±0 .2 40 .6±0 .5 38 .9±0 .4 40 .8±0 .8 39 .1±0 .2 37 .6±0 .6 38 .5±0 .4

SFA: Saturated fatty acid. MUFA: Mono-unsaturated fatty acid. PUFA: Poly-unsaturated fatty acidVacuum: surimi gel prepared in the vacuum condition. Air: surimi gel prepared in the air condition. Vacuum+Air: surimi gel prepared in the vacuum condition followed in the air condition

Table 2 . Changes in the fatty acid composition of fish oil emulsified in surimi gels prepared with different mixing speed after 30 days of storage (%).

Before 10% 14%

Storage Vacuum Air Vacuum+Air Vacuum Air Vacuum+Air

EPA 6 .7±0 .1 5 .9±0 .1 5 .4±0 .2 5 .8±0 .2 6 .3±0 .1 6 .1±0 .2 6 .2±0 .3DHA 26 .7±1 .3 25 .1±0 .3 24 .6±0 .2 24 .9±0 .1 26 .3±0 .9 26 .3±0 .1 26 .5±0 .5SFA 34 .7±0 .5 36 .5±1 .6 36 .9±0 .3 36 .6±0 .3 34 .9±0 .9 34 .5±0 .6 35 .0±0 .7

MUFA 24 .4±0 .7 24 .9±0 .7 24 .7±0 .6 24 .5±0 .2 25 .2±0 .8 24 .9±0 .6 24 .9±0 .7PUFA 40 .9±0 .2 38 .5±0 .4 36 .4±0 .7 37 .6±0 .4 39 .9±1 .1 38 .6±0 .4 39 .2±0 .3

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