Antioxidant effects of green tea extracts on lipid oxidation of half-dried eels

In order to explore the change of lipid oxidation of half-dried eels treated with green tea extracts and stored frozen, acid values, peroxide values, carbonyl values, and TBA values were measured. The eels were placed into the plastic bag and soaked for 1 hr in the solution mixed with distilled water and green tea extracts, and also vacuumed and stored frozen at –18 ∼ –20°C for 9 months after being dried with a hot-air blower for 15 hrs at 35°C (Aw 90-91). The control consisted of eels prepared in the same way without any pretreatment. At the end of the 1st month, 3rd month, 6th month, and 9th month, eel oil obtained from the samples were tested for its lipid oxidation. The activity of green tea extracts 1 mL was very similar to Vitamin C 500 μM 0.8 mL. The acid values, peroxide values, carbonyl values, and TBA values of eels treated with green tea extracts were lower than those of the control during 9 months of frozen storage (p<0.05). While the acid values and peroxide values of the control and eels treated with green tea extracts highly increased after the first month of frozen storage, the TBA values increased greatly on the third month of frozen storage. Compared to the control, the pre-application of green tea extracts to half-dried eels meaningfully prevented the generation of TBA compound during the frozen storage (p<0.01). In conclusion, pre-application of green tea extracts was effective in delaying early-stage peracid inducement and preventing the generation of secondary oxidation compounds, such as carbonyl compound and TBA compound, in the course of the drying and frozen storage.


Introduction
The concerns on heart-related diseases and cancers sparked interest in this research and relates it to the consumption of a healthy diet, including w-3 fatty acid, found in fishes. However, unsaturated fat in food downgrades the food quality due to oxidation by oxygen, moisture, heat, etc. (Cho et al., 2011). It is also related to the difficulties of processing, storing, and distribution of fish with high unsaturated fat because of their easy rancidity. In order to resolve the problems of easy rancidity, research on peroxide prevention has been undertaken in various fish, such as half-dried eel , salted mackerel (Nam et al., 2011), anchovy oil (Kang et al., 2007), saury (Cook, 1995), seasoned squid (Yang et al., 1999), white fish meat (Lee et al., 1997), and shellfish (Cho et al. 1998). Eel, one of Korea's most favourite health foods in summer, has high unsaturated fatty acid. Thanks to the increase of eel supplies based on recent developments in farming skills, not only has the annual consumption consequently increased, but HMR products containing eels have also appeared in the market. Therefore, it is essential to research how to prevent eel's peroxide activity to improve storage and distribution capabilities . Green tea has been reported in many works of literature for its high antioxidant effects based on its polyphenol compound having the antioxidant capacity (Yang et al., 1999;Son et al., 2005;Kang et al., 2007;Nam et al., 2011;Ryu et al., 2017). This research aimed to explore the antioxidant effects and storage stability of green tea extracts in regard to half-dried eel's peroxide when they were pre-applied.

Samples
Eels (Anguilla bicolor pacifica) used in the experiment were cultivated in the city of Naju, weighing around 250-300 g, eviscerated, and cleaned before use. The green tea used for extracting solutions to be preapplied to eels for the purpose of preventing eel's eISSN: 2550-2166 © 2021 The Authors. Published by Rynnye Lyan Resources FULL PAPER peroxide was dried green tea that was easily accessible in the market (Green tea, Damian, Korea). Approximately, 30 g of green tea was added to distilled water and then extracted for 6 hrs at 112°C using a pressured double boiler function in an electronic boiling pot (OC-2300R, OCOO, co., Ltd., Korea). Once the solution was obtained, it was filtered twice using gauze and filter paper (No.1, Whatman, UK), respectively.

Pre-treatment of eels and eel oil extraction
The eels were placed into the plastic bag and soaked for 1 hr in the solution mixed with distilled water and green tea extracts at a ratio of 3:2. Once treated, eels were cleaned with flowing water and were dried with paper towels. They were also dried with a hot-air blower for 15 hrs at 35°C to the extent that the water activity was maintained at 90~91 using HP23-AW water activity analyzer (Rotronic, Switzerland). Half-dried eels were cut into sizes that weighed about a 100 g and stored after being vacuumed in the freezer until the oil extracted. At the end of the 1st, 3rd, 6th, and 9th month, the antioxidant effects of eel oil obtained after 1 hr of defrosting were examined in terms of acid value and peroxide value.
Eel oil was extracted using the method specified in Folch et al. (1957). Chopped eel weighing 60 g were mixed with 300 mL of a blended solution of chloroform and methanol (2:1, v/v), then extracted using a homogenizer (SMG-G, Shinsang, Co., Ltd., Korea) and filtered using a filter paper (No.1). Another 250 mL of the blended solution of chloroform and methanol was added to the residue, then extracted using a homogenizer, which constitutes the second time of filtration. All obtained solutions were poured into a separate funnel, mixed with distilled water and left for 15 to 20 hrs. Next, the layer of chloroform was separated, dehydrated by Na 2 SO 4 and filtered. Eel oil was obtained by concentrating all filtered solutions under reduced pressure using a rotary evaporator (Rotavapor R-215, Büchi, Germany) at 40°C (Song 2019).

Acid value
Eel oil collected in 1 to 2 g using a method specified in Korean food code was placed into a flask, and 100 mL mixed solution of methanol and ether (2:1) was then added. With 1-2 drops of phenolphthalein indicator injected, pink colour was chosen as the determination of endpoint through titration by 0.1 N KOH (Kim, Lee, Ma et al., 2015; Ministry of Food and Drug Safety, 2017).

Peroxide value
After 1 to 2 g of lipid was recovered using a method specified in Korean food code, 25 mL of a mixed solution of acetic acid and chloroform (3:2, v/v) was added. On top of this, 1 mL of KI saturated solution was also added. Then, it was left in a dark room for 10 mins after being shaken for 1 min. With 30 mL of distilled water and starch indicator solution injected, the endpoint through titration by 0.01 N Na2S2O3 was observed when the solution turned colourless. (Ministry of Food and Drug Safety, 2017; Song, 2019).

Carbonyl value
Eel oil of 0.05 g was placed into a 100 mL glass bottle with a cap, along with benzene 5 mL, 0.05% 2,4-DNPH (dinitrophenyl hydrazine) benzene 5 mL, and 4.3% trichloroacetic acid 3 mL that was injected into the bottle. Then, the mixture was warmed up in a double boiler of 60°C water bath for 30 mins. After cooling it off at room temperature and popping the colour with 10 mL of 4% KOH-ethanol, the absorbance was measured at 440 nm (Choi et al., 2006;Song, 2018).

The measurement of TBA value
Mixing and sonicating 200 mg of TBA with 100 mL of 95% butanol at 60°C was done in a sonicator (Ultrasonic, JAC 4020, KODO, Korea) for 30 mins using a method specified in Korean food code. TBA (thiobarbituric acid) reagent was taken and mixed directly with glacial acetic acid at the ratio of 1:1. After the solution was cooled to room temperature the TBA value was obtained through the absorbance measurement at 530 nm, with a blend of 0.05 g eel oil, 10 mL benzene, and 10 mL of TBA reagent, which was kept for 2 hrs at 95°C in a water bath (Ministry of Food and Drug Safety, 2017; Song, 2018).

Statistical analysis
Comparative analysis among experimental groups was performed using IBM SPSS Statistics 20. After ANOVA was undertaken, Duncan's multiple range test was also performed with a 5% confidence level (α = 0.05).

DPPH scavenging effect of green tea extracts
In order to measure the DPPH radical scavenging effects of green tea extract, a Vitamin C 10 mM solution was used as the positive control (Table 1). The correlation coefficient between concentration and absorbance was more than 0.9 (r>0.9). The effects of green-tea extracts in DPPH radical scavenging were found to be dose-dependent. The activity of 1mL green tea extract yielded similar activity to 0.8 mL of Vitamin C 500 μM. The activity of Vitamin C 500 μM, the control group, was found to be 10 times higher when in 1 mL than in 0.1 mL, but green tea extracts showed about 2% difference when compared between 0.1 mL and 1 mL. Therefore, green tea extracts were not only found to be effective in terms of antioxidant effects in undiluted solutions but also diluted to some degree. Song (2018) reported that the activity of green tea extracts under 0.1 mL was similar to that of Vitamin C.

Acid value of half-dried eel oil
The acid value is one of the measures taken to analyse lipid rancidity by measuring free fatty acid values (Cho et al., 2011). While the unfrozen and halfdried eel's acid value was 2.6 mg KOH/g, the green tea extract-treated eel's acid value was 2.4 mg KOH/g. (Table 2).
The difference in acid value is believed to be inconsequential and to come from a half-day drying process prior to frozen storage through the effective antioxidant effects of green tea extract treatment applied during the drying process. According to Song (2019) and Song and Kim (2018), there were antioxidant effects during the drying process if ethanol extracts from plants were applied to eels prior to the drying. Mackerel, which was widely known for ease of lipid rancidity due to high unsaturated fatty acid such as eels, was reported to have an acid value of 2.3 mg KOH/g when pre-treated with green tea extract in the pre-refrigeration stages (Nam et al., 2011). Overall, the acid value of half-dried eels pretreated with green tea extracts was lower than half-dried eels in the control group throughout the nine months frozen in storage (p<0.05). The acid value rose steeply after the first month of storage, while the acid value of half-dried eels pre-treated with green tea extracts increased 2.5 times after the first month, and the control increased by 4 times during the same period (p<0.01). When eels pre-treated with ethanol extracts from plants, such as Hutgae, ginger, onion, green tea, etc., were refrigerated, their low acid values were reported to inhibit the increase of their free fatty acid values (Song and Kim, 2018;Song, 2019). Although the acid value was low in the case of eels pre-treated with green tea extracts (p<0.05), both testing groups showed 6 times an increase after six months of frozen in storage. However, the antioxidant effects from pre-treatment with green tea extracts were believed to be limited by the third month.

Peroxide value of half-dried eel oil
On the day zero of refrigeration, the peroxide values of eel of the control group were 13.35 meq/kg (Table 3), and those with green tea extracts were 10.03 meq/kg. Therefore, green tea extracts were effective in preventing the peroxide value of the eels during the drying process (p<0.05). During one month of frozen storage, the peroxide values of eels in the control and the eels treated with green tea extracts increased 2.4 times and 1.8 times more than the prior state. Since then, after six months of frozen storage, the peroxide values gradually increased. However, during the ninth month of frozen storage, the peroxide values decreased through re-decomposition of peroxide, which started after the sixth month of frozen storage. During nine months of frozen storage, the peroxide values of eels treated with green tea extracts were lower than those of eels in the control (p<0.05), and up to three months of frozen storage, pre-treatment of green tea extracts was comparatively effective in preventing peroxide activity (p<0.01). The researchers reported that green tea extracts effectively delayed the peracid inducement time of eel oil (Song and Kim, 2018). Additionally, the researchers found that water extract from green tea prevented the peroxide of anchovy oil (Kang et al., 2007), and green tea was used to improve the quality of sea-products in the process (Yang et al., 1999;Lee, 1999).

Carbonyl value of half-dried eel oil
Carbonyl compound, which can be generated by proteolysis and amino acid oxidation, can produce a toxic substance from the reaction to fatty acid compounds, such as malondialdehyde and 4hydroxynonenal (Berlett and Stadtman, 1997). On day zero of storage, the carbonyl values of eels in the control and the eels treated with green tea extracts were 2.26 meq/kg and 2.19 meq/kg, respectively (Table 4). During nine months of frozen storage, the carbonyl values of eels treated with green tea extracts were lower than those of eels in the control. In the case of eels treated, the lower ratio of carbonyl compound was noted, which evidently showed that the green tea extracts not only helped prevent the oxidation of lipid in the early stages but also the second stage (p<0.05). During the three weeks of the refrigeration, green tea extracts were effective in preventing the generation of the carbonyl compound, a type of lipid oxidation (Song, 2018). The generation of carbonyl compound in meat increased significantly when proteolysis activities increased (Kim, Son, Kim et al., 2015). The carbonyl values of eels refrigerated for three weeks were 2.9 meq/kg (Song, 2018), while eels treated with green tea extracts were lower than eels in the control, and eel oil stored at 37℃ were 9 meq/kg after one week of storage (Choi, 2006). Considering other research done, the carbonyl compound generation speed of green tea extracts treated, vacuumpacked, and frozen eels were quite hindered.

TBA value of half-dried eel oil
TBA value is a degree of oxidative rancidity in fats, which measures the colour of a product, a reaction between thiobarbituric acid and malondialdehyde formed by oxidation of polyunsaturated fatty acids (Kim, Son, Kim et al., 2015). The TBA values of untreated eels were 4.24 on day zero, and the TBA values of green tea extracts-treated eels were 3.87 (Table 5). During nine months of frozen storage, the TBA values of eels treated with green tea extracts were meaningfully lower than those of eels in the control (p<0.01). While the TBA values of eels in the control increased 10.6 times more than the prior state, those treated with green tea extracts increased 8.8 times more, which indicated that the treatment with green tea extracts stopped secondary oxidation of lipid. Compared to the high rise of acid values and peroxide values during one month of frozen storage, the TBA values greatly increased after three months of frozen storage. There was a finding that the TBA values of eels treated with green tea extracts and refrigerated for three weeks were similarly low to those of eels treated with the Vitamin C solution (Song, 2018).

Conclusion
In conclusion, pre-application of green tea extracts was effective in delaying the early stage peracid inducement and preventing the generation of secondary oxidation compounds, such as carbonyl compound and TBA compound, in the course of the drying and frozen storage. Especially up to three months of frozen storage, pre-application of green tea extracts was found to be highly effective in preventing the lipid oxidation of eels.

Conflict of interest
The author declares no conflict of interest.