Inhibitory Effects Of Natural Antioxidants Biology Essay

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Abstract

This study was aimed to investigate effect of aqueous citric acid and ascorbic acid on lipid oxidation and compare it with effect of vacuum packaging in order to find better treatment to delay improper changes in Persian sturgeon (Acipenser persicus) fillets during frozen storage due to lipid oxidation. In order to do that, traditional packaging, vacuum packaging, ascorbic acid solution (0.5 %) and citric acid solution (0.5 %) were considered as treatments. Rancidity development was measured by several biochemical indicators including Free Fatty Acids, Peroxide values and Thiobarbituric acid. Also pH, expressible moisture and sensory properties were measured during 6 months storage. Results showed that free fatty acid (FFA), primary and secondary oxidation products of control samples were higher than other treatments (p<0.05). Also, expressible moisture and pH value of treated samples were lower than control (p<0.05). However both antioxidants (AA and CA) extended shelf life of frozen fillets but rancidity development in CA treated samples was higher than other samples during storage. Results showed that all three treatments had significant effect on delaying lipid oxidation (p<0.05) but usage of AA and vacuum packaging had the best effect on delaying lipid oxidation and increasing shelf-life of fillets (p<0.05). Thus the employment of AA and vacuum packaging alone or in combination with other protective strategies is recommended.

Fish are considered an important part of human nutrition, because of their high content in polyunsaturated fatty acids (PUFAs), especially of the  -3 family. These unsaturated fatty acids are highly susceptible to oxidation. Deterioration of fat is not the only improper effect of oxidation and this phenomenon can cause some changes in color, texture and flavor of the product (Baker, 2001; Hamre et al., 2003). Lipid oxidation is a critical point during food processing, distribution, storage, and consumption because it decreases food quality, stability, safety, and nutritive value. Different methods have been used for extending fish products shelf life such as low temperature storage, proper packaging and glazing with solution of protecting chemicals and antioxidants (Yildiz et al., 2006; Richards et al., 1998; Lin, C. C. & Lin, C. S., 2005). Usage of antioxidants and vacuum packaging have the best influence on increasing shelf-life and delaying improper changes in sea food (Serdaroğlu & Felekoglu, 2005). Antioxidants block the formation of free radicals, stabilize hydroperoxides and thus slow down oxidation and rancidity development. Recently, the demand for novel natural antioxidants has increased; this is because of possible adverse side effects of synthetic antioxidants and beneficial effects of natural antioxidants (Benjakul et al., 2005; Sarkardei & Howel, 2006). Ascorbic acid (AA) and citric acid (CA) and their salts are widely known for their role as chelators (Oktar et al., 2001; Kim et al., 2006) in biological systems and synergists of other antioxidants. The positive effects of AA and CA on fish oil and emulsions (Osborn-Barnes & Akoh, 2003), minced fish (Stodolnik et al., 1992) and fish fillets (Badii & Howell, 2002; Aubourg et al., 2004; Pourashouri et al, 2009) have been observed. Vacuum packaging is another way for delaying lipid oxidation (auto oxidation) because of limiting oxygen molecule. As reported by Anelich et al. (2001), Fagan et al. (2004) and Perez-Alonso et al. (2004), packaging under vacuum have positive effect on extended shelf life of fish fillets. In this study we use Persian sturgeon (Acipenser persicus) fillet because it is one of the most important fish species in Caspian Sea. Most of this fillet export to different countries far from Iran, so they should be frozen before export. In order to delay improper changes of unsaturated lipid during frozen storage some antioxidants or other preservative method should be used. Thus in this study effect of ascorbic acid, citric acid (as natural antioxidants) and vacuum packaging on quality of fish fillets were investigated.

Material and method

Preparation of fish samples

Fresh Persian sturgeon (Acipenser persicus) was captured in October 2006 and kept on ice (1h) till delivery to the laboratory. Then, the fish were carefully gutted, dressed and filleted by hand. The weight of each fillet ranged from 400 g to 450 g. Fillets divided into 4 groups. Samples of the first group were left untreated (control samples; BC treatment) directly packaged traditionally in polyethylene bags and Second group were packaged under vacuum conditions (VP treatment) in polyethylene bags. Other fillets were then immersed either in 0.50% AA aqueous solution (AA treatment) and in 0.50% CA aqueous solution (CA treatment). After 5 minutes, the samples were removed out of the solutions, packaged in individual polyethylene bags. All samples were immediately frozen at -40 °C. Antioxidant concentration and dipping time were chosen according to previous related research (Chapman et al., 1993; Aubourg et al., 2004). After stored at -40°C for 24 h, all fish fillets were placed in a freezer at -18°C. Sampling was undertaken at 1, 3 and 6 months after frozen storage at -18°C and on the raw fish (initial material). For each treatment (BC, AA, CA and VP), three different fish batches (totally 48 batches of fillet) were considered and studied separately to achieve the statistical study. Chemicals (solvents and reactants) employed through the study were reagent grade (Merck, Germany).

Lipid damage measurements

Peroxide value (PV) and free fatty acid (FFA) content were determined in the lipid extract by the Egan et al. (1997) method.

a) Muscle samples (30-150 g) were blended with 250 ml of chloroform for 2-3 min and filtered through a large fluted paper. Then the samples were re-filtered through a paper containing a small amount of anhydrous sodium sulphate. Portions of this second filtrate (A) were used for other measurements ("b" to "d").

b) Weight of fat in the solution:

Transferred 10 ml of filtrate A into a weighted metal dish; removed the solvent and dried at 100oC. Then samples were cooled in deciccator and weighted. This weight was used for calculation FFA and Peroxide value in the methods below.

c) FFA:

25 ml of 95% alcohol with a few drops of 0.1 N NaOH after adding phenolphthalein was neutralized. Then this solution was added to 25 ml of "A" and titrated with 0.1 N NaOH until the pink color neutralize for 10 s. The FFA were calculated as oleic acid as a percentage of the oil. (Egan et al., 1997)

d) Peroxide value:

25 ml of filtrate "A" was transferred into a 125-ml stopper conical flask; 37 ml of glacial acetic acid and 1 ml of freshly prepared saturated potassium iodide solution were added. Allowed the solution to stand with occasional swirling for exactly 1 min, then 30 ml of water was added and titrated with 0.01 N sodium thiosulphate. Starch was used as indicator. (Egan et al., 1997)

Peroxide value was calculated as:

1) ml of 0.002 N sodium thiosulphate/g

2) Titration Ã- NÃ-1000 mequiv/kg

Wt. of sample

Where N = normality of sodium thiosulphate.

mequiv = milliequalivalants

Results were expressed as meq oxygen kg-1 lipids.

e) Thiobarbituric acid:

The thiobarbituric acid index (TBA-i) (mg malondialdehyde kg-1 flesh muscle) was determined in a 5% trichloracetic acid extract according to the method of Kirk & Sawyer (1991). Muscle samples (10.0 g) were blended with distilled water (30 ml) for 2 min. The sample was then transferred to a 500 ml distillation flask with 47.5 ml of distilled water and the pH was adjusted to 1.5 with 2.5 ml of 4 N HCL, and a drop of silicone antifoaming agent were added. The flask was connected to a distillation apparatus consisting of a Y-type connector, dropping funnel, splash head and condenser. The mixture was boiled until 50 ml of distillate was collected. In a screw capped test tube, 5 ml of the distillate was reacted with 5 ml of TBA reagent (0.02 M TBA in 90% acetic acid) and placed in a boiling water bath for 35 min. a control made up of 5 ml distilled water and 5 ml of TBA reagent was also boiled for 35 min. the tubes were cooled to room temperature and the absorbance was read at 538 nm with a DU640 UV/Vis Spectrophotometer (Beckman Coulter, Inc., Harbor Boulevard, Fullerton, CA, USA). The TBA were calculated by multiplying the absorbance reading by factor of 7.8 and expressed as mg malondialdehyde (MDA) kg-1 meat sample.

General chemical analyses

For measurement of pH, five grams of fish mince was homogenized for 1 minute with 45 ml of distilled water. pH value was measured using a standardized portable pH meter (Metrohm) (Suvanich et al., 2000). Expressible moisture content was determined by weight difference between the mussel (1-2g) of fish before and after being pressed under 0.5 and 1 kg load for 5 and 20 minutes (Parvaneh, 1998).

Sensory analyses

Sensory analyses were conducted by a taste panel consisting of five to seven panelist, according to the guidelines presented in Table 1 (DOCE, 1989; Pourashouri et al., 2009), four categories were ranked: highest quality (E), good quality (A), fair quality (B) and poor quality (C). Sensory assessment of the fish fillet included the following parameters: flesh appearance, rancid odor and flesh consistency (Table.1). At each sampling, the different fish fillets were thawed and then analyzed in the same session. The fish fillets were served to the panel members in individual polyethylene bags in which they had been kept frozen and they were scored individually. Sensory analyses were carried out at 0, 1, 3 and 6 months after storage. Three replicates were used for each experiment.

Table1. Scale employed for evaluating the sensory quality of frozen Persian sturgeon fillets

Attribute

E (Highest quality)

A (Good quality)

B (Fair quality)

C (poor quality)

Flesh appearance

Strongly hydrated and pink; myotomes totally adhered

Still hydrated and pink; myotomes adhered

Slightly dry and pale;

myotomes adhered

in groups

Yellowish and dry; myotomes totally separated

Rancid odor

Sharp seaweed and shellfish

Weak seaweed and shellfish

Slightly sour and incipient rancidity

Sharply sour and rancid

Flesh consistency

Presence or partial

disappearance of rigor

mortis symptoms

Firm and elastic; pressure signs disappear immediately

and completely

Presence of mechanical signs; elasticity notably

reduced

Important shape changes as a result of mechanical factors

*Adapted from DOCE (1989)

Statistical analysis

Data from the different quality measurements were subjected to the ANOVA one-way method. Comparisons of means after the ANOVA test were performed using a least-squares difference (LSD) method.

Results

Changes in Free Fatty Acid (FFA)

Hydrolysis development (FFA content) increased (p<0.05) in all type of samples during frozen storage. The antioxidant and vacuum packaging treatments led to lower value during the whole storage. Comparison of the different kinds of treatments led to higher (p<0.05) hydrolysis development at month 6 for BC samples while lower values were maintained throughout the whole experiment period for AA samples (p<0.05). In this study use of antioxidants and vacuum packaging decelerate the developing process of FFA production during storage; same results were reported by Aubourg et al. (2004), Pourashouri et al. (2009) and Fagan et al. (2004).

Fig. 1: FFA content of Persian sturgeon fillet during frozen storage at -18 °C.

* Bars denote standard deviation of the means.

Change in peroxide values (PV)

During frozen storage, a slow increase on the basis of primary oxidation products (PV) values was observed for each treatment, at sixth month a marked increase was observed for Blank control. In Blank control significant difference (p<0.05) were obtained at 3 and 6 months and antioxidants and vacuum packaging treatments only after 3 month had significant change(p<0.05). From the results, it is concluded that all three treatments had significant effect on delaying lipid oxidation but AA and vacuum packaging were the most effective treatments among them.

Fig. 2: PV content of Persian sturgeon fillet during frozen storage at -18 °C.

* Bars denote standard deviation of the means.

Change in Thiobarbituric acid (TBA)

PV measurements are not reliable in assessing the oxidation of highly unsaturated oils such as fish oils. This is probably because the peroxides that form initially are unstable and react quickly to form secondary oxidation products. For this reason, the PV should be used in conjunction with other methods (Sánchez-Alonso & Borderias, 2008). The thiobarbituric acid (TBA) test is among the most widely used to quantify lipid oxidation products in fish meat.

Secondary lipid oxidation products, as reported by the TBA-i, presented low values at the beginning of the study (Fig.3) and gradually increased during frozen storage (as in the case of PV). A significant increase in TBA-i value was observed for control and CA-treated samples (p<0.05) compared with the other treatments during storage. AA and vacuum packaging treatments were found to be the most effective (p<0.05) against oxidation.

Fig. 3: TBA content of Persian sturgeon fillet during frozen storage at -18 °C.

* Bars denote standard deviation of the means.

Change in pH

pH values ranged between 6.15 and 6.92 in all samples and decreased at frozen during storage but no statistical difference were obtained among the treatments and Blank control(p<0.05).

The initial pH value of treated samples was lower than their corresponding control samples and this lower value was maintained during the 3-6 months period (Fig.4). In this time, vacuum packaging (VP) treatment samples showed a lower (p<0.05) pH value when compared with other treatments samples; no significant differences were observed in the 3-6 months period among different treatments samples.

Fig. 4: pH content of Persian sturgeon fillet during frozen storage at -18 °C.

* Bars denote standard deviation of the means.

Change in Expressible moisture

Expressible moisture content showed a gradual increase for all samples during the course of the study (Fig. 5). Comparison of the different treatments revealed that the antioxidant and vacuum packaging treatments were effective at month 1, 3 and 6 as lower Expressible moisture content values were obtained compared to the Blank control but no significant differences were detected among the samples of antioxidant and vacuum packaging treatments throughout the whole experiment.

Fig. 5: Expressible moisture (%) of Persian sturgeon fillet during frozen storage at -18 °C.

* Bars denote standard deviation of the means.

Sensory analysis

At month zero (starting material) odor, taste, color and appearance of fillet was natural and fresh. But their quality deteriorated with time. Scores given to the four sensory indices (odor, color of fillet, appearance and firmness of fillet) decreased with storage time (Table 2).

Flesh appearance assessment showed a lower (P<0.05) score at month 6 for the BC samples than other treatments.

Odor analysis led to a better quality score (P<0.05) at month 3 for AA -treated samples than that for BC, CA and VP treatments. Flesh odor and flesh appearance in control samples at month 6 of storage was considered a limiting factor. Among different kinds of molecules produced as a result of lipid oxidation, secondary ones are considered the chief compounds responsible for oxidized flavors (White, 1994). A close relationship between the rancid odor development and the TBA-i assessment has been obtained in the present study.

In the end, flesh consistency assessment showed a better score at month 3 for AA- and vacuum packaging treatment samples, while at the end of the storage time no differences were obtained among the four kinds of the samples. Sensory analyses of attributes considered indicate that antioxidants particularly AA and packed under vacuum can slow down quality loss during frozen storage.

Table 2. Evolution of sensory parameters during frozen storage of Persian sturgeon fillets that were pretreated under different conditions

Frozen storage time (months)

Flesh appearance

Rancid Odor

Flesh consistency

BC

AA

CA

VP

BC

AA

CA

VP

BC

AA

CA

VP

0

E

E

E

E

E

E

E

E

E

E

E

E

1

A

E

E

E

A

E

E

E

A

A

A

A

3

B

A

B

A

B

A

B

B

B

A

B

A

6

C

B

B

B

C

B

B

B

B

B

B

B

Freshness categories: E (excellent), A (good), B (fair) and C (poor).

*All fish were category E for all attributes initially.

Discussion

Fats in fish are inclined to oxidation during storage. This is one of the most important reasons of spoilage of fish products due to formation of poisonous compounds and reduction of nutrition value (Sahoo et al., 2004). Hydroperoxides are considered as primary products of lipid oxidation. Reaction between these products with other molecules leads to "off colour" and "off odour" in fish products (Lee et al., 1998). In this study, vacuum packaging, Ascorbic acid and Citric acid (as natural antioxidants), were used as treatments. Results showed that usage of all three treatments, led to reduction of rancidity of fats in frozen Persian sturgeon fillets. As a sign of this phenomenon, primary and secondary lipid oxidation compounds formation were decreased in compare with control samples (p<0.05) and AA and vacuum packaging treatments showed the best inhibitory effect on lipid oxidation. Losada et al. (2004), Aubourg et al. (2004), Fagan et al. (2004) and Anelich et al.(2001) reported that usage of antioxidants and packaging have positive effect on delaying fat spoilage. Also effects of flaxseeds on rancidity development of frozen mackerel (Stodolnik et al., 2005), ascorbic acid on frozen herring (Hamre et al., 2003) and vitamins C & E on frozen horse mackerel (Sarkadei & Howell, 2006) were investigated and results showed that increase of peroxide value in samples which were treated by antioxidants, was significantly lower than control samples.

One of the most widely used tests to quantify lipid oxidation products in fish meat is thiobarbituric acid (TBA) test. In fact PV measurements are not reliable in assessing the oxidation of highly unsaturated oils such as fish oils. In this study, results showed that TBA-i value of control and CA-treated samples were significantly higher than AA and vacuum packaging treatments (p<0.05). Usage of AA and vacuum packaging had the best influence on delaying lipid oxidation and increasing shelf-life of fillets (p<0.05). Benjakul et al. (2005), Pourashouri et al. (2009), Sarkadei & Howell, (2008) and Sánchez-Alonso & Borderias, (2008) reported lower TBA values in samples which were treated by antioxidants in compare to control samples.

Lipolysis of fats, leads to production of free fatty acids during storage. FFA are known to undergo further oxidation to produce low molecular weight compounds that are responsible for off-flavor and undesirable taste of fish and fish products (Refsgaard et al., 2000). Also FFA has great influence on protein denaturation and texture deterioration by interaction with proteins (Lodasa et al., 2004). In this study, FFA (three glycerides and phospholipids groups) was measured in order to investigate deterioration of fats. Results showed a gradual increase in FFA formation in all samples due to hydrolysis of phospholipids and triglycerides because of lipases and phospholipases (SerdaroÄŸlu & Felekoglu, 2005) but antioxidants (particularly AA) and vacuum packaging treatments, decelerated developing process of FFA production during storage. Similar results were reported by Aubourg et al. (2002, 2005), Pourashouri et al. (2009) and Fagan et al. (2004).

In this study, some other factors such as pH, expressible moisture and sensory evaluation were investigated in order to further study on quality changes of fish fillets during frozen storage. Water holding capacity in meat tissue is strongly related to myofibril proteins. Increase of expressible moisture is a sign of reduction of water holding capacity due to denaturising of proteins (Suvanich, et al., 2000). This phenomenon leads to reduction of flavour agents and nutrition value (Reddy & Srikar, 1991). In this study expressible moisture content showed a progressive increase in all samples during frozen storage. No significant differences were detected among all three treatments throughout the whole experiment. Similar results were reported by Chen et al., 1998 (on milk fish), Pourashouri et al., 2009 (on wells catfish) and Garner et al., 2002 (on channel catfish fillets). Also Ozogol et al (2004) showed that samples which were packed under vacuum conditions had lower expressible moisture in compare with control samples. Results of PH measurements showed that pH of antioxidant treated samples was lower than control samples in fresh and frozen fish fillets during six months storage. According to other researches, frozen storage did not have significant effect on pH changes during storage period (Aubourg et al., 2004).

Results of sensory evaluation tests indicated that usage of antioxidants, particularly AA, can slow down improper changes during frozen storage. Some similar results were reported by Leaflet (2004) and Fagan et al., (2004) who found that antioxidant treatment and usage of vacuum packaging increased shelf-life and preserved sensory attributes during storage. Also previous experiments about effect of AA and CA treatments on Wells catfish fillet showed significant differences in flesh odor of treated and untreated samples at the end of storage time, although no differences were obtained for other attributes (consistency, color and flesh appearance) for both kinds of samples (Pourashouri et al., 2009).

As a conclusion, results showed that the samples which were soaked in solutions of AA and CA, had significant differences in biochemical parameters which were studied in compare to control samples at 0, 1st, 3ed and 6th months. This can be due effect of AA and CA as oxygen scavengers which can delay lipid oxidation by reducing necessary agents like oxygen and metals. Usage of AA, CA and vacuum packaging led to partial inhibition of quality loss and increased shelf-life of fillets and among all treatments (p<0.05), AA yielded the best results in preventing lipid oxidation development in frozen fillets. Thus the employment of AA and vacuum packaging alone or in combination with other protective strategies is recommended.

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