Notes
Antioxidant Activity of Alginic Acid in Minced Pork Meat
2015 Volume 21 Issue 6 Pages 875-878
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2015 Volume 21 Issue 6 Pages 875-878
In this study, the antioxidant activity of edible polysaccharides such as alginic acid, chitosan and pectin were estimated using four antioxidant assay systems: 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, ferric ion reducing activity power (FRAP), oxygen radical absorbance capacity (ORAC) assays, in minced pork meat as the storage test. Alginic acid had the strongest antioxidant ability to scavenge free radicals and reduce ferrous ion among all polysaccharides tested. Alginic acid inhibited the production of hydroperoxides and secondary oxidation products of lipids in minced pork meats during storage.
The quality of processed meat products such as sausage and ham is made inferior by the oxidation of constituent lipids during storage. Among lipid oxidation products, secondary oxidation products such as carbonyl compounds and alcohols can impart undesirable flavors to processed meat products, whereas hydroperoxides and secondary oxidation products are toxic to humans. Thus, natural and synthetic antioxidants are added to the meat products of pork, beef, chicken, and fish to inhibit the oxidation of lipids, thereby maintaining product quality. However, in general consumers prefer to limit their exposure to antioxidants, because some antioxidants may have carcinogen properties and undesirable sensory characteristics. α-Tocopherol (vitamin E) is often used as an antioxidant in meats, but its antioxidant effect depends on the variety and part of meat (Yamauchi et al., 1977). Ascorbic acid (vitamin C) as a typical antioxidant may discolor meat; thus, the use of alternative antioxidants is anticipated.
Polysaccharides such as starch, pectin and carrageenan are often used as gelling, thickening or stabilizing agents for processed meat products. It has been reported that several polysaccharides have antioxidant properties (Wang et al., 2013). Chitosan and its derivatives are potential free radical scavengers because of their hydrogen donating ability (Jarmila and Varikova, 2011). Chitosan may also have the antioxidant ability to reduce Fe3+ and chelate ferrous ions (Vairamani et al., 2013). Free radical scavenging activity was observed for polysaccharides containing uronic acid as well as glucans (Machova and Bystricky, 2013) and fructans (Peshev et al., 2013). Moreover, antioxidant activity has also been observed for xanthan oligosaccharides (Xiong et al., 2013). From these observations, some polysaccharides may be potential antioxidants for processed meat products.
In this study, we evaluated the antioxidant activity of edible polysaccharides (such as alginic acid, chitosan and pectin) commonly used in processed foods, using four antioxidant assay systems: 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, ferric ion reducing antioxidant (FRAP), oxygen radical absorbance capacity (ORAC) assays and minced pork meat.
Materials α-Tocopherol (VE) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,2'-Azobis(2-aminopropane) dihydrochloride (AAPH), DPPH, TPZT, alginic acid, chitosan, and pectin were purchased from Wako Pure Chemical Industries (Osaka, Japan). Minced pork meat was purchased from a local market.
DPPH radical scavenging activity The effect of DPPH radical-scavenging was evaluated according to the method of Chen and Ho (1995) with some modifications. To 1.25 mL of polysaccharides in ethanol solution (10 mg/mL) was added 0.25 mL of 0.5 mM DPPH solution in a 24-well plate. After 1 h incubation at room temperature, the absorbance was read at 517 nm using a Molecular Devices Spectra MAX Gemini EM microplate reader. Results were calculated as percent decrease with respect to the control values. The DPPH radical scavenging activity of polysaccharides was expressed as relative activity (%) to ascorbic acid.
FRAP assay The FRAP assay was carried out as described by Benzie and Strain (1999) with slight modifications. Briefly, FRAP reagent consisted of 10 mM TPTZ solution in 40 mM hydrochloric acid, 300 mM sodium acetate buffer (pH 3.6) and 20 mM ferric chloride (III) solution at the ratio 10:1:1 (v/v/v), respectively. A 0.1 mL volume of polysaccharide in aqueous solution (5 – 50 mg/mL) was added to 3 mL of FRAP reagent and incubated at room temperature for 3 min; absorbance at 593 nm was immediately measured using the microplate reader. The results were calculated as ascorbic acid equivalent. The reducing ability was expressed as relative activity (%) to ascorbic acid with metal chelating ability (Frankel, 1998).
ORAC assay The ORAC assay was carried out as described by Prior et al. (2003) with minor modifications. Fluorescein and AAPH were dissolved in 75 mM phosphate buffer, pH 7.4. A 25 µL aliquot of blank, Trolox standard or polysaccharide in aqueous solution was added to quadruplet wells of a clear bottomed, 96-well microplate. Then, 150 µL of 8.38 nM fluorescein was added to each well, the plate was incubated at 40°C for 10 min, and 25 µL of prepared 153 mM AAPH was added to each well. Fluorescence decay was monitored at 528 nm upon excitation at 485 nm for 1.5 h, using the microplate reader. The area under the curve for fluorescence versus time for each sample minus the area under the curve for the blank was calculated; these areas were compared to a standard curve prepared from the area under the curve for 5 to 100 µm Trolox standards minus the area under the curve for the blank. The ORAC values were expressed relative to Trolox activity.
Storage test of minced meat Alginic acid or chitosan was added to 10 g of minced pork meat at a level of 0.2 or 1.0%, as well as α-tocopherol as a positive control. Samples were prepared in duplicate under aseptic conditions on a clean bench. Minced meat samples were placed in a glass Petri dish and stored at 4°C for one week. Lipids were extracted with diethyl ether after storage of minced meat. The peroxide and carbonyl values of extracted lipids were determined by the Standard Method of Japan Oil Chemists' Society (2013). Sensory evaluation was performed for stored meat by 5 to 6 panelists. Sensory evaluation of odor was scored as follows; 5: very good, 4: good, 3: slightly bad, 2: bad, 1: very bad (inedible).
Statistical analysis Data are shown as the mean and standard deviation of triplicate values, and were analyzed using analysis of variance (ANOVA). Duncan's multiple range test was used to determine the differences among samples.
The antioxidant activities of various polysaccharides evaluated by DPPH scavenging activity assay, FRAP assay, and ORAC assay are shown in Table 1. The DPPH free radical scavenging activity assay is a simple method for evaluating the antioxidant activity of compounds. We compared DPPH radical scavenging activity of polysaccharides with that of ascorbic acid at a concentration of 10 mg/mL. As shown in Table 1, all polysaccharides we tested had strong DPPH radical scavenging activities, 90 – 70% of ascorbic acid. In particular, the DPPH radical scavenging activity of alginic acid and chitosan was almost the same as that of ascorbic acid.
| Polysaccharide | DPPH (%)a | FRAP (%)b | ORAC (%)c |
|---|---|---|---|
| Algini acid | 89±9a | 34−3a | 7.7±0.7a |
| Chitosan | 88±9a | 15±1b | 5.5±0.5b |
| Pectin | 70±7b | 3±1c | 3.0±0.3c |
Values indicate the mean ± standard deviation. Values within columns followed by different letters are significantly different (p < 0.05).
The principle of the FRAP assay is the reduction of a ferric tripyridyl-triazine complex to its ferrous form. The polysaccharides we tested had ferric iron reducing abilities, although their antioxidant activities were lower than that of ascorbic acid (Table 1). The relative FRAP value of alginic acid was 34% of ascorbic acid, the strongest among the polysaccharides tested.
The ORAC value represents the scavenging activity against the peroxyl radical induced by AAPH (Prior et al., 2003). The ORAC assay is designed to measure the antioxidant activity of foods against the peroxyl radical. A wide variety of foods have been tested using this method; therefore, ORAC is a standardized method for measuring the antioxidant potency of foods (Niki, 2010). As shown in Table 1, the polysaccharides showed considerable ORAC values. Alginic acid showed the highest ORAC value (corresponding to 7.7% of Trolox). However, the ORAC values of polysaccharides were considerably lower than that of Trolox.
Meat storage test We evaluated the stability of minced pork meat supplemented with alginic acid and chitosan as polysaccharides during storage at 4°C. Figures 1 and 2 show the changes in peroxide and carbonyl values of lipids in minced pork meat supplemented with alginic acid and chitosan at a concentration of 0.2% and 1.0%, respectively. As shown in Fig. 1, the peroxide value of lipids was temporally increased in all minced meats. Fresh meat had a peroxide value of 1 meq/kg, but the control sample showed the highest value among all samples during storage at 4°C. The peroxide value of the control sample reached 37 meq/kg after 7 days. On the other hand, α-tocopherol as a typical antioxidant retarded the increases of peroxide value of lipids in minced meat. Alginic acid and chitosan also retarded increases in peroxide value during storage. In particular, the peroxide value of lipids in minced meat supplemented with alginic acid and chitosan at 1.0% was the lowest (5 meq/kg) among all samples after 7 days. Peroxide value indicates the hydroperoxide level. This result indicates that alginic acid and chitosan could inhibit the production of hydroperoxides from lipids in minced meat during storage.
Changes in peroxide value of lipids extracted from minced pork meat with polysaccharides during storage at 4°C. ○:control △:chitosan (0.2%) ▴:chitosan (1.0%) □:alginic acid (0.2%) ■:alginic acid (1.0%) ◆: α-tocopherol (1.0%)
Changes in carbonyl value of lipids extracted from minced pork meat with polysaccharides during storage at 4°C. ○:control △:chitosan (0.2%) ◆:chitosan (1.0%) □:alginic acid (0.2%) ■:alginic acid (1.0%) ◆ :α-tocopherol (1.0%)
As shown in Fig. 2, the carbonyl value of lipids was temporally increased in all minced meats, although the carbonyl value of fresh meat was 5 µmol/g. The carbonyl value is an index of secondary oxidation products from lipid hydroperoxides. Carbonyl compounds such as aldehydes and ketones produced from lipid hydroperoxides impart undesirable flavors to processed meat products and are toxic to humans. The carbonyl value of the control sample was the highest (12.5 µmol/g) among all samples after storage at 4°C for 7 days. On the other hand, α-tocopherol retarded the increases in the carbonyl value of lipids in minced meat. The carbonyl value of meat supplemented with α-tocopherol was 8.6 µmol/g after storage for 7 days. Alginic acid and chitosan also retarded the increases in carbonyl value during storage. In particular, the carbonyl value of lipids in minced meat supplemented with 1.0% alginic acid was the lowest (8.2 µmol/g) among all samples after 7 days. This result indicated that alginic acid and chitosan could inhibit the decomposition of lipid hydroperoxides and the formation of secondary oxidation products in minced meat during storage.
The sensory evaluation of stored minced meats is shown in Table 2. The odor of all minced meats deteriorated during storage at 4°C. Sensory scores are presented as the mode. The sensory score for minced meat supplemented with alginic acid was superior to those with α-tocopherol and chitosan. Alginic acid could inhibit the flavor deterioration of minced meat due to lipid oxidation during storage.
| Day | 0 | 3 | 5 | 7 |
|---|---|---|---|---|
| Control | 5 | 4 | 2 | 2 |
| Tocopherol (1.0%) | 5 | 4 | 3 | 3 |
| Alginic acid (0.2%) | 5 | 4 | 4 | 4 |
| Alginic acid (1.0%) | 5 | 5 | 4 | 4 |
| Chitosan (0.2%) | 5 | 3 | 3 | 2 |
| Chitosan (1.0%) | 5 | 3 | 3 | 3 |
Sensory score; 5: very good, 4: good, 3: slightly bad, 2: bad, 1: very bad (inedible)
We identified the antioxidant activity of alginic acid in minced meat as a food system, although the activity has been reported in a biological system (Sarithakumari et al., 2013). The antioxidant activity of alginic acid corresponded to that of α-tocopherol in a food system. The antioxidant activity of alginic acid might be attributable to its metal chelating ability as well as free radical scavenging ability and ferric ion reducing ability, as minced pork meats are rich in Fe. In particular, pork meats contain considerable amounts of non-heme iron as well as heme iron (Kojima and Yasui, 1993). Transitional metals such as Fe are able to catalyze lipid oxidation. Ferrous ions could also decompose lipid hydroperoxides to form secondary products as carbonyl compounds and alcohols, which often impart undesirable flavors. α-Tocopherol has free radical scavenging ability but not ferric iron-reducing and chelating abilities. In contrast, excessive α-tocopherol may induce the decomposition of lipid hydroperoxides to form off-flavor compounds (Frankel, 1998). On the other hand, alginic acid may inhibit the production of off-flavor compounds during storage of mined meats. It is proposed that alginic acid may reduce or chelate ferrous iron which could decompose lipid hydroperoxides to form off-flavor compounds, since it does not have the chemical structure to scavenge peroxyl radicals such as lipid radicals. Further, alginic acid may be a potential antioxidant in processed meat products because it has no effect on the sensory quality of meats.
