Chapter 17
Natural Bioactive Antioxidants for the Enrichment of Seafood 1
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I. Medina , M. Cascante , J. L. Torres , and M. Pazos 1
IIM-CSIC, Eduardo Cabello 6,36028-Vigo, Spain University of Barcelona, Martí I Franquès 1, 08028-Barcelona, Spain IIQAB-CSIC, Jordi Girona 18-26, 08034-Barcelona, Spain
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Exploring new, effective natural antioxidants for the food industry and consumers demanding natural ingredients in foodstuffs has intensified in recent years. This has a special relevance for fatty and semi-fatty fish andfishproducts having high amounts of n-3 polyunsaturated fatty acids (PUFA), prone to undergo oxidation and rancidity. This work proposes the use of flavonoids obtained from different agrofood by -products as natural and bioactive antioxidants infishproducts. Different parameters were found relevant. Properties such as the influence of the molecular structure on the redox and chelating capacities, the distribution of the antioxidant in the oxidation sites, and the incidence of the antioxidant on the endogenous redox balance of fish muscle will be discussed. Besides increasing the shelf-life of functional seafood preparations, the addition of some of these compounds may be beneficial for the prevention of cancer due to the induction of apoptosis in different cancer cells. Thus, the product based on fish muscle supplemented with bioactive antioxidants appears to be an interesting and stable functional food offering the combined action of n-3 PUFA and natural polyphenols.
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© 2008 American Chemical Society
193 Fish products have an essential role in many traditional occidental and oriental diets due to their composition and the high number of fish species. Fish and seafood contribute significantly to "healthy diets" by their high content on n-3 polyunsaturated fatty acids n-3 PUFA, and other important components as high quality proteins, vitamins or minerals (7). However, fish products are very prone to degradation (2). Different physical and biochemical changes lead to deterioration and loss of freshness and make difficult processing and transformation. Among these changes, lipid oxidation is one of the most important. It conduces to rancid off-flavors and reduces the shelf-life of fish products especially during storage (5). Fish rancidity can not be totally avoided, but some procedures can minimise the rate of lipid oxidation. The use of antioxidants as food additives has increased as an effective methodology for controlling rancidity and its deleterious consequences (4). However, the effectiveness of these antioxidants for inhibiting lipid oxidation of a complex matrix like fish muscle is difficult to predict. The different antioxidants show different efficacy depending on the type of fat or food and even depending on the processing or manipulation (5). Antioxidants in fish can be scavengers of free radicals involved or generated in the oxidative reactions (5), can reduce the pro-oxidants present in fish muscle such as hemoglobin (6) or can reinforce the action of the endogenous antioxidant system of fish tissues (7). Synthetic phenolics as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tert-butyl hydoquinone (TBHQ) are usually employed for minimizing rancidity of foodstuffs. However, their use is lately questioned (8), and the current legislation and the restrictions and preferences of consumers limit their use in foodstuffs. In the last years, the inclusion of plant extracts in food as preservatives is a common practice in the food industry. Natural polyphenols are valuable alternatives to synthetic phenolics. They can effectively scavenge radicals resulting in the application of bioactive antioxidant compounds in foods prone to rancidity development (9).
Natural Flavonoids: Procyanidins Procyanidins, oligomeric catechins (Figure 1), are common in vegetable and forest by-products. They have demonstrated to be potent free radical scavengers increasingly appreciated as chemopreventive agents against health conditions (10-12). We have investigated the ability of procyanidins for inhibiting oxidation of chilled horse mackerel muscle stressing the factors involved. Th& compounds tested here had similar molecular structures with different polymerisation (number of phenolic residues) and galloylation degrees (gallate content). Additionally since procyanidins will be, at least in part, bioavailable in
194 the colon after ingestion of the supplemented seafood (13), their effect on colon cancer cells has been evaluated.
Procyanidin Figure 1. General structure ofprocyanidins.
Mechanism Involved in the Antioxidant Role of Procyanidins in Fish Muscle We have tested the activity of different procyanidins extracted from grape (g) and pine (p) by-products according to Torres et al (14) in minced horse mackerel muscle stored at 4°C (Table 1). Procyanidins effectively inhibited lipid oxidation of the fish homogenate by retarding the generation of peroxides and thiobarbituric acid reactive substances (TBARS) (Figure 2).
195 Table I. Polymerization and Galloylation Degrees of the Most Effective Procyanidins in Fish Muscle Procyanidin source OW grape OWpine IV Grape IV Pine a
Polymerization"
b
Galloylation 15 0 25 0
1.7 2.1 2.2 2.9 b
mean number of phenolic residues; mean percentage of gallates
Figure 2. Peroxide value and TEARSformation during chilled storage of minced fish muscle supplemented with grape (g) and pine (p) procyanidin.
196 The tested compounds significantly retarded the induction periods of oxidation and the amount of lipid oxidation by-products formed. An increment on galloylation degrees of procyanidins increased the antioxidant efficacy. With regards to polymerization, there was an optimum number of phenolic residues, around 2.2-2.4 in which the best efficiency was achieved. The fraction showing the highest antioxidant activity on fish muscle was IVg: 25% of galloylation and 2.2 degree of polymerization. IVg was also able to significantly preserve the amounts of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) during chilled storage. This fraction can donate 5.8 electrons/mol and shows a chelating capacity of 94% compared to that of ethylenediaminetetraacetic (EDTA). It is also a polar fraction showing a partitioning coefficient of 24 % oil/water. By correlating these data with the antioxidant activity found in chilled fish muscle, the capacity of procyanidins for donating electrons seemed to play the most significant role for retarding the development of rancidity in fish muscle. Instead, the properties related with the ability for chelating metals were not correlated with the inhibitory activities. The more polar procyanidins, IV and OW, were also most active for inhibiting lipid oxidation. In addition to their reducing capacities, they may establish hydrophobic and hydrophilic interactions depending on the medium and reach a higher incorporation into the oxidative sensitive-sites like fish membranes. Since phenolic antioxidants can reinforce the action of other antioxidants, we have also examined the effect of procyanidins IVg on the endogenous octocopherol of fish muscle. In vivo, fish contains an antioxidant system that stabilizes its high content of unsaturated lipids and involves a-tocopherol, ubiquinone, carotenoids, glutathione and ascorbate (5). In post mortem conditions, endogenous antioxidants are consumed sequentially. Procyanidins IVg were able to preserve a-tocopherol present in fish muscle from oxidation (Figure 3). Such preservation was strongly correlated with the inhibition of rancidity achieved by IVg on the fish homogenate. To test a possible antioxidant synergism between procyanidins and tocopherol, we have generated the atocopheroxyl radical by reaction of a-tocopherol with l,l-diphenyl-2picrylhydrazyl (DPPH) radical. The radical was unambiguously monitored by electron spin resonance (ESR) spectroscopy. ESR data showed a decrease of tocopheroxy radical in the presence of procyanidins IVg. Therefore, procyanidins can regenerate a-tocopherol from a-tocopheroxy and to provide a synergistic effect for stabilizing fish muscle. The results of this work reveal the significance of the capacity for donating electrons and the effect on the endogenous tocopherol to explain the ability of phenolic antioxidants supplemented for retarding and inhibiting lipid oxidation of fish muscle.
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Figure 3. Preservation of initial endogenous a-tocopherol in mincedfish muscle supplemented with 100 ppm ofgrape procyanidins IVg after 3 and 6 weeks of storage at 4 °C
Functional Properties of Grape Procyanidins The possible functional properties of the procyanidins IVg in the colon, their effect on the cell viability, cell cycle and apoptosis in colon cancer cells was investigated. We have used HT29 epithelial colon carcinoma immortalized cell lines and two other cell lines as control models of non-cancer colon cells. The results demonstrated that the decrease in cell number was dose-dependent and the polyphenols affected very little the cell viability in all three cell lines. There was an effect on the cell cycle in cancer HT29 cells but the cell cycle distribution in non-cancer cell lines was not affected by the procyanidin mixture. The procyanidins induced the appearance of 16% of apoptotic HT29 cells. Interestingly the pro-apoptotic effect was selective for cancer cells. These results show that the supplementation of grape procyanidins stabilizes fatty fish muscle against oxidation and the product can be an interesting functional food offering the combined action of fish and natural polyphenols
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