Antecedentes de la institución

Institute of Sciences of Food Production, Italian National Research Council, Grugliasco, Italy

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The use of natural or naturally-derived antioxidants, instead of synthetic antioxidants, to produce foods with a longer shelf life and a higher degree of safety is a growing trend. Fruit and fruit-processing by-products are considered to be an important source of bioactive molecules (vitamins C, E, carotenoids, phenolic compounds and dietary fiber) of great interest for the food industry, although their content varies greatly depending on origin, source, type of extract and concentration levels. After a brief introduction, this chapter aims to critically review the applications of fruit and pomace extracts from processing by-products of grape, pomegranate and berry fruits, in improving the safety and quality of fish products, as described in studies recently carried out worldwide. In particular, the antioxidant and antimicrobial effects of these natural food additives in the minced muscle of various marine and freshwater fish species are evaluated.

Keywords: Fish, fatty acid, grape, pomegranate, berry, oxidative spoilage, microbial spoilage.

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Fish flesh is considered to have great nutritional value due to its higher levels of long chain n-3 polyunsaturated fatty acids (PUFA), which have been shown to have various health

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benefits in human nutrition. Unfortunately, the unsaturated nature of these PUFAs makes them highly susceptible to oxidation, which is responsible for rancidity and loss of nutritive value, resulting in off-flavors and structural changes which are unappealing for consumers (Kulawik et al., 2013).

Moreover, fish products have a short shelf life compared to meat products, due to the microbiological spoilage that limits the storage, processing and use of this food source (Speranza et al., 2009). Spoilage of fish muscle results from changes brought about by biological reactions such as the metabolic activities of the microorganisms present in the aquatic environment in which fish live. The main bacterial species involved in spoilage are

Pseudomonas spp., Aeromonas spp., Vibrio spp., certain species of psychrotolerant Enterobacteriaceae, usually found in iced freshwater fish, and Shewanella putrefaciens-like

bacteria, mainly Shewanella putrefaciens, Shewanella algae, and Shewanella baltica, which are commonly found in iced marine fish (Kulawik et al., 2013).

These microorganisms generate a wide array of compounds that contribute to whole fish spoilage and produce many off-odor and off-flavor compounds such as biogenic amines (putrescine, cadaverine, and histamine), hydroxylamine, ketones, aldehydes, alcohols, and organic acids that are essentially absent or only occur at very low levels in fresh fish (Ghaly et al., 2010).

In order to contrast the chemical and microbiological deterioration of fish products, the use of natural or naturally-derived antioxidants has fostered research into identifying new low-cost antioxidants having commercial potential for the production of foods with a longer shelf life and a higher degree of safety (Corbo et al., 2008; Maqsood et al., 2013; Widsten et al., 2014). The fruit-processing industry generates large volumes of solid waste in the form of peels, kernels, and pulp, which are rich in bioactive phytochemicals, dietary fiber and unsaturated fatty acids, and hence have the potential to serve as functional food ingredients (Babbar et al., 2015).

In the following paragraphs, various studies carried out into the application of fruit and pomace extracts, by-products from the processing of grape, pomegranate and berry fruits, in limiting the oxidative and microbial spoilage of fish products, are critically reviewed.

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In the world, grape (Vitis vinifera L.) is the second largest fruit crop after oranges. During the production of wines, up to 40% of the grape ends up as by-products (Moreno-Arribas & Polo, 2005). The principal winery by-product, grape pomace, produced after pressing the crushed grapes in production of white wine or after fermentation and maceration for red wine, contains mainly pressed grape skins, pulp, seeds and stems that are rich sources of inexpensive antioxidative phenolic compounds (Cheynier, 2012; Cheynier et al., 2013). The grape seeds, which can be recovered from pressing grape pomace and after maceration during first pressing contain oils rich in linolenic acid (12–20%), protein (11%), non-digestible carbohydrates (60–70%), and phenolic and non-phenolic antioxidants (Yu & Ahmedna, 2013). Numerous studies have demonstrated that these compounds exhibit health-promoting effects, with cardioprotective, neuroprotective, and other health benefits (Pozo-Bayón et al., 2012; Del Rio et al., 2013).

The different studies investigating the properties of grape by-products carried out in fish muscle models are summarized in Table 1.

Table 1. Recent articles about the effects of grape by-products on fish products

Fish

product Scientific name

Type of ingredient Impact on product Reference Atlantic mackerel white muscle

Scomber scombrus White grape pomace ethanol extract Inhibition of muscle lipid oxidation during frozen storage by flavanol oligomers Pazos et al., 2005a,b Bonito fillets

Sarda sarda Grape seed ethanol extract Delayed lipid oxidation during frozen storage Yerlikaya & Gökoğlu, 2010a Horse mackerel fillets Trachurus trachurus Grape pomace procyanidins Inhibition of fillet lipid oxidation Pazos et al., 2006, 2010 Red grape antioxidant dietary fiber Inhibition of lipid oxidation during frozen storage Sánchez- Alonso & Borderıás, 2008 Red grape antioxidant dietary fiber Delayed lipid oxidation in 3 months of frozen storage Sánchez- Alonso et al., 2007a White grape dietary fiber concentrate Augmented aggregation of myofibrillar proteins, Enhanced water retention & improved cooking yield Sánchez- Alonso et al., 2007b White grape antioxidant dietary fiber Delayed lipid oxidation during frozen storage Sánchez- Alonso et al., 2008 Meagre sausages

Argyrosomus regius White grape antioxidant dietary fiber Lowered TBARS values; Antimicrobial effect Ribeiro et al., 2013 Rainbow trout minced muscle Oncorhynchus mykiss Red grape pomace ethanol extract Delayed lipid oxidation & cadaverine formation after refrigerated storage Gai et al., 2014 Silver carp fillets Hypophthalmichthys molitrix Grape pomace acetone extract Delayed lipid oxidation during refrigerated storage Hasani & Dughikolaei, 2014

Pazos et al. (2005a) determined the ability of white grape pomace extract fractions, containing flavanol monomers, oligomers (procyanidins) and glycosylated flavonols, to inhibit oxidation of fish lipids. The tests were conducted in mackerel (Scomber scombrus) minced fillets during frozen storage at -10 °C within 6 months. Duplicate samples of frozen muscle were taken regularly over the course of six months, and oxidation was followed by measuring peroxide value, conjugated diene and triene hydroperoxides, thiobarbituric acid- reactive substances (TBARS), fluorescence compounds and degradation of PUFA. The effectiveness of each antioxidant was evaluated by comparing the inhibition of oxidation with samples treated with synthetic antioxidant (propyl gallate) used as control. Grape phenolic fractions showed different effectiveness on decreasing the rate of oxidation and the amount of oxidation products formed; flavanol oligomers were the most potent inhibitors of oxidation in frozen fish muscle. The authors concluded that in order to obtain the highest antioxidant efficacy of grape polyphenols in frozen fish muscle, an optimal combination of procyanidin degree of polymerization and galloylation percentage should be considered.

In another similar study, Pazos et al. (2005b) investigated the capacity of a phenolic extract, obtained from pressing destemmed Parellada grapes, and of a purified fraction of procyanidins to preserve the endogenous antioxidants of minced mackerel (Scomber

scombrus) muscle and horse mackerel (Trauchurus trauchurus) fillets during frozen storage

at -10 °C for a period of 140 days. Grape polyphenols were used in muscle concentrations of 0.01% and compared with propyl gallate, as synthetic antioxidant. The results demonstrated that grape polyphenols inhibit the depletion of endogenous α-tocopherol, ubiquinone and total glutathione. Moreover, grape polyphenols showed similar efficiency to the synthetic antioxidant propyl gallate in the preservation of ubiquinone, in both minced and filleted muscle, and total glutathione, in minced muscle. Grape polyphenols more efficiently preserved α-tocopherol, an endogenous antioxidant whose depletion is highly correlated with the evolution of lipid oxidation. The development of lipid oxidation was repressed, while the concentration of α-tocopherol was not reduced up to critical levels, so the authors concluded that grape polyphenols may be an attractive potential food additive because they can stabilize frozen fatty fish and preserve an important compound such as vitamin E.

Pazos et al. (2006) carried out a study aiming to evaluate the reducing and chelating capacities, and the affinity for incorporation into frozen horse mackerel fillets stored at -10°C, of grape procyanidins isolated from grape pomace. The effects of grape procyanidin were compared against two other phenolic antioxidants, hydroxytyrosol and propyl gallate. Phenolic antioxidants were supplemented into the fillets in three ways: spraying with an aqueous phenolic solution, glazing with an aqueous phenolic solution, and pre-washing of fillets with water plus spraying with an aqueous phenolic solution. Lipid oxidation in the fish fillets was delayed by all phenolic compounds and the order of antioxidant efficiency in spraying and glazing was as follows: propyl gallate > hydroxytyrosol > grape procyanidin. As far as the reducing power of these phenolics is concerned, the order of efficiency was similar to that observed in the spraying and glazing treatments, but no correlation with their chelating capacity and their affinity to fish muscle was shown. The antioxidant activity of grape procyanidin increased synergistically where fillets were washed with water prior to spraying phenols and relative antioxidant efficiency changed to propyl gallate > grape procyanidin > hydroxytyrosol. The authors concluded that this synergism could be due to the residual water that remained on the fillet surface after washing, resulting in a better distribution of grape procyanidin across the fillet surface.

Pazos et al. (2010) evaluated the influence of polymerization and galloylation of oligomeric catechins (proanthocyanidins) obtained from grape, pine (Pinus pinaster) bark and witch hazel (Hamamelis virginiana) on their effectiveness to prevent lipid oxidation in horse mackerel muscle refrigerated at 4°C. Fractions from pine bark showed non-galloylated oligomers of catechin with diverse mean polymerization (1.9-3.4 monomeric units) while grape and witch hazel showed homologous fractions with galloylation ranging from 0.25 to <1 gallate group per molecule. The formation of lipid oxidation products, lipid peroxides, and volatiles responsible for rancidity was evaluated by comparing induction periods and inhibition percentages in order to assess the antioxidant effectiveness of the different fractions. Lipid oxidation in horse mackerel muscle was better inhibited by the proanthocyanidins of medium size (2-3 monomeric units) and low galloylation degree (0.15- 0.25 gallate group/molecule). The authors therefore concluded that their study gave information relevant for achieving optimum use of polyphenols in muscle-based foods such as pelagic fish muscle.

Neira et al. (2011) investigated the influence of different concentrations (10-100 μg/g) of polyphenolic fractions extracted from grape pomace to prevent haemoglobin-promoted lipid oxidation using in vitro experiments carried out with the pelagic fish, horse mackerel. Horse mackerel light muscles were washed, homogenized and frozen at -80°C for less than one week and then thawed for 30 min under running cold water. Horse mackerel haemoglobin was added as lipid oxidation initiator to the light muscles at a concentration of 15 μmol/kg fish. Peroxide value, TBARS index, and sensory analysis were carried out to monitor lipid oxidation. All polyphenolic fraction concentrations, except the final concentration of 10 ppm, were able to prevent haemoglobin-promoted lipid oxidation, proportionally to the amount of polyphenolic fraction added, with samples supplemented with 100 ppm showing the longest induction periods (5 days) and inhibition percentages. The same antioxidant efficiency was showed by the sensory analysis of rancid off-flavors, with the 100 ppm-supplemented group showing the greatest inhibition of rancidity. In the light of the results, the authors concluded that new antioxidant ingredients from natural sources could be useful for preventing fish lipid oxidation in which oxidation is essentially initiated by heme proteins.

Hasani & Dughikolaei (2014) determined the antioxidant effect of red grape pomace extract supplemented on fillets of silver carp (Hypophthalmichthys molitrix) during a refrigerated storage challenge. Silver carp samples were treated with different concentrations of red grape pomace extract (0%, 2%, and 4%) and stored for 15 days in a refrigerator at +4°C. At 0, 3, 6, 9, 12, and 15 days of storage, the changes in pH, peroxide value, TBARS and heme iron were measured. Results showed that samples treated with 4% of red grape pomace extract were better preserved compared with other treatments; indeed, the addition of this concentration of extract significantly delayed lipid oxidation in silver carp fillet during refrigerated storage. The authors concluded that red grape pomace extract has the potential to be used as a natural antioxidant, improving the quality and preventing the deterioration of stored fish.

Gai et al. (2014) evaluated the effects of the addition of ethanol red grape pomace extracts on the shelf life of minced rainbow trout (Oncorhynchus mykiss) muscles. Extracts were added to trout patties to give a final concentration of 0%, 1%, and 3% and after 1 and 6 days of refrigerated storage, the treated muscle samples were analyzed for their physical (pH, color) and chemical (TBARS, fatty acid and biogenic amine content) properties. Red grape pomace extracts were effective in delaying lipid oxidation and cadaverine formation in

muscle after six days of refrigerated storage, with the best results obtained in the samples treated with the 3% extract. The authors concluded that red grape pomace extract in minced trout muscle can enhance the quality and shelf life of this ready-to-cook fish-based food and simultaneously provide a functional food with natural antioxidants that are beneficial for consumers.

Wine by-products are also rich in dietary fiber. Saura-Calixto (1998) proposed to define antioxidant dietary fiber as a natural product that combines the beneficial effects of dietary fiber and natural antioxidants, such as polyphenol compounds. This dietary fiber can also be an effective tool in seafood processing for improving functional properties, such as water binding, gelling etc.

Sánchez-Alonso et al. (2007a) studied the effect of red grape antioxidant dietary fiber addition, at concentrations of 0%, 2%, and 4% to horse mackerel (Trachurus trachurus) minced fillets on flesh lipid stability during a 6-month frozen storage experiment. Red grape antioxidant dietary fiber was characterized in terms of dietary fiber, total polyphenols and antioxidant capacity, and multifunctional antioxidant assays were carried out on all the minced fillets immediately after the preparation of samples and at 30, 60, 90, 120, 150 and 180 days of storage at -20 °C. The formation of conjugated dienes and trienes (products of lipid oxidation) was monitored during frozen storage and the samples with added dietary fiber exhibited reduced formation of dienes and trienes until 90 days compared with the control lot. Moreover, TBARS values were lower in the samples with dietary fiber added for most of the storage period. The rate of inhibition at 90 days of frozen storage was 57.3% and 54.1% for the treated sample groups, respectively. At this time of frozen storage, antioxidant action was not significantly different for the two levels of dietary fiber. The authors concluded that the addition of red grape fiber considerably inhibits oxidation in minced fillets during the first 3 months of frozen storage, indicating that red grape antioxidant dietary fiber could be used as an ingredient to prevent oxidation in minced fillets during frozen storage. A possible reason for this effect could be the chelating action of fiber on some pro-oxidant metals or the action of grape flavonoids associated with dietary fiber.

In another similar study, Sánchez-Alonso & Borderias (2008) studied the effect of adding red grape antioxidant dietary fiber to horse mackerel (Trachurus trachurus) minced fillets as a technological ingredient in a frozen storage trial lasting 6 months. Concentrations of 0, 2 and 4% of red grape antioxidant dietary fiber were added to minced fish muscles. Sensory analyses, protein solubility, water retention, color, mechanical properties and lipid oxidation analyses were carried out immediately after preparation of samples and after every 30 days of storage at -20°C. The presence of fiber significantly increased the water retained after physical stress while it reduced thawing drip in minced products over the entire storage period. Texture profile analysis indicated that changes in fiber content significantly affected the textural characteristics, with lower textural parameters found in the samples with the highest concentrations of fiber. Regarding the lipid oxidation products formed from PUFAs, lower levels of conjugated hydroperoxides, dienes and trienes were found in samples with added red grape antioxidant dietary fiber (2% and 4%) over a period of 6 months at -20 °C. At 90 days of storage, inhibition of oxidation development was significant in samples with added red grape antioxidant dietary fiber; development of conjugated dienes was inhibited by 18.2% and by 26.7% in the 2% and 4% extract groups, respectively. As regards sensory analysis, the panellists detected no significant changes in flavor over the course of frozen storage. Overall, the sample with 2% fiber addition received the best ratings followed by the

control group and the 4% extract group. In conclusion, in the light of the chemical, physical and sensory analyses, the authors suggest that red grape antioxidant dietary fiber is a highly active technological ingredient in frozen dark minced fish.

In another study, carried out again in horse mackerel, Sánchez-Alonso et al. (2007b) investigated the functional properties of a white grape (var. Airén) dietary fiber concentrate obtained from peels and seeds. This product was added at 2% and 4% to minced fish muscle and stored for 6 months at -20 °C. Every month, samples were analyzed for their physical and mechanical properties, sensory and color attributes, microscopic and electrophoretic profiles. White grape dietary fiber concentrate showed good functional properties, high water and oil retention capacity, and considerable swelling properties. Its addition augmented aggregation of myofibrillar proteins and made samples softer and less springy and cohesive in the course of frozen storage. Moreover, addition significantly enhanced water retention and improved cooking yield. In sensory evaluation, according to the hedonic analysis, all samples were considered acceptable with 4% samples scoring the lowest values. The minced fish muscle with 2% concentrate scored slightly higher for flavor than the 4% and control groups. Any odd flavors, throughout frozen storage, were detected by the panellists while some differences, albeit insignificant, were perceived in the texture. The results indicate that white grape dietary fiber concentrate could be proposed as a new ingredient in minced fish muscle, not only for its nutritional and antioxidant properties but also for its technological properties.

In a similar experimental design, Sánchez-Alonso et al. (2008) carried out a study evaluating the antioxidant properties of white grape dietary fiber concentrate in horse mackerel minced muscle. The evaluation of this by-product‘s antioxidant capacity when added to frozen minced muscle over 6 months of storage was similar for all the methods followed. Addition considerably delayed lipid oxidation in minced fish muscle during frozen storage. These results indicate that white grape dietary fiber concentrate could be used as a natural ingredient to prevent oxidation in minced fish during frozen storage and could be used as a functional ingredient in the design of healthy foods.

Ribeiro et al. (2013) evaluated the effect of antioxidant grape dietary fiber addition in sausages made with farmed meagre (Argyrosomus regius), during a 98-day storage experiment at a refrigeration temperature of 2±2°C. Physical, chemical, nutritional, microbiological and sensory assessment were performed in two experimental groups, one contained 3.9% of inner pea dietary fiber and the other group contained 0.9% inner pea dietary fiber plus 3.0% of antioxidant grape dietary fiber. The latter group had an effective antioxidant capacity, proven not only by the radical scavenging activity and reducing power measurements, but also by the lower TBARS values over storage time. Moreover, antioxidant grape dietary fiber seemed to have some antimicrobial effects. Formulation had an effect on