Effect of Red Grape Pomace Extract on the Shelf Life of Refrigerated Rainbow Trout (Oncorhynchus mykiss) Minced Muscle
The use of naturally-derived antioxidants to produce ready-to-cook fish-based foods is a growing trend. Grape pomace is a rich source of phenolic compounds with antioxidant and antimicrobial effects. The effects of red grape pomace (RGP) extracts on the shelf life of minced rainbow trout muscles were evaluated. Extracts were added to trout patties to give a final concentration of 0, 1 and 3%. RGP extracts were effective in delaying lipid oxidation and cadaverine formation in minced trout muscle after six days of refrigerated storage. It is concluded that RGP extract in minced trout muscle can enhance the quality and shelf life of this ready-to-cook fish-based foods and simultaneously provide a functional food with natural antioxidants beneficial for consumers.
Keywords minced trout muscle, red grape pomace extract, antioxidant effect, fatty acid, flavonoid,
Introduction
Polyunsaturated fatty acids (PUFAs) present in marine and freshwater fish have been shown to have various health benefits, as recently reviewed by Davidson et al. (2011). The unsaturated nature of these PUFAs makes them highly susceptible to oxidation, resulting in off-flavors and structural changes which are unappealing to consumers. This limits the storage, processing and use of such fish, in particular in ready-to-cook fish-based foods (Speranza et al., 2009). Spoilage of fish muscle results from changes brought about by biological reactions such as the metabolic activities of microorganisms involved in the production of biogenic amines (BAs) (Gram and Dalgaard, 2002). BAs, such as putrescine, cadaverine and histamine, are essentially absent or occur at very low levels in fresh fish. Consequently, they are considered indicators of spoilage and, more importantly, of safety in seafood (Rossi et al., 2002).
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 (Corbo et al., 2008). One group of active molecules of great interest to the food industry are the phenols extracted from fruit, vegetables and food processing by-products. Among the latter group, grape skins and seeds (pomace) are grape by-products providing a rich source of various high-value products such as ethanol, tartrates and malates, citric acid, grape seed oil, hydrocolloids and dietary fibre, that are increasingly being used to obtain functional ingredients (Goñi et al., 2005;). Grape extracts are in general expected to have a high content of proanthocyanidins (Gharras, 2009). The class of proanthocyanidins arises from a subgroup of the flavonoids, namely the flavanols. The monomeric flavanol units are the building blocks, which form the oligomeric and polymeric proanthocyanidins. Typical flavanols, which are expected to make up the majority of the building blocks are (+)-catechin and (-)-epi(+)-catechin, constituting the group of procyanidins (Gharras, 2009; Pascual-Teresa
et al., 2010). Qualitative and quantitative distribution of polyphenols in red grape pomace may
show significant differences, depending on different factors, such as varietal differences of the grapes, climate, location of the cultures and wine-making procedures, as reported by Ruberto et al.
(2007). This chemical composition makes the grape pomace extracts interesting to be used in the production of phytochemicals (González-Paramas et al., 2004) and as potential food ingredient in meat (Sagdic et al., 2011) and in seafood products (Sánchez-Alonso et al., 2007a, 2007b). Recent studies, carried out in seafood products, showed that phenolic compounds from grape by-products were effective at delaying lipid oxidation in frozen horse mackerel (Trauchurus trauchurus) fillets (Pazos et al. 2006a). In another study, Pazos et al. (2005a) demonstrated that grape polyphenols inhibit the depletion of endogenous α-tocopherol, ubiquinone-10 and total glutathione in minced mackerel (Scomber scombrus) muscle and horse mackerel fillets. In addition, other authors have reported high levels of inhibition of lipid oxidation in minced horse mackerel during frozen storage when red (Pazos et al., 2005a; Sánchez-Alonso et al., 2007b; Sánchez-Alonso and Borderías, 2008) and white (Sánchez-Alonso et al., 2008) grape dietary fibre concentrate was added, respectively. All of these studies have been carried out in very fatty marine fish species while, to our knowledge, there are no studies about the possible utilization and the effects of grape pomace extracts in the minced flesh of a freshwater fish species such as the rainbow trout (Oncorhynchus mykiss), that represent the most important product of aquaculture in Northern Italy generating a considerable volume of business (API, 2011). Although this fish has marked differences than those of the marine species, in fact its flesh showed a lower content of fat and polyunsaturated fatty acids as well as for BAs precursor amino acids, in the form of minced product is itself susceptible to oxidation and consequently have a minor shelf life as happens in the seafood products. Considering these assumptions, the aim of this study was to evaluate if the addition of red grape pomace (RGP) extract, at different concentrations, in rainbow trout minced muscles would further enhanced its health features with adding beneficial effects for the consumer, such as improving the shelf life and consequently the reduction of lipid peroxidation and of BAs formation. Moreover the RGP use could provide fillets with natural antioxidants which are known as beneficial for consumers.
Red grape pomace and preparation of extracts
The RGP samples from wine grape (Vitis vinifera L.) “Barbera” cultivars were provided by a local wine processing plant located in the North West of Italy. RGP consisting of skins, seeds, and a small amount of stem was frozen immediately after pressing and then freeze-dried and ground in a Cyclotec mill to pass a 1 mm screen. The powders obtained were vacuum-sealed in oxygen barrier bags (Platone, Torino, Italy) and stored at -20°C for later analysis. RGP ethanol extracts were prepared according to the method described by Yilmaz and Toledo (2004). Briefly, 1g of freeze dried powder was mixed with absolute ethanol at a ratio of 1:10 (w/v). The mixture was sonicated for 15 min, and shaken for 30 min at room temperature, then centrifuged at 4 °C for 20 min at 5000 g. Supernatant was filtered through Whatman filter paper (Whatman No:4) and then evaporated under vacuum at room temperature using a Speedvac (SC210A; Savant Instruments, Farmingdale, NY, USA). The residue was solubilised in a mixture of distilled water-ethanol (1:1 v/v) in order to reach a final concentration of 10 mg/ml.
Preparation of minced fish muscle samples
Minced trout muscle (MTM) was prepared from ice-stored rainbow trout bought in a local supermarket. Fish were filleted and after removing the skin and the bones, the muscle was ground through a grinder (Platone, Torino, Italy). MTM samples were shaped into patties 11 cm in diameter and 1.5 cm thick using a glass shaper. For each group, a total of 6 patty samples were produced. To study dose-dependent efficacy, RGP extracts were added separately to each MTM portion to give a final concentration of 0% (control, 5 mL distilled water-ethanol mixture per 100 g MTM), 1% (1 mL RGP extract + 4 mL distilled water per 100 g MTM) and 3% (3 mL RGP extract + 2 mL distilled water per 100 g MTM). In order to compare the effectiveness of the RGP extract, a further group, containing a synthetic antioxidant, was prepared in the same manner as the control group using 5 mL of 0.5% (v/w) of a vitamin E analogue (Trolox™, (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, Sigma-Aldrich, Milan, Italy) solution.
The patty groups were then wrapped in plastic film and stored in a refrigerated room (4 ±1°C). After 1 and 6 days of refrigerated storage the treated MTM samples were analysed for physical and chemical analysis.
pH measurement
The pH measurement was performed according to Bao et al. (2009). A portion (3 g) of MTM was homogenized with 20 mL of distilled water. The homogenate was centrifuged at 10000 rpm for 10 min at 4 °C and the supernatant was collected. The pH of the supernatant was then measured with a Crison pH meter, model MicropH 2001 (Crison Instruments, Barcelona, Spain).
TBARS measurement
Lipid oxidation was determined on the MTM after different days of storage at +4 °C using modified thiobarbituric acid (TBA) analysis according to the iron-induced thiobarbituric acid reactive substances (TBARS) procedure described by Sárraga et al. (2006). The assay was performed at 30 min of incubation, and absorbance was read at 532 nm. Liquid malonaldehyde bis–(diethyl acetal) (MDA) (Aldrich Chemical Co. Ltd., Dorset, UK) was used as standard to determine the linear standard response and recovery. The TBARS values were expressed as mg of MDA per kilogram of muscle tissue. All measurements were performed in triplicate.
Colour measurements
MTM colour was measured using a bench colorimeter Chroma Meter CR-400 Konica Minolta Sensing (Minolta Sensing Inc, Osaka, Japan) in the CIELAB colour space (CIE, 1976). Lightness (L*), redness (a*), and yellowness (b*) were recorded. Three readings, with measurements standardized with respect to the white calibration plate, were taken on the surface of the MTM and averaged.
Sample preparation for HPLC-MS/MS analyses
Two extraction procedures were carried out on each MTM lyophilized sample. In order to measure BAs concentration, 300 mg of freeze-dried flesh were extracted using 3 mL of 5 mM heptafluorobutanoic acid in water – methanol 1:1 v/v, centrifuged (10000 rpm) and spiked with synephrine as internal standard (final concentration 100 µg/L). Flavonoids were extracted by treating 100 mg of MTM with 3 mL of water – methanol 9:1 v/v without internal standard. Each extraction was conducted in triplicate.
Determination of flavonoids
The chromatographic separations were run on a Dionex Ultimate 3000 HPLC coupled with a LTQ-Orbitrap ion trap mass spectrometer (Thermo, Bremen, Germany) through an atmospheric pressure interface and an ESI ion source. Samples were analyzed in order to detect flavonoids using a RP C18 column (Phenomenex Luna 150 × 2.1 mm, 3 μm particle size) at 200 μL/min flow rate. Gradient mobile phase composition was adopted: 95/5 to 0/100 in 30 minutes acetonitrile/0.05% formic acid in water. Injection volume was 20 μL. We used a procedure described in literature (Wang et al., 2010) with slight modifications. The analyses were run using MS/MS acquisition. The tuning parameters adopted for the ESI source were as follows: source voltage 4.5 kV, capillary voltage 10 V, tube lens 80 V, capillary temperature 265°C.
The transitions were as follows: (-) 291 to 123 m/z (collision energy 30) for (-) epicatechin, (+) 291 to 123 m/z (collision energy 30) for catechin, (+) 319 to 273 m/z (collision energy 30) for myricetin, (+) 303 to 257 m/z (collision energy 30) for quercetin. The retention times of the analytes were 8.04 min epicatechin, 8.80 min catechin, 12.53 min myricetin, 14.38 min quercetin. The method show a satisfactory linearity in the range 0.01 (LLOQ) – 500 μg/g.
Fatty acid (FA) composition was determined on the MTM lyophilized samples. The lipid extraction of the samples was performed according to Peiretti and Meineri (2008); the extract was expressed as crude fat and used for the trans-methylation of the FAs. The FA methyl esters in hexane were then injected into a gas chromatograph (Dani Instruments S.P.A.GC1000 DPC; Cologno Monzese, Italy) equipped with a flame ionisation detector (FID). The separation of the FA methyl esters was performed using a Famewax™ fused silica capillary column (30 m x 0.25 mm [i.d.], 0.25 µm) (Restek Corporation, Bellefonte, PA, USA). The peak area was measured using a Dani Data Station DDS 1000. Each peak was identified and quantified on the basis of pure methyl ester standards (Restek Corporation, Bellefonte, PA, USA). All analyses were performed in triplicate.
Determination of biogenic amines (BAs)
In order to measure BAs concentration, 300 mg of freeze-dried MTM were extracted using 3 mL of 5 mM heptafluorobutanoic acid in water – methanol 1:1 v/v, centrifuged (10000 rpm) and spiked with synephrine as internal standard (final concentration 100 µg/L). The chromatographic separations were run on a Varian 920-LC HPLC coupled with a triple quadrupole mass spectrometer 320-MS (Varian, Leinì, Italy) through an atmospheric pressure interface and an ESI ion source. Samples were analyzed for detecting BAs using a RP C18 column (Phenomenex Luna 150 × 2.1 mm, 3 μm particle size) at 200 μL/min flow rate. Gradient mobile phase composition was adopted: 95/5 to 40/60 in 25 minutes 5 mM heptafluorobutanoic acid/methanol. Injection volume was 20 μL. We used a procedure described in literature (Calbiani et al., 2005) with slight modifications. The analyses were run using MS/MS acquisition. The tuning parameters adopted for the ESI source were: source voltage 4.5 kV, capillary voltage 82 V, shield voltage 450 V, drying gas temperature 300°C. The followed transitions were 89 to 72 m/z (collision energy 7.5 V) for putrescine, 103 to 86 m/z (collision energy 6.5 V) for cadaverine, 146 to 129 m/z (collision energy 8.5 V) for spermidine, 138 to 121 m/z (collision energy 6.5 V) for tyramine, 203 to 129 m/z (collision energy 8.5 V) for spermine, 112 to 95 m/z (collision energy 7.0 V) for histamine, 154 to
137 m/z (collision energy 10.5 V) for dopamine and 168 to 150 m/z (collision energy 5.5 V) for synephrine. The retention times of the analytes were 15.8 min (putrescine), 16.2 min (cadaverine), 22.1 (spermidine), 15.9 (tyramine), 24.8 (spermine), 13.4 (dopamine), 15.6 (histamine) and 12.4 min (synephrine). The method show a satisfactory linearity in the range 0.01 (LLOQ) – 500 μg/g .
Statistical Analysis
Data were evaluated by means of the GLM procedure of the SPSS software package (version 11.5.1 for Windows, SPSS Inc., USA) and considering the treatment, days of storage, and their interaction as the main effects. The data were presented as the means of each group and the standard error of the means (SEM) together with the significance levels of the main effects and interactions. Significance was established at P < 0.05.
Results and Discussion
Table 1 shows the results of the pH, TBARS and colour variables of trolox-treated fish flesh after 1 and 6 days of refrigerated storage. None of the parameters differed significantly. Considering the control and RGP-treated groups (Table 2), differences due to the treatment appeared in pH, TBARS and flesh colour parameters. Days of storage influenced all freshness indicators, with the exception of pH, while an interaction was recorded only for TBARS and redness (a*) values.
pH was about 6.0–6.5 for fresh fish, and increased during storage. The limit of pH acceptability is usually 6.8–7.0. During storage at 4 °C, the pH values of the RGP0 and RGP1 group samples decreased, while they increased slightly for the RGP3 group. Similar values of pH were found by Erkan et al. (2011) in bluefish (Pomatomus saltatrix) treated with thyme and laurel essential oil after 9 days of iced storage, while lower levels of pH (around 6.2) were found in cod (Gadus morhua) Frankfurter sausages containing different levels of food additive ingredients (Cardoso et al., 2009).
TBARS were measured as an indicator of secondary oxidation products. The samples treated with RGP were more stable to oxidation than the control sample. Flesh supplemented with RGP showed TBARS values 4 times lower than the control group (RGP0 = 2.86, RGP1 = 0.70, and RGP3 = 0.71, respectively) regardless of the level of inclusion of RGP, after 6 days of refrigerated storage. It is interesting to note that after the same period of storage the TBARS values in the groups treated with RGP were also lower than those treated with Trolox, a synthetic compound with known antioxidant effects. Numerous studies carried out in marine fatty fish, such as mackerel, have reported high levels of inhibition of lipid oxidation due to the use of grape antioxidants (Pazos et al., 2005a, 2010; Sánchez-Alonso et al., 2007b; Sánchez-Alonso and Borderías, 2008). Similar effects were found by Yerlikaya and Gokoglu (2008), who showed that grape seed extracts (GSE) were effective in retarding the increase of TBA levels in bonito (Sarda sarda) fillets during frozen storage. Özalp Özen et al. (2011) observed that the formation of lipid hydroperoxides and TBARS was significantly inhibited by the addition of GSE in chub mackerel (Scomber japonicus) minced muscle during frozen storage.
Colour parameters (lightness, redness and yellowness) were evaluated to detect whether RGP addition caused any changes in the treated samples. The control sample (RGP0) showed the highest values for lightness (L), while this parameter decreased with the increase in RGP inclusion level. The lowest L levels were recorded after six days of refrigerated storage (RGP0 = 61.0, RGP1 = 51.3, and RGP3 = 47.1, respectively). A similar trend of colour parameter values was also detected in the minced muscle of chub mackerel added with GSE (Özalp Özen et al., 2011). These authors found that GSE added samples had the highest redness and the lowest lightness and yellowness values, whereas a different trend was observed in a physical study carried out on minced horse mackerel muscle with a white-grape by-product (WGBP) added as an ingredient (Sánchez-Alonso et al., 2007a). These authors reported that the control sample score was highest for lightness throughout the study, though the differences were not significant, while slight differences between the samples with WGBP were detected in the course of storage.
In our study, redness values (a*) increased with the increase of the addition of RGP (RGP0 = 5.1, RGP1 = 6.0, and RGP3 = 7.0, respectively) after one day of refrigerated storage. This increase is related to the incorporation of red pigments present in the RGP. Similar redness values were also reported with the addition at concentrations of 0.2 and 4% of red grape antioxidant dietary fibre in horse mackerel minced muscle (Sánchez-Alonso and Borderías, 2008). On the other hand, after six days of conservation, the a* values decreased for all treatments and in particular in the control group (RGP0 = 0.1, RGP1 = 2.6, and RGP3 = 4.7, respectively). This decrease in redness could be related to fish flesh lipid oxidation, in fact decreases in redness are an indirect means of following haemoglobin-mediated lipid oxidation in fish muscle (Undeland et al., 1998) (haemoglobin being a well-known activator of lipid oxidation). This trend confirms the efficacy of RGP in delaying lipid oxidation, as previously demonstrated in a study carried out by Pazos et al. (2005b), where grape phenolics were added to mackerel minced muscle during frozen storage at -10 °C. Eymard et al. (2010) evaluated and compared the antioxidant activities of different synthetic and natural antioxidants added to horse mackerel minced muscles. They reported that mince washed with propyl gallate (PG) had a high a* value, indicating a redder colour, and also had lower b* and L* values compared with the control group. This could indicate that PG, as well as grape polyphenols, prevented the oxidation of the heme proteins, hemoglobin and myoglobin, which are red in their reduced form and brown in their oxidized ferric form. Many plant extracts have a dark colour and may affect the visual perception of final products, altering the fillet colour, as demonstrated in herring fillets marinated with different solutions containing berry powder (Sampels et al., 2010).
In the present study, yellowness values (b*) decreased significantly with the increase in RGP supplementation after one day of storage (RGP0 = 14.1, RGP1 = 8.5, and RGP3 = 5.2, respectively), while lower values were recorded in the RGP1 and RGP3 groups (RGP1 = 5.2 and RGP3 = 3.7, respectively) at six days of storage. On the other hand, our results were in contrast with those reported utilising white grape antioxidant dietary fibre (WGDF) in minced horse
mackerel muscle during frozen storage (Sánchez-Alonso et al., 2008). In this work, the authors observed that yellowness did not change significantly in samples with WGDF, but did increase significantly in samples without WGDF during frozen storage. The authors hypothesised that this trend could be related to loss of redness, since oxygenated heme proteins confer a bright colour, while oxidized pigments are brown (met-heme proteins).
As a general categorization proposed by Shi et al. (2003), phenolic compounds in grape can be divided into two groups: phenolic acids (precursors of flavonoids) and flavonoids. In this study, flavonoids were analytically investigated both in the RGP extract and in the MTM samples. The four most relevant flavonoids [(+)-Catechin, (-)-Epicatechin, Myricetin, and Quercetin] were evaluated using the HPLC-MS system. The standard mixture separation together with a sample analysis chromatogram are shown in Figure 2. Amongst these flavonoids, (+)-catechin was the major compound detected in the RGP extract, with an average value of 227.53 µg/g followed by (-)-epicatechin with 127.63 µg/g and myricetin with 14.96 µg/g, while quercetin content was not detectable, being below the instrument limit of detection (LOD) corresponding to 0.01 µg/g. The same analyses were also carried out in MTM samples treated or not with RGP extracts during refrigerated storage and the results are illustrated in Figure 1. No flavonoids were detected in the control groups (RGP0) as was of course to be expected, while proportional concentrations of (+)-catechin and (-)-epicatechin were found in the RGP1 and RGP3 groups, whereas myricetin was undetectable (below LOD). (-)-Epicatechin showed concentrations 3-4 times higher than (+)-catechin, probably due to the high oxidative susceptibility of the latter, that has been found to be the most powerful scavenger among the different classes of flavonoids (Perumalla and Hettiarachchy, 2011). As far as storage time is concerned, it is interesting to note that neither (+)-catechin nor (-)-epicatechin were detectable in the RGP1 group after one day of storage, while detectable levels were found after six days of storage. Different parameters such as polarity, solubility, diffusion, and affinity to protein or lipid of antioxidants might also need to be considered when working with complex food matrices. Interaction between phenols and protein has been
reported to be related not only to their polarity (Heinonen et al., 1998), but also to the amino acid composition and the structure of the protein involved (Kroll and Rawler, 2001). Different affinity for muscle protein of the various antioxidants retained in the mince has been demonstrated in minced horse mackerel (Eymard et al., 2010). In a glassing experiment, phenolics extracted from grape have been shown to be quickly transferred from the aqueous phase into fish muscle and to have a high affinity for the fish muscle membrane (Pazos et al., 2006b).
As far as FA composition of treated or untreated MTM is concerned, C18:0, C20:4n6 and C22:1n9 contents were influenced by RGP treatment, while all other FAs were not statistically affected by RGP treatment. On the other hand, none of the FAs detected were affected, neither by conservation time nor treatment and time interaction (Table 3). The present study showed that the addiction of RGP extracts did not promote or protect the oxidation of the major fish n3 PUFAs (C20:5n3, C22:5n3 and C22:6n3), which have been shown to have various health benefits. By contrast, in a study carried out by Pazos et al., (2005b), a significant degradation of n3 PUFA was observed after 10 weeks of freezer storage in untreated mackerel muscles, while upon extended freezer storage a significant protection of n3 PUFA was observed using grape fractions as antioxidants. The differences obtained in the two studies could be due to the different cultivars of grapes used and/or the purity of RGP extract, which is reflected in a different polyphenol content.
The European Union (EU) has established regulations for histamine levels, such that they should be below 100 ppm in raw fish, and below 200 ppm in salted fish for species belonging to the
Scombridae and Clupeidae families, while in the USA the Food and Drug Administration (FDA,
1998) sets the limit of histamine tolerance in fresh fish at 50 ppm.
In this study, the BA concentration showed significant differences due to the antioxidant treatment (decreased with antioxidant addition), storage time (increased with time) and their
interaction only in MTM cadaverine (CAD) content (Table 4). The standard compounds and a
detected in fish flesh, but their levels did not differ amongst the different groups. By contrast, tyramine, histamine and dopamine were not detected. The analytical determination of BAs suggested a partial significant protective effect of RGP treatment in the degradation of proteins. CAD is liberated from lysine by the action of bacterial decarboxylases and increases with the disintegration of sample proteins. Therefore, given that in our study, CAD content was reduced proportionally to RGP quantity used, an antimicrobial effect against the food spoilage microorganisms due to the grape pomace flavonoids could be supposed, as previously reported in beef patties (Sagdic et al., 2011). PUT and CAD are usually the predominant compounds produced during storage, as reported in four commercially important fish species (hake, mackerel, sea bass and sea bream) processed to produce fresh-fish based hamburgers (Barbuzzi et al., 2009). Considering the use of natural antimicrobials and antioxidants, Özogul et al. (2011) showed that the use of rosemary and sage tea extracts significantly reduced PUT and CAD contents in sardine (Sardina pilchardus) fillets stored at 3 ± 1 °C. Comparing our results, similar levels of PUT and higher CAD contents were found in non-vacuum-packed flesh of carp after six days of storage at 3°C, while the application of vacuum packaging prolonged shelf life by about 4–5 days (Křížek et al., 2004). Similar levels of PUT and spermidine were found in a work carried out on the effects of vacuum packaging followed by high-energy electron beam irradiation on the shelf life of fillets of rainbow trout after 7 days of refrigerated storage at 3.5°C (Křížek et al., 2012). During that storage time, the irradiated samples did not show detectable levels of CAD. The use of classical and new preservation techniques such as vacuum packaging and irradiation, respectively, could enhance the effect of natural antimicrobials and antioxidants and help to extend the shelf life of fish-based foods.
In the light of the experimental results, it might be concluded that the Red Grape Pomace extracts were effective in delaying lipid oxidation and cadaverine formation in minced trout muscles after six days of refrigerated storage. The use of RGP therefore may enhance the shelf life
and the safety of rainbow trout minced muscles and simultaneously provide the fillets with natural antioxidants beneficial for consumers at higher concentration levels.
Acknowledgments
Financial support for this work was provided by the Ministero delle Politiche Agricole Alimentari e Forestali, Italy - Project title: Azione concertata per l’identificazione di contributi scientifici per lo sviluppo dell’acquacoltura biologica in Italia – Acquacoltura biologica. The authors thank Dr. Ilaria Barchi for her aid during flavonoid and biogenic amine analysis and Mr. Luciano Sola for providing the red grape pomace samples. All the authors contributed equally to the work described in this paper.
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Table 1
pH, thiobarbituric acid reactive substances (TBARS, as mg MDA/kg of muscle) and colour variables (means ± standard error) of minced trout muscle treated with 5 mL of 0.5% (v/w) of
Trolox as an antioxidant, after 1 and 6 days of refrigerated storage. Trolox Significance Days 1 6 pH 6.67±0.14 6.62±0.01 0.755 TBARS 1.28±0.23 0.99±0.14 0.348 L 69.3±1.2 65.3±0.9 0.055 a* 4.65±0.24 3.53±0.45 0.095 b* 13.2±0.1 13.1±0.7 0.879
Table 2
pH, thiobarbituric acid reactive substances (TBARS, as mg MDA/kg of muscle) and colour
variables of minced trout muscle treated with red grape pomace (RGP) extracts at three inclusion
levels (0%, 1% and 3%) ranged according to treatment and time
RGP0 RGP1 RGP3
SEM Significance
Days 1 6 1 6 1 6 Treatment Time Interaction
pH 6.78 6.64 6.76 6.61 6.47 6.56 0.01 0.007 0.168 0.078
TBARS 1.07 2.86 1.07 0.70 1.06 0.71 0.07 0.000 0.016 0.000
L 65.5 61.0 56.2 51.3 52.0 47.1 2.49 0.000 0.000 0.956
a* 5.13 0.07 5.95 2.56 6.96 4.70 0.24 0.000 0.000 0.001
Table 3
Fatty acid (FA) composition (% total fatty acids) of minced trout muscle treated with Red Grape Pomace (RGP) extracts at three inclusion levels (0%, 1% and 3%) ranged according to treatment
and time
RGP0 RGP1 RGP3
SEM Significance
Days 1 6 1 6 1 6 Treatment Time Interaction
C14:0 2.50 2.30 2.47 2.40 2.77 2.53 0.05 0.152 0.140 0.795 C16:0 16.8 16.9 16.6 16.8 16.4 16.5 0.20 0.253 0.571 0.964 C16:1n7 3.57 3.40 3.50 3.37 3.90 3.53 0.07 0.183 0.100 0.718 C18:0 5.10 5.43 4.90 4.80 4.33 4.43 0.10 0.002 0.474 0.519 C18:1n9 15.4 15.2 15.9 15.1 16.8 15.8 0.97 0.239 0.183 0.751 C18:1n7 2.47 2.50 2.53 2.47 2.60 2.53 0.01 0.363 0.504 0.634 C18:2n6 12.3 11.5 12.5 12.0 13.8 12.7 0.82 0.068 0.078 0.849 C18:3n3 1.40 1.30 1.43 1.37 1.53 1.43 0.09 0.084 0.069 0.940 C18:4n3 0.40 0.33 0.53 0.50 0.60 0.57 0.04 0.176 0.631 0.985 C20:1n9 1.67 1.60 1.73 1.63 2.00 1.77 0.03 0.058 0.122 0.676 C20:2n6 0.80 0.77 0.73 0.77 0.80 0.80 0.003 0.344 1.000 0.619 C20:3n3 0.37 0.37 0.17 0.33 0.50 0.50 0.06 0.260 0.645 0.803 C20:4n6 1.27 1.27 1.13 1.23 1.10 1.13 0.01 0.020 0.251 0.546 C20:4n3 0.43 0.43 0.70 0.40 0.70 0.67 0.07 0.290 0.385 0.568 C20:5n3 5.77 5.53 5.57 5.60 5.47 5.47 0.07 0.519 0.614 0.663 C22:1n9 1.13 1.10 1.20 1.13 1.37 1.33 0.01 0.012 0.439 0.961 C22:5n3 3.00 3.07 3.03 3.07 2.90 2.93 0.01 0.116 0.410 0.956 C22:6n3 24.6 26.0 25.1 26.2 22.2 24.5 4.39 0.168 0.139 0.885
Table 4
Biogenic amine contents (μg/g) of minced trout muscle treated with Red Grape Pomace (RGP) extracts at three inclusion levels (0%, 1% and 3%) ranged according to treatment and time
RGP0 RGP1 RGP3
SEM Significance
Days 1 6 1 6 1 6 Treatment Time Interaction
Putrescine 4.21 4.25 4.58 5.87 3.95 4.38 3.65 0.328 0.365 0.712 Cadaverine 1.10 3.29 0.79 1.76 0.30 0.63 0.12 0.000 0.000 0.000 Spermidine 2.86 2.34 3.39 2.97 2.51 3.61 3.09 0.701 0.922 0.481 Spermine n.d. n.d. 1.10 0.57 0.52 0.57 0.09 0.143 0.224 0.145 Tyramine n.d. n.d. n.d. n.d. n.d. n.d. - - - -Histamine n.d. n.d. n.d. n.d. n.d. n.d. - - - -Dopamine n.d. n.d. n.d. n.d. n.d. n.d. - - -
-Figure 1. Concentration of (+) Catechin (A) and (-) Epicatechin(B) in minced trout muscles (n=3)
treated with Red Grape Pomace (RGP) extracts at three inclusion levels (0%, 1% and 3%) after one day and six days of storage (4 ±1°C), respectively (error bars show standard deviation)
RT:0,00 - 30,00SM:7G 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min) 0 20 40 60 80 100 R el at iv e Ab un da nc e 0 20 40 60 80 100 R el at iv e Ab un da nc e 0 20 40 60 80 100 R el at iv e Ab un da nc e 8,80 7,97 12,53 14,31 NL: 3,30E6 m/z= 123,0368-123,0492 F: FTMS + c ESI Full ms2 291,00@cid30,00 [80,00-305,00] MS MIX_STD_FLAVO_5ppm NL: 2,00E6 m/z= 273,0248-273,0522 F: FTMS + c ESI Full ms2 319,00@cid30,00 [85,00-325,00] MS MIX_STD_FLAVO_5ppm NL: 2,00E6 m/z= 285,0239-285,0525 F: FTMS + c ESI Full ms2 303,00@cid30,00 [80,00-315,00] MS MIX_STD_FLAVO_5ppm RT:0,00 - 30,00SM:7G 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min) 0 20 40 60 80 100 R el at iv e Ab un da nc e 0 20 40 60 80 100 R el at iv e Ab un da nc e 0 20 40 60 80 100 R el at iv e Ab un da nc e NL: 3,30E3 m/z= 123,0368-123,0492 F: FTMS + c ESI Full ms2 291,00@cid30,00 [80,00-305,00] MS V3AT6 NL: 2,00E3 m/z= 273,0248-273,0522 F: FTMS + c ESI Full ms2 319,00@cid30,00 [85,00-325,00] MS V3AT6 NL: 2,00E3 m/z= 285,0239-285,0525 F: FTMS + c ESI Full ms2 303,00@cid30,00 [80,00-315,00] MS V3AT6
Figure 2. SRM traces by HPLC-MS2 analysis of a trout extract sample (3% RGP extract treated, 6
day storage) (on the right) and of a flavonoid standard mixture (1 mg/L concentration) (on the left). Analytes are reported in elution order: epicatechin, catechin, myricetin, quercetin. HPLC and SRM conditions are reported in the experimental section.
Figure 3. SRM traces by HPLC-MS2 analysis of a trout extract sample (3% RGP extract treated, 6
day storage) (on the right) and of a standard mixture of amines (1 mg/L concentration) (on the left). Analytes are reported in elution order: synephrine, dopamine, putrescine, tyramine, cadaverine, histamine, spermidine, spermine. HPLC and SRM conditions are reported in the experimental section.
