Influence of fat content on physico chemical and oxidative stability of foal liver pâté

The effect of fat content on physicochemical properties and lipid and protein stability of foal liver pâté was studied. For this purpose, two batches (10 units per batch) of foal liver pâté with different pork back fat content 30% (30F) and 40% (40F) were manufactured. 30F foal liver pâté was darker (lower L value, P > 0.05), redder (higher a, Pb0.001) and harder (higher hardness value; Pb0.001) compared to those with 40F. Fat level was closely related to the calorific value of foal liver pâté, being more calorific in those with higher fat contents (352 kcal100 g; Pb0.001). Regarding total Fe content, 30F foal liver pâté showed the higher value (4.19 mg100 g; Pb0.01). Oxidative stability of foal liver pâté was influenced by fat level. 40F foal liver pâté presented higher TBARS and lower carbonyl contents compared to 30F ones (Pb0.001). Finally, foal pâtés with the two different fat contents had significantly (Pb0.001) different n−6n−3 ratios, foal liver pâtés with 30F showed the lowest values (9.97) compared to those with 40F content

Influence of fat content on physico-chemical and oxidative stability of foal liver pâté José M. Lorenzo ⁎ , Mirian Pateiro Centro Tecnológico de la Carne de Galicia, Rúa Galicia Nº 4, Parque Tecnológico de Galicia, San Cibrán das Viñas, 32900 Ourense, Spain abstractarticle info Article history: Received 7 February 2013 Received in revised form 10 April 2013 Accepted 13 April 2013 Keywords: Fat content Foal liver pâté Lipid and protein oxidation Physico-chemical properties The effect of fat content on physico-chemical properties and lipid and protein stability offoalliver pâté was stud- ied. For this purpose, two batches (10 units per batch) of foal liver pâté with different pork back fat content [30% (30F) and 40% (40F)] were manufactured. 30F foal liver pâté was darker (lower L* value, P > 0.05), redder (higher a*, P b 0.001) and harder (higher hard- ness value; P b 0.001) compared to those with 40F. Fat level was closely related to the calorific value of foal liver pâté, being more calorific in those with higher fat contents (352 kcal/100 g; P b 0.001). Regarding total Fe con- tent, 30F foal liver pâté showed the higher value (4.19 mg/100 g; P b 0.01). Oxidative stability of foal liver pâté was influenced by fat level. 40F foal liver pâté presented higher TBARS and lower carbonyl contents compared to 30F ones (P b 0.001). Finally, foal pâtés with the two different fat contents had significantly (P b 0.001) different n− 6/n− 3 ratios, foal liver pâtés with 30F showed the lowest values (9.97) compared to those with 40F content (13.41). © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Liver pâté, a traditional cooked meat product consumed in many countries, particularly in Europe (Denmark, France, Germany, Spain), is usually considered to be of high quality (Le Ba & Zuber, 1996). It con- sists of minced liver, fat and meat mixed with water and different addi- tives, and is packed in glass containers and thermally treated. This product is characterized by its high iron content, in some cases provid- ing up to 40% of daily requirements (Mataix & Aranceta, 2002). Due to its chemical composition (high amounts of fat and non-heme iron, and low content of natural antioxidants) and its manufacturing process, this product is susceptible to lipid oxidation (Estévez, Ramírez, Ventanas, & Cava, 2007; Russell, Lynch, Lynch, & Kerry, 2003). Horse meat is characterized by low fat (6.63 g/100 g) and cholester- ol contents (61 mg/100 g), and is rich in iron (3.89 mg/100 g) and vita- min of the B group (Badiani, Nanni, Gatta, Tolomelli, & Manfredini, 1997). This meat has a favorable dietetic fatty acid profile, with a high content of unsaturated fatty acids relative to saturated acids and con- tains a greater proportion of components from the α-linolenic fatty acid family (Lorenzo, Fuciños, Purriños, & Franco, 2010; Sarriés, Murray, Troy, & Beriain, 2006; Tateo, De Palo, Ceci, & Centoducati, 2008). These nutritional characteristics mean that this type of meat may be considered as an alternative meat (Robelin, Boccard, Martin-Rosset, Jussiaux, & Trillaud-Geyl, 1984). Consumption has in- creased in recent years, with Spain being the fourth major producer of horse meat in the U.E. in 2009 with 6400 tons (FAOSTAT, 2009), but is still not comparable to the consumption of other meats such as beef, chicken or pork (Franco et al., 2011; Lombardi-Boccia, Lanzi, & Aguzzi, 2005). These increases might be due to changes in attitude towards this type of meat and the wish of consumers to taste new meat products (Hoffman & Wiklund, 2006; Sarriés et al., 2006). The meat is mainly consumed as fresh meat however, it is starting to be used in the manu- facture of meat products, such as dry-cured sausages (Lorenzo, Temperán, Bermúdez, Cobas, & Purriños, 2012). Although, foal meat represents a good alternative for these products, some manufactures add pork fat to compensate for its low fat content. Several studies have evaluated the meat quality of foal fresh meat (Franco et al., 2011; Juárez et al., 2009; Lanza, Landi, Scerra, Galofaro, & Pennisi, 2009; Lorenzo et al., 2010; Sarriés & Beriain, 2005, 2006; Sarriés et al., 2006; Tateo et al., 2008), but there is little information about the physico-chemical and nutritional quality of products made withthis meat (Lorenzo et al., 2012).These types of productswould uti- lize the parts of the carcass that have less value for fresh consumption. Some studies concerning the physico-chemical characteristics of pork, duck, goat and ostrich liver pâté (Dalmás, Bezerra, Morgano, Milani, & Madruga, 2011; Delgado-Pando, Cofrades, Rodríguez-Salas, & Jiménez-Colmenero, 2011; Estévez, Morcuende, Ramírez, Ventanas, & Cava, 2004; Estévez, Ventanas, & Cava, 2005; Estévez, Ventanas, Cava, & Puolanne, 2005; Fernández-López, Sayas-Barberá, Sendra, & Pérez-Álvarez, 2004; Russell et al., 2003) have been carried out. Howev- er, foal meat has never been used in the production of these products. Furthermore, the level of fat has been demonstrated to influence the nutritional and sensory characteristics of these products (M. Estévez, S. Ventanas et al., 2005; M. Estévez, J. Ventanas et al., 2005). The aim of this work is to develop a new value-added foal pâté and study the Meat Science 95 (2013) 330–335 ⁎ Corresponding author. Tel.: +34 988 548 277; fax: +34 988 548 276. E-mail address: jmlorenzo@ceteca.net (J.M. Lorenzo). 0309-1740/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2013.04.045 Contents lists available at SciVerse ScienceDirect Meat Science journal homepage: www.elsevier.com/locate/meatsci effect of different fat levels on the physico-chemical and oxidation sta- bility characteristics of this product. 2. Material and methods 2.1. Manufacture of the foal liver pâté Two different formulations of foal liver pâté were considered, differ- entiated in terms of fat content [30% fat (30F) and 40% fat (40F)]. The pâtés were prepared in the pilot plant of the Meat Technology Center of Galicia. The composition of each sample is presented in the Table 1. Foal liver, foal meat (from the hind quarter, composed principally of gluteus medius,semitendinosus and semimembranosus muscles) and subcutaneous fat from commercial slaughter pigs was used as the main ingredients. Foal meat used to the manufacture of pâté had a chemical composition of 76.3% moisture, 20.8% protein and 1.5% fat, while foalliver had values of 69.9%, 23.6% and 1.0% for moisture, protein and fat, respectively. The fatty acid profile of liver, muscle and adipose tissue is shown in Table 2. The day before the preparation, liver and foal meat were ground through 10 mm diameter mincing plate in a cooled chopped (La Minerva, Bologna, Italy) at 4 °C and mixed with the nitrificant ingredients (sodium chloride, sodium nitrite and sodium ascorbate). This blend was kept in darkness and refrigerated until the following day. On the day of manufacture, the fat was chopped using the same conditions used for the meat and liver, and heated in water to 65 °C. Then, the remaining ingredients were added,sodium caseinate to the heated fat, and water, milk powder and potassium phosphates to the meat mixture. Finally, both mixtures were blended to obtain a ho- mogeneous raw paste. The liver pâtés were packed in glass containers prior to thermal treatment (80 °C/30′). The samples were cooled in a blast chiller (−21 °C/30′) and then analyzed. 2.2. Analytical methods 2.2.1. Physico-chemical analysis The pH of samples was measured using a digital pH-meter (Thermo Ori on 710 A+, Cambridgesh ire, UK) equipped wit h a penetration pro be. Color measurements were carried out using a CR-600 color- imeter (Minolta Chroma Meter Measuring Head, Osaka, Japan ). Three measurements were p erformance for each sample. CIELAB space (CIE, 1976): lightness, (L*); redness, (a*); yellowness, (b*) were obtained. Before each series of measurements, the instrument was calibrated using a white ceramic tile. Hue (h ab ) and chroma (C*) were calculated from the a* and b* values according to the formula: C à ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi a à ðÞ 2 þ b à ðÞ 2 q and h ab ¼ acr tan b à a à : Moisture, fat, protein (Kjeldahl N × 6.25) and ash were quanti- fied according to the ISO recommended standards 1442:1997 (ISO, 1997), 1443:1973 (ISO, 1973), 937:1978 (ISO, 1978), and 936:1998 (ISO, 1998), respectively. Briefly, moisture percentage was calculated by weight loss of the sample (5 g) maintained in an oven (Memmert UFP 600, Schwabach, Germany) at 105 °C, until constant weight. Ash percentage was calculated by weight loss of the sample (5 g) in a muffle furnace (Carbolite RWF 1200, Hope Valley, Englan d) in a porcelain capsule at 600 °C until constant weight. For fat content samples (2 g) were subjected to a liquid–solid extraction using petroleum ether in an extractor (Ankom HCI Hydrolysis Syste m, Macedon NY, USA) for 90 min. Fat content was calculated by gravimetric differ- ence. Protein content was determined according to Kjeldahl total ni- trogen method, multiplying the total nitrogen content by 6.25. A sample (1 g) was reacted with sulfuric acid (cupric sulfate was employed as a catalyst) in a digester (Gerhardt Kjeldatherm KB, Bonn, Germany). Organic nitrogen was transformed to amm onium sulfate, which was distilled in alkali conditions (Gerhardt Vapodest 50 Carrousel, Bonn, Germany). 2.2.2. Total Fe content Five gram (5.000 ± 0.001 g) samples were weighed into porce- lai n crucibles for total Fe analysis. The foal pâté samples were incin- erated in a furnace at 450 °C for 12 h. The ash was dissolved in 10 mL of 1 M HNO 3 . For the determination of Fe this soluti on was used directly. The quantifica tion of total Fe content was performed by induc- tively coupled plasma-optical emission spectrosc opy (ICP-OES), using a Thermo-Fisher ICAP 6000 plasma emission spectrometer (Thermo-Fish er, Cambridge, UK), equipped with a radio frequency source of 27.12 MHz, a peristaltic pump, a spraying chamber and a concentric spray nebulizer. The system was totally controlled by ICP software using 99.996% liquid argon plasma gas (Praxair, Madrid, Spain). Operating conditions of the ICP-OES equipment were: reflected power, 1150 W; nebulizer gas flow, 0.7 L/min; auxiliary argon flow, 0.5 L/min; main argon flow, 12 L/min; background correction, 2 points; Table 1 Recipe used for the preparation of foal liver pâtés with different fat contents. Ingredients (%) 30F 40F Foal meat 20 10 Foal liver 33 33 Porcine back fat 30 40 Water 11.5 11.5 Sodium chloride 2 2 Milk powder 2 2 Sodium caseinate 1 1 Potassium phosphate 0.5 0.5 Sodium nitrite 0.05 0.05 Sodium ascorbate 0.025 0.025 Table 2 Fatty acid composition (means ± SD) of liver, muscle and adipose tissue. Liver (from foal) Muscle (from foal) Adipose tissue (from pork) P value SEM C14:0 0.45 ± 0.03 a 2.29 ± 0.09 c 1.24 ± 0.01 b 0.000 0.33 C15:0 0.21 ± 0.02 b 0.31 ± 0.02 c 0.00 ± 0.00 a 0.001 0.06 C16:0 16.58 ± 0.27 a 25.07 ± 0.21 c 23.24 ± 0.12 b 0.000 1.63 C16:1cis-9 1.28 ± 0.03 a 2.55 ± 0.07 b 2.84 ± 0.01 c 0.000 0.30 C17:0 0.63 ± 0.03 c 0.51 ± 0.01 b 0.25 ± 0.03 a 0.002 0.07 C17:1cis-9 0.20 ± 0.01 0.51 ± 0.19 0.26 ± 0.01 0.124 0.07 C18:0 20.88 ± 0.09 c 6.20 ± 0.08 a 11.36 ± 0.07 b 0.000 2.72 C18:1cis-9 9.85 ± 0.92 a 15.94 ± 0.30 b 43.40 ± 0.15 c 0.000 6.52 C18:2n−6 31.64 ± 0.06 c 16.88 ± 0.75 b 14.64 ± 0.13 a 0.000 3.37 C20:0 0.12 ± 0.02 b 0.02 ± 0.02 a 0.09 ± 0.02 ab 0.045 0.02 C20:1 0.17 ± 0.01 a 0.21 ± 0.02 a 0.80 ± 0.01 b 0.000 0.12 C18:3n−3 12.05 ± 0.11 b 25.31 ± 0.46 c 0.86 ± 0.02 a 0.000 4.47 C20:2 0.29 ± 0.01 b 0.24 ± 0.01 a 0.60 ± 0.01 c 0.000 0.07 C20:3n−6 0.44 ± 0.01 c 0.28 ± 0.02 b 0.05 ± 0.02 a 0.001 0.07 C20:3n−3 0.49 ± 0.03 b 0.68 ± 0.01 c 0.10 ± 0.01 a 0.000 0.11 C20:4n−6 3.84 ± 0.09 c 1.82 ± 0.22 b 0.24 ± 0.01 a 0.000 0.66 C20:5n−3 0.12 ± 0.16 0.13 ± 0.09 0.00 ± 0.00 0.505 0.04 C22:6n−3 0.47 ± 0.03 c 0.27 ± 0.03 b 0.00 ± 0.00 a 0.001 0.08 SFA 38.89 ± 0.48 c 34.78 ± 0.24 a 36.18 ± 0.23 b 0.003 0.77 MUFA 11.59 ± 0.95 a 19.36 ± 0.41 b 47.30 ± 0.14 c 0.000 6.86 PUFA 49.51 ± 0.47 c 45.85 ± 0.66 b 16.51 ± 0.09 a 0.000 6.60 TUFA 61.10 ± 0.48 a 65.21 ± 0.24 c 63.81 ± 0.23 b 0.003 0.77 Σn−6 36.38 ± 0.13 c 19.45 ± 0.99 b 15.54 ± 0.11 a 0.000 4.05 Σn−3 13.12 ± 0.33 b 26.39 ± 0.33 c 0.97 ± 0.02 a 0.000 4.64 n− 6/n− 3 2.77 ± 0.06 b 0.73 ± 0.05 a 16.05 ± 0.28 c 0.000 3.03 SFA/TUFA 0.64 ± 0.01 b 0.53 ± 0.01 a 0.56 ± 0.05 a 0.004 0.02 Results expressed as percentage of total fatty acid analyzed. SEM: Standard error of mean. PUFA = Σ (C18:2n−6 + C18:3n− 3 + C20:2 + C20:3n6 + C20:3n3 + C20:4n− 6 + C20:5n3 + C22:6n3). MUFA = Σ (C16:1cis-9 + C17:1cis-9 + C18:1cis-9 + C20:1). TUFA = Σ MUFA + PUFA. SFA = Σ (C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + C20:0). Σn− 6=Σ(C18:2n− 6 + C20:3n− 6 + C20:4n−6). Σn− 3=Σ(C18:3n− 3 + C20:3n− 3 + C20:5n3 + C22:6 n−3). 331J.M. Lorenzo, M. Pateiro / Meat Science 95 (2013) 330–335 integration and reading time, 5 s; replicate number, 3; height of vertical observation, 19 mm; nebulizer pressure, bar and radial torch configura- tion. The operating wavelength was 259.940 nm. Stock solution (1000 mg/L; SCP-SCIENCE, Countaboeuf, France) was used for prepar- ing the standard solutions in 4% HNO 3 , v/v. The concentration range was 0.01 to 50 mg /kg of Fe. The final value was the average of three de- terminations. The results were expressed in mg/100 g of pâté. 2.2.3. Analysis of heme iron Total heme pigments in the samples were determined as hemin after extraction with acidified acetone solution (Hornsey, 1956). Briefly, fresh meat samples (5 g) were dissolved in 1 mL deionized water, 0.5 mL concentrated HCl (37%) and 20 mL of acetone in glass test tubes. The tubes were sealed to reduce evaporation, held at room tem- perature in darkness to minimize pigment fading during the 24 h ex- traction and then filtered through 0.45 μm (Filter Lab, Spain). The absorbance was measured (Agilent 8453, Waldbronn, Germany) at 512 nm. The heme iron content was calculated with the factor 0.0882 μgiron/μg hematin. All samples were assayed in duplicate. The results were expressed as mg/100 g of pâté. 2.2.4. Analysis of non-heme iron The non-heme iron was determined by the ferrozine method (Purchas, Simcock, Knight, & Wilkinson, 2003). Briefly, dry samples of meat (500 mg) were ground using a mortar and pestle, dissolved in a mixture of 3 mL of 0.1 M citrate phosphate buffer (pH 5.5) and 1 mL of 2% ascorbic acid (as reducing agent) in 0.2 M HCl and left to stand at room temperature for 15 min before adding 2 mL of 11.3% trichloroacetic acid. After centrifugation at 3000 g for 10 min the su- pernatant was removed. To 2 mL of the supernatant, 0.8 mL of 10% ammonium acetate and 0.2 mL ferrozine reagent (40 mM) were added and the absorbance was measured at 562 nm. Concentrations were obtained using a standard curve from 0 to 5 mg of iron/L made with ferrous sulfate heptahydrate (Panreac Química S.L.U., Bar- celona, Spain). All samples were assayed in duplicate. The results were expressed in mg/100 g of pâté. 2.2.5. Gross energy determination The gross energy (the heats of combustion of protein, fat and car- bohydrate) was determined in duplicate using an automatic adiabatic bomb calorimeter (Parr 6100, Parr Instrument Company, USA), cali- brated with benzoic acid. The process essentially involves measuring the energy evolved on total combustion of the sample in a stream of oxygen. The results were expressed in kcal/100 g of pâtés. 2.2.6. Texture measurement The Texture Analyzer (TA-XT.plus, Stable Micro Systems, Vienna Court, UK) was used (Bourne, 1978). The penetration test was carried out at room temperature (22 °C) and performed with a 6 mm diam- eter penetration probe linked to a 5 kg cell at a velocity of 0.8 mm/s and for a distance of 8 mm. Hardness (kg/cm 2 ), cohesiveness, springi- ness, gumminess (kg/cm 2 ) and chewiness (kg) were obtained using the available computer software (TEE32 Exponent 4.0.12. Stable Micro Systems, Vienna Court, UK). 2.2.7. Lipid oxidation Lipid stability was evaluated using the method proposed b y Vyncke (1975).Briefly, a meat sample (2 g) was dispersed in 5% tri- chloroacetic acid (10 mL) and homogenized in an Ultra-Turrax (Ika T25 basic, Sta ufen, Germany) for 2 min. The homogenate was maintained at − 10 °C for 10 min and centrifuged at 2360 g for 10 min. The supernatant was filtered through a Whatman No. 1 fil- ter paper. The filtrate (5 mL) was reacted with a 0.02 M TBA solu- tion (5 mL) and incubated i n a water bath at 96 °C for 40 min. The absorbance was measured at 532 nm. Thiobarbituric acid reac- tive substances (TBARS ) values were calculated fro m a standard curve of malonaldehyde with 1,1-3,3 tetraetoxipropane (TEP) and expressed as mg MDA/kg sample. 2.2.8. Protein oxidation Protein carbonyls, as measured by the total carbonyl content, were quantified as described by Oliver, Ahn, Moerman, Goldstein, and Stadtman (1987). Meat samp les were homogenized in 20 mL of 0.15 M KCl buffer for 60 s using an Ultra-Turrax (Ika T25 b asic, Staufen, Germany). Two aliquots of homogenate (0.1 mL) were transferred to Eppendorf vials. Then, proteins were p recipitated in both aliquots by 10% trichloroacetic acid (TCA) (1 mL) and centri- fuged for 5 min at 5000 g. One pellet was treated with 1 mL of 2 N HCl (protein quantification) and the other with 1 mL of 2 M HCl containing 0.2% 2,4-dinitrophenyl hydrazine (DNPH) (carbonyl con- tent). Both samples were incubated for 1 h at room temperature (shak- en every 20 min). After incubation, 10% TCA was added (0.8 mL). The samples were vortexed for 30 s, centrifuged for 5 min at 5000 g and the supernatant removed. The pellet was washed three times with 1 mL of ethanol-ethyl acetate (1:1 v/v) and then was dried under N 2 gas. Finally the pellet was dissolved in 2 mL of 6 M guanidine HCl in 20 mM sodium phosphate buffer (final pH 6.5), stirred and centrifuged for 2 min at 5000 g to remove insoluble fragments. Protein concentra- tion was calculated from the absorbance at 280 nm using bovine serum albumin (BSA) as standard. The amount of carbonyls was expressed as nmol of carbonyl per milligramofprotein using an adsorp- tion coefficient of 21.0 mM −1 cm −1 at 370 nm for protein hydrazones. 2.2.9. Analysis of fatty acid methyl esters Fat was extracted from 5 g of foal pâté, according to Folch, Lees, and Stanley (1957). Lipid extracts were evaporated to dryness under vacuum at 35 °C and stored at − 80 °C until analysis. Lipids were transesterified with a solution of boron trifluoride (14%) in methanol, as described by Carreau and Dubacq (1978). Fifty milli- grams of the extracted lipids were esterified and the FAMEs were stored at − 80 °C until chromatographic analysis. Separation and quantification of FAMEs was carried out using a gas chromatograph, GC-Agilent 6890N (Agilent Technologies Spain, S.L., Madrid, Spain) equipped with a flame ionization detector and an auto- matic sample injector H P 7683, and using a Supelco SPTM-2560 fused silica capillary column (100 m, 0.25 mm i.d., 0.2 μm film thickness, Supelco Inc., Bellafonte, PA, USA). Chromatographic conditions were as follows: initial oven temperature 120 °C (held for 5 min), first ramp at 2 °C/min to 170 °C (held for 15 min), second ramp at 5 °C/min to 200 °C (held for 5 min) and third ramp at 2 °C/min to a final tempera- ture of 235 °C (held for 10 min). The injector and detector were maintained at 260 and 280 °C respectively. Helium was used as carrier gas at a constant flow-rate of 1.1 mL/min, with the column head pres- sure set at 35.56 psi. 1 μL of solution was injected in split mode (1:50). The fatty acids were quantified using nonadecanoic acid methyl ester at 0.3 mg/mL, as internal standard, it was added to samples prior to fat extraction and methylation. Identification of fatty a cids was performed by comparison of the retention times with those of known fatty acids and the results expressed as a percentage of total fatty acids identified. The proportion of polyunsaturated (PUFA), monounsaturated (MUFA), total unsaturated (TUFA) and saturated (SFA) fatty acid contents, and SFA/TUFA, n− 6/n− 3 and nutritional ratio were calculated. 2.3. Statistical analysis An analysis of variance (ANOVA) of one way using SPSS package (SPSS 19.0, Chicago, IL, USA) was performed for all variables in the study. The least squares mean (LSM) were separated using Duncan's t-test. All statistical tests of LSM were performed for a significance level α b 0.05. Correlations between variables were determined by correlation analyses using Pearson's linear correlation coefficients using the above statistical software package. 332 J.M. Lorenzo, M. Pateiro / Meat Science 95 (2013) 330–335 3. Results and discussion 3.1. Effect of fat content on chemical composition of foal liver pâtés The results for the foal pâtés manufactured with different fat levels are presented in Table 3. As expected, the differences in formulation (Table 1) produced significant changes in the proximate composition of the pâtés. 30F foal pâtés had a higher water content than the 40F batch (54.04 vs. 52.48%, P = 0.055). Pearson correlation indicated that moisture contents were positively related to the color attribute, a* (r = 0.53, P b 0.01), in agreement with M. Estévez, S. Ventanas et al. (2005) and Delgado-Pando et al. (2011) who found higher water levels in low-fat pâtés. Fat and protein content were also significantly affected by the fat level showing the opposite behavior. Protein c ontent followed the decreasing order: 30F > 40F (P b 0.001). In contrast, 40F foal pâtés had a higher fat content (26.33 g/100 g foal pâtés) than 30F (23.20 g/100 g foal pâtés) (P b 0.001). These results agree with M. Estévez, S. Ventanas et al. (2005) who found higher protein content and lower fat contents in low-fat pâtés. Regarding ash content, fat level did not significantly affect the batches (3.25–3.26 g/100 g foal pâtés; P = 0.932). Pinho, Ferreira, Casal, Oliveira, and Ferreira (1998) found in15 brands of bovine liver pâtés on sale in Portugal, a moisture content of 53.4%, similar to the present study. The same authors found 11.8% protein, 29.4% fat and 2.6% ash. In comparison to bovine pâtés, the 30F foal pâtés had higher ash and protein contents, and lowervalues for lipids, showing that the 30F foal pâtés could have consumer appeal, being healthier, low-fat meat products. With reference to the chemical forms of iron, significant differ- ences (P ≤ 0.001) between groups were found (Table 3). The total Fe content was higher in pâtés with 30F (4.19 mg/100 g) compared to those with h igh-fat content (3.61 mg/100 g) (P = 0.001). The re- sults agree with M. Estévez, S. Ventanas et al. (2005), who found higher levels of total Fe in pâtés samples with low-fat content. The content of heme iron was si gnificantly (P = 0.000) higher in 30F foal pâtés samples (2.50 mg/100 g) as compared to those with high-fat content (2.22 mg/100 g). On the other ha nd, the levels of non-heme iron were sig nificantly (P = 0.001) higher in 40F foal pâtés samples (1.14 mg/100 g) than the other one (1.01 mg/100 g). These results are similar to those reported by M. Estévez, S. Ventanas et al. (2005), who observed that fat reduction causes increased heme iron and decreased non-heme iron. Fe deficiency is the most prevalent nutritional disorder in the world, especially in developing countries. Knowledge of the levels of total, heminic and non-heminic Fe in meat is of importance, because of the differences in bioavailability of these forms of Fe (Lombardi-Boccia, Martínez-Domínguez, & Aguzii, 2002). Foal pâtés from the two different fat content presented signifi- cantly different caloric values, bein g highe r in those with high-fat content (40F: 352.55 kcal/100 g pâtés and 30F: 315.88 kcal/100 g pâtés, P = 0.000). Thes e results are logical in view of the different fat contents and agree with those reported by (Delgado-Pando et al., 2011; M. Estévez, S. Ventanas et al., 2005) who found lower energy con- tents in the low-fat pâtés. The caloric values were positively related (P b 0.01) to fat level (r = 0.63) and negatively related (P b 0.01) to protein content (r = − 0.67). 3.2. Effect of fat content on physical properties of foal liver pâtés Table 4 shows the physical properties of foalliver pâtés with the two different fat contents. No significant differences were found in pH. 40F foal pâtés samples presented higher pH values and are in agreement with those reported by M. Estévez, S. Ventanas et al. (2005).Thevalues obtained (6.68–6.69) were slightly higher to those described for this type of product (Delgado-Pando et al., 2011; M. Estévez, S. Ventanas et al., 2005; Fernández-López et al., 2004; Hong, Lee, & Min, 2004). pH was positively correlated with fat content (r = 0.44; P b 0.05). Color parameters were affected by fat content (Table 4). These re- sults were expected because the color of pâtés is closely relat ed to the color properties of raw material used for the formulation (M. Estévez, J. Ventanas et al., 2005) and therefore , changes in the pro- por tions of the ingredients might lead to different colors. The results indicated that higher contents of fat and lower contents of meat, in- creased lightness and reduced redness in the samples. The 40F foal pâtés samples were lighter than the 30F samples (54.21 vs. 52.97; P > 0.05, respectively) and was positively correlated with fat con- tent (r = 0.39; P b 0.05) and negatively related with prot ein con- tent (r = −0.48; P b 0.01). 30F foal pâtés samples were redder than 40F samples (a* values: 9.27 vs. 8.06, P b 0.001). a* values were negatively related (P b 0.01) to fat content (r = -0.53) and positively correlated with protein content (r = 0.58). Consequently, 30F foal pâtés samples had a more intense color (Chroma values: 16.33 vs. 15.55; P b 0. 01) with lower values of hue (55.37 vs. 58.78; P b 0.001) compared to foal pâtés with higher fat levels. As expected, the manufacture of foal pâtés with increasing fat levels resulted in products with different textural properties (Table 4). Fat re- duction increased hardness (0.22 vs.0.37kg;Pb 0.001 for 40F and 30F groups, respectively), chewiness (0.11 vs. 0.19 kg mm; P b 0.001 for 40F and 30F groups, respectively), gumminess (0.11 vs.0.21kg; P b 0.001 for 40F and 30F groups, respectively) and cohesiveness (0.49 vs.0.54;Pb 0.01 for 40F and 30F groups, respectively). Fat con- tent was negatively correlated (P b 0.01) with hardness (r = −0.54), chewiness (r = − 0.51) and gumminess (r = − 0.53); and protein content was positively related (P b 0.01) with hardness (r = 0.85), chewiness (r = 0.82) and gumminess (r = 0.83). This behavior is in agreement with M. Estévez, S. Ventanas et al. (2005) in liver pâtés, who observed that fat reduction i ncreased hardness. Howe ver, o ther a uthors (Delgado-Pando et al., 2011; Viana, Silva, Delvivo, Bizzotto, & Silvestre, 2005)foundthatfatre- duction decreased (P b 0.05) penetration force, or had no effect on Table 4 pH, color parameters and instrumental texture of foal liver pâtés from foal with differ- ent fat levels (means ± SD). Fat content SEM P-values 30F 40F pH 6.68 ± 0.02 6.69 ± 0.01 0.01 0.281 Color parameters Luminosity (L*) 52.97 ± 2.01 54.21 ± 1.37 0.40 0.127 Redness (a*) 9.27 ± 0.41 8.06 ± 0.32 0.16 0.000 Yellowness (b*) 13.43 ± 0.48 13.30 ± 0.54 0.11 0.564 Chroma (C*) 16.33 ± 0.51 15.55 ± 0.59 0.15 0.006 Hue (h ab ) 55.37 ± 1.31 58.78 ± 0.73 0.45 0.000 TPA test Hardness (kg) 0.37 ± 0.07 0.22 ± 0.05 0.02 0.000 Springiness (mm) 0.91 ± 0.03 0.94 ± 0.04 0.01 0.171 Chewiness (kg mm) 0.19 ± 0.05 0.11 ± 0.03 0.01 0.000 Gumminess (kg) 0.21 ± 0.05 0.11 ± 0.03 0.01 0.000 Cohesiveness 0.54 ± 0.03 0.49 ± 0.02 0.01 0.005 Table 3 Chemical composition and energy content of foal liver pâtés with different fat levels (means ± SD). Fat content SEM P-values 30F 40F Moisture (%) 54.04 ± 2.24 52.48 ± 0.86 0.41 0.055 Fat (%) 23.20 ± 0.92 26.33 ± 1.53 0.45 0.000 Protein (%) 16.16 ± 0.58 14.99 ± 0.59 0.18 0.000 Ash (%) 3.25 ± 0.20 3.26 ± 0.06 0.03 0.932 Total iron (mg/100 g) 4.19 ± 0.28 3.61 ± 0.39 0.10 0.001 Heme iron (mg/100 g) 2.50 ± 0.18 2.22 ± 0.09 0.04 0.000 Non-heme iron (mg/100 g) 1.01 ± 0.11 1.14 ± 0.03 0.02 0.001 Energy content (kcal/100 g) 315.88 ± 12.48 352.55 ± 3.60 4.65 0.000 333J.M. Lorenzo, M. Pateiro / Meat Science 95 (2013) 330–335 the textural parameters (Lurueña-Martínez, Vivar-Quintana, & Revilla, 2004; Ordónez, Rovira, & Jaime, 2001). 3.3. Effect of fat content on oxidative stability of foal liver pâtés The oxidative stability of foal liver pâtés, as measured by TBARS from lipid oxidation and carbonyl content from protein oxidation, is shown in Fig. 1. The fat level presented significant differences in lipid oxidation since 40F foal pâtés showed significantly higher TBARS values, compared to pâtés with lower fat content (0.54 vs. 0.46 mg MDA/kg pâtés; P b 0.001). These results were expected be- cause TBARS are derived from lipid oxidation and thus pâtés with higher fat content would yield higher amounts of oxidation products. TBARS values were positively correlated (P b 0.01) with fat content (r = 0.64), agreeing with M. Estévez, S. Ventanas et al. (2005). Foal pâtés with higher TBARSvalues had lowercontents ofcarbonyls from protein oxidation (Fig. 1). The carbonyl contents were higher in pâtés with 30F (14.70 nmol carbonyls/mg protein) as compared to those with high-fat content (11.48 nmol carbo nyls/mg protein) (P b 0.001). Among amino a cids, cysteine, tyrosine, phenyl alanine, tryptophan, histidine, proline, arginine, lysine and methionine have been desc ribed as particularly susceptible to R OS (reactive oxygen species ) (Davies & Dean, 2003). Th e nature of the Pox products formed is highly dependent on the amino acids involved and how the oxidation process is initiated. The side chains of some particular amino acids such as arginine, lysine and proline are oxidized through metal-catalyzed reactions into carbonyl residues while others such as cysteine or methionine are involved in cross-linking or yield sulfur- containing derivatives (Lund, Heinonen, Baron, & Estévez, 2011). Car- bonyl content was positively related (P b 0.01) to protein content (r = 0.67) and negatively related (P b 0.01) to fat level (r = − 0.57). These results disagree with M. Estévez, S. Ventanas et al. (2005) who reported higher carbonyl contents in pâtés manufactured with high-fat levels. 3.4. Effect of fat content on fatty acid composition of foal liver pâtés The fatty acid composition of foal liver pâtés of two diffe rent fat contents is shown in Table 5.Thefattyacidprofiles were dominated by MUFA “appro ximately 44.6% of total m ethyl esters,” followed by SFA “approximately 36.9% of total methyl esters” and finally PUFA “approximately 18.5% of total methyl esters” ( Table 5). These results are in agreement with other authors for liver pâtés (Estévez et al., 2004; M. Estévez, J. Ventanas et al., 2005; Ordóñez, D'Arrigo, Cambero, Pinc, & de la Hoz, 2003) since MUFA were the most abun- dant o f fatty acids. As expected, the fatty acid profile of the foal liver pâtés reflected the fatty acid composition of the porcine adipose tissue ( Table 2) as the proportion of lard in the recipe was the highest of all ingredients. The higher MUFA contents observed in foal liver pâtés were very significantly (r = 0.97, P b 0.01) correlated with C 18:1cis-9 content and significantly (r = 0.58, P b 0.01) with C 16:1cis-9 content. The fat level showed significant differences in MUFA content (44.30 vs. 44.86%, P = 0.000; for 30F and 40F groups, respectively) and in PUFA (18.82 vs. 18.23%, P = 0.001; for 30F and 40F groups, respectively). Within the MUFA, oleic was the most abundant with significant dif fere nces between batches (40.64 vs. 41.03%, P = 0.003; for 30F and 40F groups, respectively), followed by palmitoleic acid . These re- sults are in agreement with those of Estévez et al. (2004) who found that oleic acid was the predominant fatty acid in liver pâtés. Within SFA, the main fatty a cid was palmitic, which did not differ signifi - cantly between grou ps (22.86 vs. 22.96%, P = 0.188; for 30F and 40F batches, respectively) , in agreement with Estévez et al. (2004), M. Estévez, J. Ventanas et al. (2005) who observed simila r percent- ages in liver pâtés. Finally, within PUFA, linoleic acid was predomi- nan t but did not differ significantly between groups (15.91 vs . 15.82%, P = 0.451; for 30F and 40F batches, respectively), followed by linolenic acid, which was higher in pâtés with 30F (1.59%) com- pared to those of higher fat content (1.26%) (P b 0.001). The nutritional ratio (C 14:0 +C 16:0 )/(C 18:1cis-9 +C 18:2n−6 ), which indicates the healthiness of the diet with regard its lipid content, is im- portant (Estévez et al., 2004). In the present study, fat content did not affect (P > 0.05) this ratio. Finally, foal pâtés with the two different fat contents presented significantly different n−6/n−3 ratios, those of low-fat content showed the lowest values (9.97) compared to those with higher fat content (13.41) (P = 0.000). These values were higher than the nutritional recommendations of the British Department of Health (1994) and FAO (2010) for the human diet, it should not exceed 4.0. 40F30F Fig. 1. Lipid and protein oxidative stability of foal liver pâté with different fat content as assessed by TBARS (mg MDA/kg foal pâté) and carbonyl (nmol carbonyls/mg protein) contents, respectively (mean ± SD). Table 5 Fatty acid composition (means ± SD) of foal liver pâtés from foal with different fat levels. Fatty acid Fat content SEM P values 30F 40F C14:0 1.18 ± 0.02 1.18 ± 0.02 0.01 0.928 C16:0 22.86 ± 0.15 22.96 ± 0.17 0.04 0.188 C16:1cis-9 2.53 ± 0.04 2.56 ± 0.04 0.01 0.157 C17:0 0.34 ± 0.01 0.33 ± 0.02 0.00 0.183 C17:1cis-9 0.32 ± 0.01 0.31 ± 0.01 0.00 0.584 C18:0 12.32 ± 0.23 12.24 ± 0.14 0.03 0.359 C18:1cis-9 40.64 ± 0.33 41.03 ± 0.14 0.05 0.003 C18:2n−6 15.91 ± 0.25 15.82 ± 0.26 0.06 0.451 C20:0 0.13 ± 0.00 0.13 ± 0.02 0.00 0.891 C20:1 0.80 ± 0.01 0.82 ± 0.02 0.00 0.100 C18:3n−3 1.59 ± 0.13 1.26 ± 0.04 0.03 0.000 C20:2 0.65 ± 0.01 0.66 ± 0.01 0.00 0.513 C20:3n−6 0.10 ± 0.00 0.10 ± 0.00 0.00 0.272 C20:3n−3 0.13 ± 0.00 0.14 ± 0.00 0.00 0.000 C20:4n−6 0.43 ± 0.04 0.39 ± 0.03 0.00 0.011 SFA 36.87 ± 0.24 36.89 ± 0.16 0.05 0.838 MUFA 44.30 ± 0.37 44.86 ± 0.16 0.06 0.000 PUFA 18.82 ± 0.33 18.23 ± 0.29 0.08 0.001 TUFA 63.12 ± 0.24 63.10 ± 0.16 0.05 0.838 Σn−6 17.10 ± 0.28 16.97 ± 0.27 0.06 0.304 Σn−3 1.72 ± 0.13 1.26 ± 0.04 0.04 0.000 n− 6/n− 3 9.97 ± 0.79 13.41 ± 0.42 0.28 0.000 SFA/TUFA 0.59 ± 0.00 0.59 ± 0.00 0.00 0.476 Nutritional ratio 0.43 ± 0.00 0.43 ± 0.00 0.00 0.673 Results expressed as percentage of total fatty acid analyzed. SEM: Standard error of mean. PUFA = Σ (C18:2n− 6 + C18:3n− 3 + C20:2 + C20:3n6 + C20:3n3 + C20:4n− 6). MUFA = Σ (C16:1cis-9 + C17:1cis-9 + C18:1cis-9 + C20:1). TUFA = Σ MUFA + PUFA. SFA = Σ (C14:0 + C16:0 + C17:0 + C18:0 + C20:0). Σn− 6=Σ (C18:2n− 6 + C20:3n−6 + C20:4n−6). Σn− 3=Σ (C18:3n− 3 + C20:3n−3). Nutritional ratio = (C14:0 + C16:0)/(C18:1cis-9 + C18:2n−6). 334 J.M. Lorenzo, M. Pateiro / Meat Science 95 (2013) 330–335 4. Conclusions As expected, fat level affected most of the physico-chemical proper- ties. Higher fat levels in foal liverpâtés,decreasedrednessand hardness. Concerning protein and lipid oxidation increased fat level encouraged the production of TBARS and decreased carbonyl contents. On the other hand, 30F foal pâtés had a better n−6/n−3 ratio compared to pâtés of higher fat levels. Using meat and liver from foals and back fat from pigs for the manufacture of pâtés results in a high quality product in which the iron is highly bioavailable. 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Effect of fat content on fatty acid composition of foal liver pâtés The fatty acid composition of foal liver pâtés of two. 2013 Keywords: Fat content Foal liver pâté Lipid and protein oxidation Physico-chemical properties The effect of fat content on physico-chemical properties and lipid and protein stability offoalliver pâté