Effect of pasture on chestnut or acorn on fatty acid composition and aromatic profile of fat of Cinta Senese dry-cured ham By

El objetivo de este estudio fue la evaluación de la composición de los ácidos grasos y de los compuestos volátiles del jamón curado Toscano obtenido de cerdos Cinta Senese alimentados con bellota, castaña o pienso. Quince cerdos de raza Cinta Senese fueron criados en extensivo: 5 fueron criados en bosque de bellota (AC), 5 en bosque de castaña (CH) y 5 en un cercado y alimentados con pienso (MI). El día posterior al sacrificio se curaron los jamones. La composición de los ácidos grasos fue estadísticamente diferente entre los tipos de alimentación. La grasa de AC presentó el mayor porcentaje de AGMI total en comparación con CH y MI (51.3 y 53.9 vs 49.5% respectivamente) gracias al mayor porcentaje de ácido oleico (48.4 y 50.8 vs 46.2%, respectivamente), y la menor de AGPI total (13.8 vs 15.4 y 16.3%, respectivamente). En relación a los compuestos volátiles, la grasa de AC, en comparación con CH y MI, presentó mayores valores de aldehídos (44.1 vs 30.3 y 33.5% respectivamente) y éteres (4.04 vs 1.15 y 1.71% respectivamente).


INTRODUCTION
The Tuscan dry-cured ham is a traditional pork meat product from Central Italy with a Protected Denomination of Origin (PDO) registration.Nowadays an increasing number of Tuscan hams is produced with the local Cinta Senese pig and consumers confer to those hams a consistent added value (Franci, 2004).The farming system used with the Cinta Senese is typical of the extensive pig production in the Mediterranean area.The characterization of meat from the extensive rearing system of the local breed could be exploited to increase the commercial value of the ham, similarly to the Iberian pig experience (Lopez-Bote, 1998).Dry-curing of Cinta Senese hams is a traditional process that leads to a food product with unique flavour.The aroma is due to chemical and biological changes and lipids serve several roles in flavour development (Calkins and Hodgen, 2007) giving rise to a large number of volatile compounds.Several studies on the flavour of raw ham of Mediterranean area have been dealing (Dirinck et  al., 1997).Buscailhon et al. (1994) and Careri et al. (1993) studied the relations between compositional traits and sensory qualities of French and Italian dry-cured ham, while López et al. (1992) and Carrapiso et al. (2002) described the volatile compounds of Iberian dry-cured ham as affected by rearing system.Compounds coming from feeds contribute to the final flavour of dry-cured meat products.Also the quality of Cinta Senese meat strongly depends on rearing and feeding system (Pugliese and Bozzi, 2004) then, in the future, the commercial value of dry-cured products of this breed could be linked to a particular rearing system.So, the aim of this study was to evaluate the effect of pasture on oak plantation and on chestnut grove on chemical and aromatic traits of dry-cured ham.

Animals and diets
Fifteen Cinta Senese barrows, weighing about 124 kg, were randomly allotted to three groups (5 pigs per group) according to the type of feeding during the finish-fattening period (last 90 days prior to slaughter).One group (MI) was reared in confinement (paddock of 1 ha) and had free access to appropriate diet (3 kg/pig/d).The other two groups were raised under extensive production system according to the traditional free-range conditions: pasture and acorn (AC) for the group reared in oak plantation (8 ha) (Quercus cerris and Quercus pubescens); pasture and chestnut (CH) for the group reared in chestnut grove (8 ha) (Castanea sativa).Chemical composition, tocopherol content and fatty acid composition of experimental mixture, acorn and chestnut are shown in table 1.Before the experimental period, animals were reared together, under a confinement system, and fed the same feedstuff.Pigs were slaughtered at the weight of 148 kg, on average.

Materials and technology
At slaughtering thighs were removed from the right half-carcasses and processed according to the dry-curing method of Tuscan ham (Table 2).At the end of seasoning period, a sample-slice, about 3 cm thick, was taken transversally from the caudal portion of the cured ham in the middle of the area where the femur was removed, immediately vacuum-packed and frozen at Ϫ80 °C until the analysis.

Chemical analysis
The chemical analysis of food was carried out following the methods of A.O.A.C. (1990) and tocopherol analysis were performed according to the method described by Rey et al. (2006).
The following analyses were carried out on the subcutaneous fat: 1) moisture; 2) total lipids extracted according to Folch et al. (1957); 3) fatty acid profile of total lipids.Fatty acid methyl esters were prepared by esterification in presence of sulphuric acid (Morrison and Smith, 1964), and were analysed by gas chromatography, using a DANI 86.10 apparatus equipped with a flame ionisation detector (FID).Fatty acids were separated on a capillary column coated with FFAP-TPA stationary phase (30 m length; 0.32 mm internal diameter; 0.25 mm film thickness).Temperature of the column started at 160 °C and reached 220 °C, with 2 °C/min.increase.Temperature of the detector was set at 260 °C.Methyl esters were identified by their retention time and expressed as percentage of total detected methyl esters.

Volatile compounds analysis
Volatile compounds of seasoned fat were extracted by headspace-SPME (Ruiz et al., 2001).One gram was weighed into a 4 ml vial and screwcapped with a Teflon-silicone disk.A SPME (Supelco, Bellefonte, PA, USA) fibre coated with carboxen/poly (dimethylsiloxane) (75µm thickness) was inserted into the sample vial and exposed to the headspace.Extraction was performed at 37 °C for 30 min in a water bath.Before the analysis the SPME fibre was preconditioned at 280 °C for 50 min.in the gas chromatograph injection port.Volatile compounds analysis was performed using a HP-6890-GC series II gas chromatograph (Hewlett-Packard) coupled to a mass selective detector (HP-5973 Network, Hewlett-Packard).Volatiles were separated using a 5% phenylmethylsilicone (HP-5) bonded phase fused-silica capillary column (50 m 0.32 mm i.d., film thickness 1.05 µm, Hewlett-Packard).Carrier gas was helium at a flow rate of 1.45 mL min Ϫ1 .Compounds were identified by comparing their mass spectra with those contained in the NIST and Wiley libraries and by comparison of Kovats indexes with those reported in the literature (Kondjoyan and Berdagué, 1996).

Statistical analysis
The effect of feeding system was tested by analysis of variance (ANOVA), using GLM procedure (SAS, 2007).Data were analysed with the following linear model: where Y ij is the l th observation; µ is the overall mean; D is the i th diet; the b i term is the regression coefficient on cured ham weight (X), e ij is the residual error.For the proportion of identified volatiles compounds only the effect of feeding system was tested.

Trimming
Ham is defatted and trimmed, by leaving 10 cm of lean beyond the femur knob.In the hams of Cinta Senese, foot is not removed.

Salting
Ham is covered with salt and a mixture of natural and typical Tuscan aromas, then they were placed in a salting cellar for 18-21 days, at 4 °C and 90% relative humidity.In the rule of Tuscan ham the use of nitrite is admitted, but in this case no preservatives were used.

Resting
Ham is first brushed to remove superficial salt, than it is kept for 40 days in a chamber temperature varied between 2-4 °C, 60% relative humidity.

Greasing
Six month after starting the processing (salting), the lean portions of hams are covered with minced pork fat with the addition of salt and pepper.This treatment avoid that the outer muscular layers are dried out in relation to the internal ones.

Seasoning
Ham is kept in a cellar for 300 days.The conditions in the cellar are highly constant: temperature varies between 17-18°C, 60 % relative humidity.

Fatty acid composition
In table 3 the chemical composition of fat of drycured ham is reported.The pigs pastured on acorn showed the highest content of lipid, while no differences were found between the other two groups.Given that moisture was analogous among the groups, this result is probably linked to the high content of N-free extractive of acorn.As reported by Edwards (2004), the high quality of dry-cured ham in Mediterranean silvopastoral system is associated to a relatively high level of intramuscular fat resulting from access of local breeds to acorn which is high in starch.It is likely that in subcutaneous fat the high energy level of acorn lead to higher synthesis of lipids as well.
Fatty acids percentage (Table 3) is significantly affected by feeding system.Pigs fed acorn showed the lowest content of SFA and the highest percentage of MUFA, oleic acid prevalently.Significant enrichment in MUFA in hams from pigs raised extensively with free availability of acorn and pasture has been reported by several Authors (Flores et al., 1988;Cava et al., 2000;Carrapiso et al., 2003).This results can be explained by a ready incorporation of dietary fatty acids that occurs during fattening (Fontanillas et al., 1998).In fact, as reported in table 1, acorn showed a very high concentration of oleic acid (nearly 50%), and a more limited concentration of linoleic acid, in relation to the other two feedstuffs.These results are in agreement with previous findings on fresh fat of the same animals (Pugliese et. al., 2006).Pigs reared on chestnut showed lower content of MUFA than pigs fed acorn but higher than the ones fed mixture.Also in this case the quality of lipids is strongly related to the feeding conditions.Indeed also chestnut is characterized by higher content of oleic acid respect to concentrate (Table 1).The higher MUFA percentage of CH pigs respect to MI pigs is in general agreement with findings of Sirtori et al. (2008) on Cinta Senese reared in confinement and fed chestnut and findings of Coutron-Gambotti et al. (1998) on subcutaneous and intramuscular fat of Corsican pigs.As regard PUFA content AC group showed the lowest value while no differences between the other two groups were found.In pigs reared outdoor the variability of PUFA content, mainly depend on grass availability.As reported by Lopez-Bote (2000), the grass is the main source of long chain PUFA (n-3 prevalently) but, in extensive conditions, it is very difficult to quantify the intake of grass, so in this trial it is probable that the pasture on chestnut wood, which was more rich in grass than those of oak wood, leaded to higher accumulation of PUFA n-3.However, the picture relative the PUFA n-6 content appears in good agreement with the content of the same fatty acids in the original feedstuff.
No differences among groups were found in MDA content that, however, resulted very low.This confirms previous results on cured lard of the same animals (Pugliese et al., in press) and the positive effect of pasture on wood on oxidative stability of pig.

Volatile profile
About 60 volatile compounds were identified and assigned to the respective chemical families.are derived from the autoxidation of unsaturated fatty acids, although some could also derive from amino acids, having origin from Strecker degradation (Ruiz et al., 1999).According to research on Iberian dry-cured ham (Andrés et al., 2002), a high number of acids has been detected.Some of these, as acetic acid, are originated from the microbial metabolism of glucides (Kandler, 1983).The other acids could derive from the hydrolysis of triglycerides and phospholipids and from lipid oxidation reactions (Ruiz et al., 2002).Among short chain acids, 3-methylbutanoic acid and 2-methylbutanoic acid have been detected.These acids, as reported by Muriel et al., (2004), play an important role in aroma development.Aldehydes compounds represent the most important chemical family in Cinta Senese ham, hexanal being the major one.Hexanal derives from the oxidation of n-6 fatty acids, heptanal arises from oleic or linoleic autooxidation and, finally, octanal comes from oleic acid oxidation.Other compounds from lipid oxidation were also aliphatic hydrocarbons (as pentane and hexane), furans (as furan-2-ethyl) and ketones (as 2-pentanone, 2-propanone, 2,3-pentanedione).About aromatic hydrocarbons, methylbenzene (toluene) could derive from cyclation of unsaturated carboxylic chains produced by lipid degradation (Min et al., 1977).The major ester found was ethyl acetate, which is formed through esterification reactions between ethanol and carboxilic acids.It leads to fruity notes and it has a low odour threshold (Muriel et al., 2004).Other chemical families, such as sulfur compounds (i.e.carbon disulfide) and nitrogen compounds (i.e.2,6-dimethylpirazine and pyridine), derive from Maillard reaction (Ruiz et al., 2001).Pirazine has characteristic walnut, toasted or potato odours (Meynier et al., 1999).The main terpen found is linalool that leads to flowery notes.The accumulation of terpens in fat depots has two possible origins: from animal feeding or from spices added during processing (Muriel et al., 2004).As regard the rearing system effect the significance was found only for ethers compounds and for total aldehydes percentage, highest in AC group.The latter results is in agreement with those reported by Muriel et al. (2004) on Iberian drycured loin and by López et al. (1992) on Iberian dry-cured ham.Hexanal is the main compound derived from the oxidation of n-6 fatty acids, heptanal arises from oleic or linoleic acid while octanal and nonanal comes from oleic acid oxidation.A relationship between these volatile compound and fatty acids percentage seems to be only for heptanal, higher in pigs fed acorn that showed also the highest content of oleic acid.On the other hand hexanal, highest in AC group, is associated to green, fruity and acorn-like odour (Carrapiso et al., 2002).For the other compounds, the high variability found among data, didn't allowed to reach the significance threshold, nevertheless the high differences in absolute values.Apart of the significance level it is possible to underline the highest percentages of ketons, as 2-propanone, 2,3-pentanedione and aliphatic hydrocarbons, showed by pigs fed chestnut.

CONCLUSIONS
The pasturing on wood, especially on acorn wood, affected the fatty acid composition of cured subcutaneous fat.In particular the highest level of oleic acid found in ham from pigs fed acorn could be an important item of characterization of Cinta Senese dry-cured ham.These differences in acidic composition seem to be related to the same differences in volatile compounds, even if only for aldehydes and ethers compounds statistic differences among groups were achieved.

Table 1 Composition of diets Chestnut Acorn Mixture
CAROLINA PUGLIESE, FRANCESCO SIRTORI, JORGE RUIZ, DIANA MARTIN, SILVIA PARENTI AND ORESTE FRANCI EFFECT OF PASTURE ON CHESTNUT OR ACORN ON FATTY ACID COMPOSITION AND AROMATIC…

Table 3 Fatty acid composition of seasoned fat (% total fatty acid)
a, b within criterion means different (P Ͻ 0.05).
The main compounds are shown in table 4. Most of them CAROLINA PUGLIESE, FRANCESCO SIRTORI, JORGE RUIZ, DIANA MARTIN, SILVIA PARENTI AND ORESTE FRANCI

Table 4 Percentage of volatile compounds
* Expressed as area percentage of total volatile compounds identified.** KI ϭ Kovats index in agreement with literature.