Fatty Acid Composition of Salami Made by Meat from Different Commercial Categories of Indigenous Dairy Cattle

Simple Summary The Cinisara is a Sicilian breed raised on pasture to produce the Caciocavallo Palermitano cheese. Even if it is penalized by competition with meat breeds, characterized by higher growth rate and yield, the production of fresh meat represents a considerable added value for the smallfarms. The meat of Cinisara is not appreciated, despite having a high content of iron, vitamin E, conjugated linoleic acid (CLA), and low content of lipids and cholesterol, above all due to incorrect management of the supply chain phases that negatively affects the quality of the final product. Alternative production such as bresaola and salami could contribute to the enhancement of Cinisara meat. The present study investigated the fatty acid profile of salamis produced by processing the meat of young bulls and adult cows with the addition of lard pork to provide additional information to that reported in a previous experiment on physicochemical and sensory properties. The results suggest the possibility of producing cured meats with Cinisara meat, even if the addition of pork lard mitigates some favorable effects deriving from the livestock system of this breed, based on grazing. Further studies should be conducted to investigate the possibility of making cured meats with beef only. Abstract In autochthonous dairy cattle farms, the production of salami could represent an alternative commercial opportunity. Therefore, a study was carried out to investigate the fatty acid (FA) composition of salami made using the meat from grazing (GB) or housed (HB) young bulls and grazing adult cows (AC) of Cinisara breed. The products were manufactured by adding 20% of pork lard. Animal category influenced the FA composition, although the addition of lard mitigated the differences found in fresh meat. The salami from GB showed higher polyunsaturated FA content (p ≤ 0.01) and, in particular, a higher level of linoleic acid (p ≤ 0.05), than from other animal categories. Salami made from AC meat showed lower polyunsaturated/saturated FA ratio (p ≤ 0.05), but a better n-6/n-3 ratio compared to HB (p ≤ 0.05), due to the lower content of linoleic acid. Multivariate analysis showed an important influence of animal category on FA composition due to age, feeding system and meat fat content of animals, despite the addition of lard.


Introduction
Cinisara cow, a Sicilian autochthonous dairy breed, is traditionally reared with a feeding system based on the prevalent exploitation of natural pasture, and its milk is used to produce the Caciocavallo Palermitano cheese [1][2][3]. The manufacture of processed products, such as salami and bresaola, an alternative to fresh meat, could increase the economic profit of farms, diversifying the offer in the market [4][5][6][7]; in particular, some cuts of the posterior quarter (semimembranosus, semitendinosus and quadriceps femorismuscles) and

Meat and Salami Manufacturing
Carcasses of animals of Cinisara breed, selected on the basis of age and the livestock system (use or non-use of pasture) were used, and in the specific: n. 4 grazing young bulls (GB-18 months old), fed pasture-based diets from a 6-month age until slaughtering, with hay and concentrate supplements in the final phase (16-18 months); n. 4 housed young bulls (HB-18 months old), fed pasture-based diets from a 6-month age, whereas housed and fed a diet of only hay and concentrate in the finishing phase (16-18 months); n. 4 adult cows at the end of their productive life (AC-10 years old), fed pasture-based diets from 6 months of age until slaughtering, receiving hay and concentrate supplements from the beginning of the reproductive career (24 months).
The animals were slaughtered at an EU-licensed abattoir, according to the standard handling procedures with respect to EU regulations [31] on the protection of animals at the time of slaughter. The carcasses were stored in a cooling room at 4-8 • C for a 7-day ageing period. After, they were dissected (day zero), and the meat from the forequarters of each carcass was minced separately with a 6 mm plate; 50 kg of minced meat of each carcass was used.Each batch of minced meat was supplemented with pork lard (20% w/w) from Nebrodi Black Pig (Sicilian native pig breed) and other ingredients (sodium chloride, 2.5%; sucrose, dextrose and maltodextrin,0.35%; sodium ascorbate, 0.016%; sodium nitrate, 0.01%; potassium nitrate, 0.051%),obtaining in a blender a raw mixture for each animal (4 for each animal category, 12 total mixtures). Subsequently, each raw mixture was separated into two batches; one part fermented spontaneously, while the other one was inoculated with freeze-dried cultures, containing Staphylococcus xylosus and Pedioccocuspentosaceus (Tec-AL. s.r.l., Traversetolo, Italy; final concentration of approximately 107 CFU g −1 ).

Sampling
For each carcass, a sample of minced meat (before the addition of other ingredients) and pork fat was collected.
The salami were sampled at day 0 after mixing all ingredients (raw mixture) (12 samples, 1 for each animal) and at day 45 (end of the maturing process) (12 samples of uninoculated salami and 12 samples of inoculated salami). The samples, placed in sterile vacuum containers, were refrigerated at 8 • C for the transfer to laboratory where they were homogenized (2 min at maximum speed) by stomacher (LAB Blender 400, Seward Medical, London, UK) and then freeze-dried for successive analysis as described by Alabiso et al. [10] (SCANVAC Coolsafe 55-9, Labogene Aps, Lynge, Denmark).

Fatty Acids Composition
Fatty acids (FA) were extracted according to the method developed by O'Fallon et al. [32]. The total FA quantification was performed using C23:0 (Sigma-Aldrich, Darmstadt, Germany) as internal standard (0.5 mg/g freeze-dried sample). Each sample (1 µL) was injected by autosampler into an HP 6890 gas chromatography system equipped with a flameionization detector (Agilent Technologies Inc., Santa Clara, CA, USA). For separation of FA methyl esters from each sample, a capillary column (100 m length, 0.25 mm i.d., 0.25 µm; CP-Sil 88; Chrompack, Middelburg, the Netherlands) was used. Temperatures of the injector and the detector were kept at 255 • C and 250 • C, respectively, using H 2 flow of 40 mL/min, air flow of 400 mL/min and He flow of 45 mL/min. The oven temperature was held for 1 min at 70 • C, increased at 5 • C/min to 100 • C, held for 2 min, increased at 10 • C/min to 175 • C, held for 40 min and finally increased at 5 • C/min to 225 • C, held for 45 min. The carrier gas was He, used with a head pressure of 158.6 kPa and a flow rate of 0.7 mL/min (linear velocity of 14 cm/s). The identification of each FA was performed as described by Alabiso et al. [4].

Statistical and Explorative Multivariate Analysis
Data were statistically processed using the SAS 9.2 software [35]. FA composition was analyzed according to a MIXED model including the fixed effects of animal category (A, with three levels: GB, HB and AC), product (P, with three levels: raw mixture RM, inoculated salami IS and uninoculated salami US) and the animal within category as a random effect. The interactions between the effects A × P was removed from the model since it was always not significant. Tukey's test was used to compare means when the effects were significant (p < 0.05).
The principal components analysis (PCA), performed using the PRINCOMP SAS procedure, was based on FA composition in order to assess its specific contribution in explaining the differences among salami type, due to the different animal category. The variables used in the analysis were identified on the basis of a stepwise selection using the STEPDISC SAS procedure, after they were standardized by multiplying them by the inverse of standard deviation (1/SD). The number of main components was selected according to Kaiser's criterion and only those with Eigen values above 1.00 were retained. Table 1 shows the FA composition of meat of each animal category and the pork lard. AC is characterized by a higher percentage content of monounsaturated fatty acids (MUFA) and a lower presence of polyunsaturated fatty acids (PUFA) compared to the levels found in GB, similar to what was found in bresaola obtained from cows at the end of their career [4]. Intermediate features were found in HB.

Fatty Acids Composition
Pork lard showed a high MUFA content, consisting of 87% of oleic acid (OA, C18:1 n-9), and a low PUFA content possibly related to direct deposition of MUFA from acorns present in the diet to the fat depots. The FA composition of pork lard is comparable to that found by Pugliese et al. [36] in outdoor rearing system. The FA composition of pork lard used suggests that it derived from animals that evidently were reared outdoors with a feeding integration to the natural pasture, according to the farming system typically used for the Nebrodi Black Pig.
In general, the processed products show fat quantity and profile depending on the ingredients of the mixture. In this study, the same receipt was used for all products, changing only the kind of meat. Therefore, the differences in fat content and FA composition should be mainly attributable to beef rather than to lard and other ingredients. The results indicate mean values of three measurements performed on each of the four carcasses for animal category and for each sample of lard. a Animal category: GB = grazing young bull; HB = housed young bull; AC = adult cow; PL = pork lard. b DM = dry matter; c SFA = saturated fatty acids; d MUFA = monounsaturated fatty acids; e PUFA = polyunsaturated fatty acids; f HPI = health promoting index; g TI = thrombogenic index. Table 2 shows FA profile in relation to the animal category and the product type. The total FA content ( Table 2) was higher in AC than in GB and HB (p < 0.05), confirming that meat of adult cows is characterized by a major fat content compared to young animals [5] and that fat and proportions of FA change depending on the age of the animal and its diet [4]. The saturated fatty acids (SFA) ( Table 2) did not significantly change among animal categories, while MUFA were higher in AC (p ≤ 0.05) compared to GB. HB showed intermediate values (differences not statistically significant). PUFA were higher in GB than in HB, which yet registered higher values compared with AC (p ≤ 0.01).
The animal category influenced the n-6 and n-3 contents, the PUFA/SFA and n-6/n-3 ratio, as well as HPI (Table 2). GB showed higher values of both n-6 (p ≤ 0.01) and n-3 (p ≤ 0.05) compared to AC and HB, and higher HPI (p ≤ 0.05) compared to AC. The ratio PUFA/SFA was lower in AC compared to GB and HB (p ≤ 0.05), while the ratio n-6/n-3 was higher in HB compared to AC (p ≤ 0.05). The FAO/WHO report indicates an optimal n-6/n-3 ratio of 4.5-5 [37]. A diet contrasting various "lifestyle diseases", such as coronary heart diseases and cancers, requires a ratio of PUFA/SFA above about 0.45 and a ratio of n-6/n-3 below 4.0 [38]. Therefore, AC show an unfavorable PUFA/SFA ratio compared to GB and HB. However, despite the lower PUFA content, AC showed a health favorable n-6/n-3 ratio, especially due to the lower linoleic acid (LA) level. Table 3 shows the SFA composition of raw mixtures and ripened salami. Significant differences were found for palmitic acid (C16:0), higher for HB and AC compared to GB (p ≤ 0.01), for C15:0 and C17:0, lower in GB, and for stearic acid (C18:0) higher in GB compared to AC (p ≤ 0.05), as found in bresaola by Alabiso et al. [4]. The effect of the product type emerged for C17:0, lower in the raw mixtures than in salami.  Table 4 shows the MUFA composition of raw mixtures and ripened salami. In particular, the C14:1 and especially OA were higher in AC than in GB (p ≤ 0.05), while HB showed intermediate values (not statistically significant); as in bresaola, OA was the most representative of the MUFA and its content was influenced by the age of the animals [4,29]. The raw mixtures showed the lower level of C17:1.  Table 5 shows the PUFA composition of raw mixtures and ripened salami. In all animal categories, the most represented among PUFA was linoleic acid (LA, 18:2 n-6), and was lower in AC compared to HB and GB (p < 0.01). In accordance with Wood et al. [29], the LA content was negatively related to the trend of salami fat. In fact, total FA content ( Table 2) was higher in AC than in HB and GB. Furthermore, the arachidonic acid (AA, C20:4 n-6), derived from LA through the action of ∆5 and ∆6 desaturase enzymes and elongase [4], was on average lower in AC compared to HB and GB (p < 0.05), similar to its precursor. Moreover, eicosapentaenoic (EPA, C20:5 n-3) and docosapentaenoic (DPA, C22:5 n-3) acids were on average higher in GB than HB and AC (p < 0.05 and p < 0.01, respectively). Feeding systems based on grazing have been known to have a positive effect on PUFA content of beef, and in general, on products obtained from ruminants [39][40][41]. Indeed, the use of grazing until slaughter resulted in a higher PUFA content of GB compared to HB.AC, despite being grazed until slaughter, showed lower PUFA concentrations than GB.These trends can be linked to the older age of AC, confirming the results obtained in bresaola, in which beef had higher concentrations of fat and lower PUFA levels than those of younger subjects, fed under the same conditions. Analogous differences were found between HB and GB which differed in the feeding pattern [4]. Table 5. Effects of animal category and product on polyunsaturated fatty acids (% of total FA) of salami fat.  The overall concentration of rumenic acid (RA), the main of CLA isomers, in ripened salami was higher in AC (p < 0.05), although the differences between the commercial categories of salami were smaller with respect to those found in bresaola [4], probably due to the mitigating effect of pork lard addition.

Product (P) b Animal Categories (A) a SEM c Significance
Since the addition of pork lard did not induce any positive effects due to the diet and farming system of pigs, the FA composition was better than that found on salami produced with only Nebrodi Black Pig [42].
The fermentation processes in both inoculated and uninoculated salami did not show appreciable differences in FA composition at the end of the maturation. A significant increase in the raw mixture in comparison with the ripened salami was found only for C17:0, C17:1 and others, C18:2 (p < 0.05). As found in Gaglio et al. [7], the microbiological evolution may have been comparable between inoculated and uninoculated samples, especially with regard toits role in modifying the FA profile, probably as a consequence of microbial contamination of raw meat during slaughter and subsequent processing stages.

Multivariate Analysis
The plot generated by PCA is shown in Figure 1, where the length of each vector measures the contribution of each variable to the main components.

Conclusions
The origin of meat, with regard to the animal category, differing for age and feeding system, influenced the FA composition of salami. The addition of pork lard reduced the initial differences between the animal categories.
In bulls, the use of pasture up to slaughter improved the FA profile, determining an increase in n-3 PUFA content. Adult cows, despite benefiting from the pasture until slaughter, showed lower PUFA contents than young bulls, due to the higher fat content. However, the lower LA level resulted in a more favorable n-6/n-3 ratio in AC than HB. The higher age also determined an increase of OA in AC.
On the basis of these results, salami made with Cinisara meat could be an alternative  The first two principal components accounted for 90.23% of the total variance.With 59.46% of the total variance, the first principal component was able to discriminate the AC salami from those of GB and HB on the basis of the main contributions of total FA, SFA, C18:1n-9 and MUFA.Instead, with 30.77% of the total variance, the second main component was able to discriminate GB salami from HB salami, especially for the contribution of total FA, SFA, C18:0, MUFA and C16:0. Overall, the animal category, which associates the effect of age and feeding system, influenced the qualitative traits of salami more markedly than the inoculated starter culture, since the effect of the inoculation did not allow a clear distinction of the salami within the animal category.

Conclusions
The origin of meat, with regard to the animal category, differing for age and feeding system, influenced the FA composition of salami. The addition of pork lard reduced the initial differences between the animal categories.
In bulls, the use of pasture up to slaughter improved the FA profile, determining an increase in n-3 PUFA content. Adult cows, despite benefiting from the pasture until slaughter, showed lower PUFA contents than young bulls, due to the higher fat content. However, the lower LA level resulted in a more favorable n-6/n-3 ratio in AC than HB. The higher age also determined an increase of OA in AC.
On the basis of these results, salami made with Cinisara meat could be an alternative product, even if the addition of pork lard mitigated the favorable effect of the livestock system based on grazing in increasing the PUFA content and thus in improving the FA profile.
In salami, fermentation processes and the addition of pork lard would allow one to appreciate only significant differences linked to the animal categories. Further studies should be conducted in which salami is made only with beef, considering also the consumers who do not eat pork.  Data Availability Statement: All data included in this study are available upon request by contacting the corresponding author.

Conflicts of Interest:
The authors declare that there is no conflict of interest regarding the publication of this article.