In the last few years, concerns over adverse health effects of synthetic additives such as synthetic antioxidants caused and increased consumers demand for food products containing natural ingredients. Synthetic antioxidants have long been used to delay quality deterioration in meat and meat products induced by oxidation processes, while in the recent years their use has been discouraged due to their potential genotoxic effects (Jiang Jaing et al., 2016). Therefore, the current industrial food trend has shifted towards different natural plant extract as sources of bioactive substance for food preservation (Burt, 2004; Zheng et al., 2001). Several studies demonstrated that extracts obtained from fruit, vegetables, spices and herbs such as rosemary, sage, oregano and other, thanks to their high content of phenolic compounds, exert high antioxidant activities able to improve oxidative stability in food (Akarpat et al., 2008; Burt, 2004; Huang et al., 2011; Srinivasan, 2014; Zheng et al., 2001). Naturals antioxidants are also included in animal diets for their positive effects on animal performance, health and welfare. Recently, several animal feeding strategies have been investigated to enhance nutritional value, improve the organoleptic properties and the health benefit of meat and meat products. Nieto et al. (2010) observed that dietary rosemary supplementation in pregnant ewes delayed the microbial growth, decreased the lipid oxidation and improved colour and odour of subsequent lamb meat during storage. Luciano et al. (2011) reported that a tannin enriched diet extended colour stability and increased total phenols content and antioxidant capacity of lamb meat. Moreover, Ranucci et al. (2015) obtained interesting results on cooked ham including oregano essential oil and sweet chestnut wood extract in pig diets. Products during storage showed an increased oxidative stability, antioxidant capacity and better values in colour scores but not modification were recorded for microbiological characteristics. However, despite several studies reported the effect of feeding natural antioxidants on meat characteristics, limited data on processed meat, such as salami, are available in literature.
Thus, the aim of the present study was to evaluate the influence exerted by oregano extract dietary supplementation in pigs on the microbiological profile, chemicalphysical characteristics and sensory properties of Fabriano salami.
Materials and Methods
In this study, 32 Duroc x Large White pigs, with an initial live weight of 35 Kg, were bred. At the beginning of the trial, the animals were divided into two experimental groups of 16 subjects each and fed with a commercial pelleted feed (CTRL) or the same feed supplemented with 0.2% of oregano (Origanum vulgaris L.) extract (OR). Both diets were isonitrogenous and isoenergetic and in finishing stage (from 120 Kg live weight to slaughtering) were supplemented with 0.5% commercial source of conjugated linoleic acid (CLA) (LodeStarTM CLA, Berg + Schmidt GmbH & Co., Hamburg, Germany). When the pigs reached a live weight of 170 Kg were regularly slaughtered in a local slaughterhouse and the carcasses were transported to an industrial processing plant located in the province of Ancona (Central Italy) for the salamis production. The animal care procedures were in accordance with the European recommendations (European Parliament and the Council of the European Union, 2010) for the protection of animals used for scientific purposes. Fabriano salami was produced from meat derived from each dietary group (CTRL and OR) according to traditional procedures (Rea et al., 2005). Briefly, the mixture was prepared using refrigerated meat from pork shoulders (67%) and scraps from thigh and loin processing (33%). After grinding the mixture, loin lard (10% of meat weight) cut into small cubes of 10 mm, salt (2.6%), black pepper (0.4%), starter cultures, and preservatives were added to the mixture (E301 – sodium ascorbate: 0.050 Kg/q of meat; E252 – potassium nitrate: 0.0020 Kg/q of meat). The products were dried in a drying room at progressively decreasing temperature, from 23° to 17°C, and relative humidity (RH) progressively increasing from 40 to 80% and then ripened in conditioned rooms at 14°C and 80% RH for 45 days. Ten salamis per group were collected at the beginning of processing phases (T0) and after 7 (T1), 20 (T2) and 45 (T3) days of ripening and then transferred to the laboratory for the following microbiological and physic-chemical analysis: total viable count (TVC) on Plate Count Agar (PCA; CM0325, Oxoid, Basingstoke, UK) aerobically incubated at 30°C for 48h; Enterobacteriaceae count using Violet Red Bile Glucose Agar (VRBG; CM1082, Oxoid) aerobically incubated at 37°C for 24h; enumeration of Enterococcus spp. on Slanetz Bartley (4020472, Biolife) with 2,3,5-Triphenyltetrazolium chloride 1% solution (42111801, Biolife), incubated at 37°C for 48 h; coagulase negative and positive staphylococci were enumerated on Baird Parker agar (4011162, Biolife) with Rabbit Plasma Fibrinogen Supplement II (423102, Biolife) incubated at 37°C for 48 h; Lactobacillus spp. were counted on de Man, Rogosa and Sharpe (MRS) agar (CM0361, Oxoid) anaerobically incubated at 37°C for 48; Lactococcus spp. on M17 agar (CM0785, Oxoid) aerobically incubated at 37°C for 48h. After counting, the data were reported in Log Colony Forming Units (cfu)/g and the mean and standard deviation were calculated; presence of Salmonella spp. and Listeria monocytogenes were tested using the criteria set by ISO 6579 (ISO, 2004) and ISO 11290-1 (ISO, 2005) respectively; water activity (aw) was determined through an AquaLab CX-3 (Decagon Pulman, WA, USA); pH determination using a penetrating electrode connected to a portable pHmeter (Mod SG2, Mettler Toledo AG, Schwerzenbach, CH); colour coordinates (CIE, 1986). Only at the end of the ripening period (T3), the samples were sliced and immediately analysed for the following determination: chemical composition (AOAC, 1990); total antioxidant capacity using the oxygen radical absorbance capacity method (ORACFL) based on the fluorescence decay rate of a probe in the presence of a radical oxygen species (ROO°) compared with a reference standard (Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman- 2-carboxylic acid, Sigma- Aldrich, Steinheim, Germany), as reported by Branciari et al., 2015b; lipid oxidation assessed using the thiobarbituric reactive substances test (TBARS) according to Tarladgis et al. (1960) and values were expressed as mg malondialdehyde (MDA)/kg salami; for the determination of total phenolic content (TPC), the extraction of polyphenols was carried out using method described by Branciari et al., 2015b with some modification: 1 g of sample was homogenised with 20 mL of ethanol 80% (w/v), the homogenate was vortexed and centrifuge for 30 min 6000 rpm at 35 °C. For evaluating the phenolic content using the Folin–Ciocalteu method (Singleton et al., 1999) 20 μl of the surnatant were transferred into tube containing 1.58 mL of H2O2, 100 μL of Folin-Ciocalteu phenol reagent (Sigma-Aldrich, St. Louis, MO, USA) was added and mixed. 20% (w/v) of Na2CO3 solution (300 μL) was added and mixed. The solution was immediately transferred to an incubator and leave at 40°C for 30 min. The absorbance of the sample was measured at 765 nm using an Ultrospec 2100 pro UV/visible spectrometer (Amersham Pharmacia Biotech, Buckinghamshire, UK). For the quantitative determination of TPC, a Gallic acid (Sigma-Aldrich, St. Louis, MO, USA) standard calibration curve (y = 0.0011x + 0.023 R² = 0.9998), corresponding to a concentration range of 0.05-0.75 mg/mL. The TPC concentration was expressed as mg gallic acid equivalents (GAE) per g. After 45 days (T3), when Fabriano salamis were ready to be commercialized, in line with standard production methods, a series of consumer tests were performed at the Department of Veterinary Medicine, University of Perugia. Consumers were asked to complete a questionnaire including information regarding their age, sex and frequency of salami consumption (Branciari et al., 2012). The consumer tests were performed in three sessions under different conditions (blind, expected and informed), one week apart. For each session, 95 regular salami consumers (aged 20-60, 45 females and 50 males) were used (regular consumers were those who had a consumption frequency of at least once every two weeks). A practicing session was performed before the test to allow consumers to become familiar with the use of a nine point hedonic scale (from 1, dislike extremely to 9, like extremely). In the first session, 1 sample/group was monadically served on white plastic plates identified by three random digit codes. Consumers received no information (blind experimental condition) and were asked to rate sensory attributes using the nine-point hedonic scale for appearance, texture, taste, and overall liking. In the second session, the participants were asked to assess on the same hedonic scale their liking expectation from salami (expectation test) when given the following information regarding animal diet: i) salami from pigs fed a standard diet and ii) salami from pigs fed a standard diet enriched with oregano extract, a natural antioxidant with potential positive effect on the product. In the third session the participants rated the samples under informed condition as already performed in blind test, but in this case samples were accompanied by the same information provided for the expectation test on animals dietary treatments.
The data were analysed using Statview (SAS Institute inc., Cary, NC, USA) program. The dietary effects on the same sampling time was evaluated using the unpaired t-test and the differences of the means were considered to be significant when P<0.05.
The results of the microbiological analysis performed on Fabriano salami at each time considered are reported in Figure 1. The trend of microbial flora was consistent between groups during the whole ripening period. No Salmonella spp. and L. monocytogenes were isolate from the samples and coagulase positive staphylococci were below the limit of detection (<Log 2 cfu/g) since T0. Furthermore, also chemical composition, aw and pH values did not differ (P>0.05) between groups (Tables 1 and 2). As for the colour parameters, no significant differences in the L* values between the groups at each time considered (data not reported) were recorded, while after 20 days of ripening (T2) the a* values for the OR salami were higher (P<0.01) compared to CTRL samples (ORT2: 16.88±0.91 vs CTRLT2: 14.35±1.12; ORT3: 15.86±0.86 vs CTRLT3: 12.96±1.26), which were also yellower (b* value, P<0.01) at the end of ripening period (OR: 7.78±0.35 vs CTRL: 9.56±0.80). Furthermore, the lipid oxidation (TBARS values) of meat was lower (P<0.01) in OR than in the CTRL group (Table 2). Differences between groups were recorded also for antioxidant activity and polyphenols content as evidenced by ORACFL and TPC values, higher (P<0.05) in OR then CTRL salami (Table 2). With respect to sensorial analysis, the results of consumer tests performed under blind, informed and expectation conditions are reported in Table 3 and Figure 2. The two groups of salami received the same scores in the blind test, whereas when the samples were accompanied by a label (informed condition), the consumers gave a higher (P<0.05) score to OR salami.
The improvement of meat and meat products hygienic characteristics due to animal diet supplementation with plant extracts reported by several authors (Bañón et al., 2012; Nieto et al., 2010; Serrano et al., 2014) has not been recorded in this study. This result is in agreement with previous studies from the same team in which natural preservatives feed supplementation did not affect microbiological characteristics of animal food product (Branciari et al. 2015a, 2015b, 2016; Ranucci et al., 2013, 2015). Moreover, the antimicrobial activity of these substances assessed in vitro, or when directly added to food, is widely demonstrated in literature (Chaves-López et al., 2015; Fasolato et al., 2015; Miraglia et al., 2016), even though the antimicrobial activity is related to the plant extract concentration which could affect sensory characteristics of the product (Giarratana et al., 2013, 2016). The use of natural substances in vivo as feed additives could avoid unpleased organoleptic traits in the product, however the antimicrobial efficacy is controversial. Probably other factors, such as primary production hygiene, process hygiene and process technology exert a great influence on microbial characteristics of the product. The dietary integration with oregano extract did not affect starter bacteria growth, therefore the salami from both experimental groups showed equivalent microbial evolution and fermentative processes. Neither pH and aw have been influenced by diet and at the end of ripening values were in line with those typical for this type of products. Nevertheless, in this study the introduction of bioactive substances in the diet was responsible for the effects on the other chemical and physical parameters such as oxidative stability and the colour. Lower content of MDA was found in OR salami, denoting an antioxidant effect exerted on the lipidic component (Table 2). Also the increase in b* values for CTRL salami could be a consequence of the oxidative reactions of lipids, which, not protected from dietary polyphenols, became more yellowish (Nieto et al., 2010). An antioxidant effect has also been registered for myoglobin, responsible for the red colour of meat, as highlighted by the higher values of a* in OR salami compared to CTRL ones. These results are in agreement with several authors, claiming that polyphenols may delay oxidation of fats and myoglobin, therefore stabilizing meat colour (Balentine et al., 2006; Luciano et al., 2011; Nieto et al., 2010; Ranucci et al., 2015; Ripoll et al., 2011). Apart from improving the oxidative status, the presence of oregano in the diet has significantly increased the polyphenolic concentration in OR salami, enhancing antioxidant power (Table 2). These results confirm that dietary inclusion of bioactive substances could represent an effective solution for the achievement of enriched meat and meat products (Moñino et al., 2008), increasing their nutritional value. Valenzuela et al. (2003) reports that the consumption of meat products rich in natural antioxidants reinforce the endogenous antioxidant efficacy against oxidative stress and ROS-induced tissue damage and degenerative diseases. With respect to sensorial analysis, consumers were positively affected by the information, giving a higher score to OR samples for all attributes. Furthermore, consumers showed a higher expectation for the OR salami compared with the CTRL ones. Results regarding consumer behaviour obtained in the present study are in line with Branciari et al. (2016), who showed how the effect of the label modifies consumers acceptability of the products.