Use of dietary rosemary diterpenes to extend the preservation of sulphited-lamb products

Small Ruminant Research 123 (2015) 269–277

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Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres

Use of dietary rosemary diterpenes to extend the preservation of sulphited-lamb products ˜ Rafael Serrano, Sancho Banón ˜ ∗ Jordi Ortuno, Department of Food Science and Technology and Nutrition, Faculty of Veterinary Science, University of Murcia, Espinardo, 30071 Murcia, Spain

a r t i c l e

i n f o

Article history: Received 11 April 2014 Received in revised form 10 September 2014 Accepted 18 December 2014 Available online 31 December 2014 Keywords: Carnosic acid Carnosol Lamb Endogenous Antioxidants Antimicrobials

a b s t r a c t The use of dietary antioxidants is proposed for enhancing the preservative effects of sulphite in minced lamb products. Lamb diet was supplemented with 400 mg rosemary diterpenes (carnosic acid plus carnosol at 1:1 w:w ratio) per kg feed during the fattening stage. The patties were formulated combining meat from different sources (lambs given feed supplemented with rosemary extract and control lambs) and SO2 addition levels (0, 150, 300 and 450 mg kg−1 ). Several physical–chemical (reflectance, pH, WHC, carbonyls and volatiles from lipid oxidation), microbial (viable and lactic acid bacteria and coliforms) and sensory (appearance and odor) traits were determined in patties kept at 2 ◦ C and packed under 70/30 O2 /CO2 atmospheres. Dietary antioxidants extended the shelf life from 7.9 to 12.3 days in patties made with 450 mg kg−1 SO2 , but had little effect at lower SO2 doses. Greater inhibition of browning, lipid oxidation, odor deterioration and rancidity was achieved by using supplemented lamb. The processing of lamb meat reinforced with endogenous diterpenes from rosemary seems to be a promising strategy for manufacturing sulphited raw products. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Worldwide food safety strategies aim at linking the entire chain of food production and consumption. The use of natural antioxidants in animal feeding is considered as part of the “farm-to-fork” strategy involving animal production, meat processing, sale and consumption. Dietary antioxidants may be effective at improving the antioxidant status of meat, contributing to color and flavor stabilization and rancidity prevention, among other benefits. Dietary antioxidants are metabolized and deposited in

∗ Corresponding author at: Department of Food Science and Technology and Human Nutrition, Veterinary Faculty, University of Murcia, Campus Espinardo, 30071 Murcia, Spain. Tel.: +34 868 888265; fax: +34 868 884147. ˜ E-mail address: [email protected] (S. Banón). http://dx.doi.org/10.1016/j.smallrumres.2014.12.006 0921-4488/© 2015 Elsevier B.V. All rights reserved.

muscle, especially tissue membranes, where their antioxidant actions are more effective (Botsoglou et al., 1994). Another possible application of dietary antioxidants might be in reducing potentially toxic preservatives, in particular, sulphites or nitrites, in meat products. Both preservatives degrade more slowly in substrates containing antioxidants and so lower concentrations may be required to achieve the same degree of preservation in meat products (Roller ˜ et al., 2007). et al., 2002; Banón Sulphur dioxide (SO2 ), generally known as sulphite, is widely used as a preservative in minced raw meat due to its antimicrobial, antioxidant and, in particular, myoglobin stabilizing properties (Shayne, 2005). However, the potentially allergic and respiratory reactions associated with SO2 ingestion, along with the increasing awareness of consumers as regards food safety, make necessary to reconsider its use as meat preservative. The Food and Agriculture and World Health Organisations established a maximum

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permissible daily ingestion of 0–0.7 mg SO2 per kg of body weight and considered especially important to minimize SO2 in food with a high thiamine content, such as red meat (FAO/WHO, 1986). European Community Directive (95/2/EC) authorizes a maximum of 450 mg SO2 per kg in meat products. Phytochemicals, particularly, phenolic compounds, are being used as dietary antioxidants in different livestock species. Rosemary (Rosmarinus officinalis, L.) is recognized as a source of bioavailable polyphenols, such as carnosic ˜ acid and carnosol, for sheep (Monino et al., 2008). Rosemary essential oils (Vasta et al., 2013; Smeti et al., 2013) ˜ oil-free leaf (Nieto et al., 2010) and oil-free extracts (Banón ˜ et al., 2014) have et al., 2012; Morán et al., 2012; Ortuno been tested as dietary supplements for sheep. Depending on the method used, rosemary-based diets could induce antioxidant and, to a lesser extent, antimicrobial effects on lamb meat. Among rosemary derivatives, the use of typified dietary rosemary extracts (DRE) avoids the problems derived from the heterogeneity found in the phenol content of raw rosemary (Sotomayor et al., 2009). Supplementing the lamb diet with 200–400 mg carnosol-enriched extract per kg feed was seen to be effective in improving the antioxidant status of lamb meat (Jordán et al., 2014). Several ingredients, such as green tea, grape seed, ascorbate or chitosan, have been successfully tested for reducing the quantity of SO2 required for preserving minced raw meat, although certain sensory limitations were reported ˜ ˜ et al., 2007; Serrano and Banón, 2012). On the other (Banón hand, different rosemary extracts or oleoresins showed antioxidant and/or antimicrobial effects when they were added as ingredients to raw ground beef and pork (Rojas and Brewer, 2008), lamb patties (Baker et al., 2013) or buffalo and chicken patties (Naveena et al., 2013). The possibility of reducing SO2 was not considered in the above studies. Indeed, no dietary studies on reduced-SO2 meat products are available, although the dietary use of vitamin E was seen to be effective in extending the shelf-life of reduced-nitrite cooked ham (Dineen et al., 2001). Thus, dietary strategies involving rosemary could also help to limit the use of SO2 in raw meat products. The aim of the present work was to study the possibility of enhancing the preservative actions of sulphites in lamb patties through supplementation with a DRE containing carnosic acid and carnosol. The possibility of reducing the SO2 dose required to preserve the patties was also considered. 2. Materials and methods 2.1. Experimental design The shelf life of lamb patties from two combined treatments (Dietary Rosemary Extract “DRE” × SO2 added at different concentrations) was compared. Lamb diet was supplemented or not with carnosic acid and carnosol mixes during the fattening stage. Two homogeneous groups of nine lambs per each dietary treatment were selected and slaughtered to obtain the meat. Meat from leg was processed. The patties were formulated combining meat from two different sources (lambs given feed supplemented or not with 400 mg DRE kg−1 ) and four different SO2 addition levels (0, 150, 300 and 450 mg kg−1 ). Nine patty manufacturing trials including the eight treatment levels were made. One boned leg from each diet group (Control “C” and DRE “R”) was sampled during nine different trials (18 lamb legs in total). The resulting minced meat from each lamb leg was mixed and divided into four homogeneous batches to prepare the

samples with different SO2 addition levels. Ninety-six patties (15 ± 0.5 g) from each leg were used for sampling (6 patties × 4 SO2 levels × 4 control days). The patties were packed and kept in retailing conditions. Several physical–chemical (Instrumental color, pH, Water Holding Capacity “WHC”), total carbonyls and volatiles from lipid oxidation), microbial (total viable bacteria, lactic acid bacteria and total coliforms) and sensory (appearance and odor) quality traits were determined. 2.2. Diets and animal production Lamb diet was supplemented or not with DRE during the fattening stage. A typified DRE containing 0.3 kg diterpenes (carnosic acid and carnosol at 1:1 w:w) per kg extract was used (Nutrafur-Furfural, Murcia, Spain). The DRE was incorporated to the feed for lambs during pelletizing process (1.8 kg DRE per 1000 kg feed). After the pelleting process, the ˜ carnosic acid and carnosol contents were quantified by HPLC-MS (Monino na et al., 2008) in 186 and 213 mg kg−1 feed, respectively. The lambs (Segure˜ breed) were fattened in different pens located in the same farm during two different time periods (from November to March). Nine lambs per each dietary treatment were randomly selected from a larger group and were slaughtered (25 ± 1 kg slaughter weight) to obtain the meat. For ˜ more detailed information on DRE, diets and sheep rearing, see Ortuno et al. (2014). 2.3. Meat processing and sampling The lambs were slaughtered in a local abattoir according to EC Regulations and the carcasses were chilled at 2 ◦ C for 72 h in a cooling room. The legs were removed from the carcasses, boned by a professional butcher, vacuum packed, frozen and stored at −20 ◦ C for a maximum of 3 months. The frozen boned legs were thawed at 2 ◦ C overnight and minced (2 mm plate) using a P3298 cutter (Braher International, San Sebastian, Spain). Minced meat from gluteus, quadriceps, biceps femoris, semimembranosus, semitendinosus, adductor and other minor leg muscles were mixed for 5 min using an RM-60 mixer (Mainca Granollers, Spain). Twenty grams of sodium chloride salt per kg meat were added to the minced meat. Eight different patty formulations were established combining the two diets (C and R) and four SO2 addition levels (0, 150, 300 or 450 mg SO2 kg−1 meat). The resulting treatments were denominated as C0, C150, C300, C450, R0, R150, R300 and R450. SO2 was added in the form of sodium metabisulphite (Juan Martinez Perez, Murcia, Spain) using 5 mL of deionized water to dissolve it. The same amount of water was added to the non-sulphited patties. The mix, containing minced meat, sodium chloride, sulphite solution (or equivalent of water) was mixed for 1 min using an RM-60 mixer and then 15 ± 0.5 g patties were formed manually. The patties were assigned to different control days and packed in polystyrene trays, B5-37 Aerpack (Coopbox Hipania, Lorca, Murcia, Spain), covered by COMBIVAC 95 bags (Alcom Food Packaging, Girona, Spain) composed of polyamide with a polyethylene sealing layer. Gas permeability of bag was: 50, 150 and 10 cm3 mL2 per 24 h bar for oxygen, carbon dioxide and nitrogen, respectively (measured at 23 ◦ C and 75% R.H.). The samples were packed in a modified atmosphere (MAP) composed of 70% O2 and 30% CO2 (EAP20, Carburos Metálicos, Barcelona, Spain) in a discontinuous INELVI VISC 500 packer (Industrial Eléctrica Vilar, Barcelona, Spain). The meat/gas ratio was approximately 0.03 kg meat per liter O2 /CO2 . After sealing, the atmosphere inside the bags was checked using a Dan Sensor gas meter (WITT Gasetechnik, Witten, Germany). No significant variation in the gas mixture was detected during storage. The packed patties were kept at 2 ± 1 ◦ C for 0, 4, 8 or 12 days in a Climacell 707 display cabinet (MMM Medcentre Einrichtungen, München, Germany) continuously illuminated with white fluorescent light (800 lx), simulating retail display conditions. Samples were analyzed in triplicate. 2.4. Physical–chemical analysis Patties spoilage was monitored by reference to different physical–chemical parameters related with meat discoloration (instrumental color and pH), exudation (WHC) and oxidation (total carbonyls and volatile lipid oxidation markers). Objective color was measured using a CR-200/08 Chroma Meter II (Minolta Ltd., Milton Keynes, United Kingdom) with illuminant D65, 2◦ observer angle, and aperture size of 50 mm and calibrated against a standard white tile. Reflectance measures were taken directly on the meat surface after a

J. Ortu˜ no et al. / Small Ruminant Research 123 (2015) 269–277 Table 1 Target ions, linear retention indexes and secondary ions used to determine Volatile Oxidation Compounds (VOCs). VOCs

LRI

TI

Q1

Q2

Hexanal Heptanal 2-Pentylfuran Octanal Hexanoate vinyl ester 1-Hexanol Nonanal (E)-2-Octenal 1-Octen-3-ol 2-Octen-1-ol

761 835 877 936 979 1007 1045 1083 1116 1296

67 70 81 56 99 69 67 83 57 81

72 55 82 84 55 56 81 55 72 87

82 81 138 110 71 99 95 70 85 105

LRI, linear retention index; TI, target ion used for identifying and quantifying VOCs; Q1/Q2, secondary ions used for identifying VOCs.

blooming time of 30 min. The results were expressed as CIELAB values: lightness (L*), redness (a*), yellowness (b*) (CIE abbreviation means “Commission Internationale de l’Éclairage or “International Commission on Illumination”). The Chroma (C*) and Hue angle (H*) (expressed as √ sexagesimal degrees) values were calculated as follows: C* = (a*2 + b*2 ); −1 H* = tan (b*/a*). Twelve replicate measurements were taken for each sample. The pH was determined using a micropH 2001 pHmeter (Crison, Barcelona, Spain) equipped with a combined electrode Cat. No. 52–22 (Ingold Electrodes, Wilmington, USA) (ISO 2917:1999). Water holding capacity (WHC) was calculated using the method described by Grau and Hamm (1953) and expressed as g per 100 g sample. Protein oxidation (POx) was estimated as total carbonyls. Carbonyl groups were detected by their reactivity with 2,4-dinitrophenylhydrazine (DNPH) to form protein hydrazones according the method of Oliver et al. (1987) with ˜ et al., 2014). The results were expressed as slight modifications (Ortuno nmol DNPH fixed per milligram of protein. Volatile lipid oxidation markers (VOCs) were determined by Headspace-Solid Phase Microextraction (HS-SPME), using a SPME device (Supelco Co., Bellefonte, PA) containing a fiber coated with (CAR-PDMS-DVB) carboxen-poly(dimethylsiloxane)-divinylbenzene (50/30 lm thickness). The samples were analyzed using an Agilent 7890A GC series (Agilent, Avondale, USA) coupled to an Agilent IonTrap GC Mass Spectrometer (Agilent, Avondale, USA). The analytes were separated using a VF-WAXMS (30 m × 0.5 ␮m (0.25 mm)) column. For more details, ˜ et al. (2014). Table 1 includes the target ions, linear retention see Ortuno indexes and secondary ions used to determine the VOCs. 2.5. Microbiological analysis Total Viable Counts (TVC) and Lactic Acid Bacteria (LAB) were determined on Plate Count Agar (Oxoid CM0325) (ISO 4833:2003) and MRS agar (Cultimed-Panreac 413784) (ISO 15214:1998) under aerobic and anaerobic conditions respectively, after incubation at 30 ◦ C for 72 h, in an ST 6120 culture incubator (Heraeus S.A., Boadilla, Madrid, Spain). The total coliform counts (TCC) were determined using a chromogenic Escherichia coli/coliform medium (Oxoid CM956, Basingstoke, Hampshire, United Kingdom) (ISO 4831:2006). Plates were incubated for 24 h at 37 ◦ C in a ST 6120 culture incubator (Heraeus S.A., Boadilla, Madrid, Spain). All the microbial counts were expressed as log CFU g−1 . 2.6. Sensory analysis A Quantitative Descriptive Analysis was performed to assess lamb meat quality (ISO 4121:2003). Eight experienced pane lists were specifically trained in six training sessions (ISO 8586:2012). In the first two sessions, the color and/or descriptors of raw lamb meat were studied; the next two sessions were concerned with identifying, selecting and quantifying attributes to evaluate raw lamb spoilage under retail display conditions. The final two sessions were concerned with quantifying appearance, odor and freshness. The sensory descriptors selected were color (lean and fat), and odor (meaty, rancid, putrid and acid). Intensity scales graduated in one-point intervals (1: absent; 2: slight; 3: moderate; 4: intense; 5: very intense) were used to quantify these descriptors. Different freshly and strongly spoiled (stored at 4 ◦ C under 70% O2 and 30% CO2 atmosphere for 21 days) lamb samples were used as extreme references for the scales

271

of color and odor, except for putrid odor, for which air-packed samples stored at 4 ◦ C for 7 days were used. Finally, the panel scored the freshness (overall quality) using the same five-numeric scale according to the losses of appearance and odor. The maximum score of freshness corresponded to fresh by made lamb patties, while the minimum score of freshness corresponded to the above mentioned spoiled lamb patties. Fresh made lamb patties were characterized by lean redness, fat whiteness, slight serum-metallic odor and absence of off-flavors. Strongly spoiled lamb was characterized by lean browning, grayish fat, loss of the typical serummetallic odor and intense off-flavors (rancid, putrid and/or acid). 2.7. Statistical analysis The model design was completely random and the diet and the SO2 addition levels were considered as the main treatments. In order to clarify the influence of both treatments, all the variables were subjected to a two-way multivariate analysis of variance for each day of control. Average values from patty triplicates (9 lambs × 2 diets × 4 SO2 addition levels) were analyzed at the same storage time (n = 72 average values per each control day). A Least Significant Difference (LSD) test was used to compare the Least Significant Means, which were considered to be statistically different when P < 0.05. The data were analyzed using the IBM SPSS Statistics 19 (IBM Software group, Chicago, IL, USA). The shelf life of lamb was calculated by means of polynomial regression equations between average freshness and storage time (Excell 2010, Microsoft Corporation, Redmond, WA, USA). Polynomial regression was chosen because provided the best R2 coefficients compared with other non-linear regressions models used for predictive calculations. The loss of half of the initial freshness was used to establish the maximum shelf life of the lamb.

3. Results Table 2 shows the effects of the preservative treatments (DRE × SO2 ) on CIELAB color and pH of lamb patties kept in retailing conditions. Gradual meat discoloration was found in terms of instrumental color. Increases in L*, b* and H* (associated with decreases in a* and C*) reflected meat browning during the retailing period. Changes in H*, C* and a* were more evident than changes in L* and b*. DRE delayed (P < 0.05) meat browning from day 4 (a* and C*), 8 (H*) or 12 (L*) onwards, while b* was not affected (P > 0.05) by DRE. The addition of SO2 affected CIELAB color differently, depending on the dose used. Meat discoloration was less intense in the patties with 300 or 450 mg kg−1 SO2 . The effects of combined treatment were only evident at the end of storage; for example, R300 and C450 treatments provided similar protection against discoloration, while average H* was higher (P < 0.05) in C450 than in R450 patties at day 12. According to the reflectance values, DRE would contribute to stabilizing color in patties with high levels of SO2 . The pH stabilized throughout the retailing period and was not affected by any preservative treatment at any storage time (P > 0.05). Table 3 shows the effects of the preservative treatments (DRE × SO2 ) on WHC, POx and Volatile lipid oxidation compounds (VOCs) values of lamb patties kept in retailing conditions. WHC values were not affected (P > 0.05) by any preservative treatment at any control day. On the other hand, early oxidation was evident, probably triggered by the patty manufacturing process. Gradual oxidation (POx and VOCs increases) occurred in the patties kept under retail display conditions. Both DRE and SO2 treatments inhibited (P < 0.05) POx formation during all the retailing period. There were lower (P < 0.05) average values of POx on day 12 in R than in C patties for the four SO2 levels tested. In addition, similar (P > 0.05) POx levels were found in R0

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Table 2 Effects of diet and SO2 addition level on CIELab color and pH of lamb patties kept in retail conditions for up to 12 days. Lightness L*

Redness a*

Hue angle H*

Treatment/storage day

0

4

8

12

0

4

8

12

0

4

8

12

C0 R0 C150 R150 C300 R300 C450 R450 SEM

41.6 42.3 43.3 42.9 42.5 43.6 42.5 42.6 1.12

41.1ab 41.6ab 42.1b 41.3ab 39.9ab 40.7ab 39.5a 41.4ab 0.85

45.9c 43.6bc 44.8bc 44.0bc 42.7ab 42.4ab 42.5ab 40.8a 0.86

46.1cd 46.9d 46.5cd 45.4bcd 45.2bcd 43.3ab 44.3bc 42.9a 0.85

17.7 17.4 17.1 17.4 17.2 17.2 17.7 17.5 0.66

11.5b 13.5c 9.32a 11.6b 12.2bc 13.5c 13.1bc 13.1bc 0.66

6.39bc 7.52b 3.94a 6.20ab 7.04bc 9.30cd 8.88cd 11.1d 0.82

3.56ab 3.79abc 2.64a 3.24a 3.61ab 5.33c 5.02bc 8.34d 0.62

21.7 20.8 22.0 20.1 21.9 20.9 21.5 21.1 1.08

27.3ab 25.8a 33.8b 27.7ab 25.9a 23.2a 23.9a 22.8a 2.40

50.1bc 43.4cd 60.5d 51.0cd 40.9bc 36.1ab 34.7ab 26.9a 3.90

68.3d 66.8d 73.5d 69.4d 66.1cd 56.0b 57.2bc 39.6a 3.49

P-SO2 P-DRE P-SO2 × DRE

N.S.

*

**

***

N.S.

***

***

***

N.S.

*

***

***

N.S.

N.S.

N.S.

*

N.S.

**

***

***

N.S.

N.S.

*

***

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

*

N.S.

N.S.

N.S.

*

Yellowness b*

Chroma C*

pH

Treatment/storage day

0

4

8

12

0

4

8

12

0

4

8

12

C0 R0 C150 R150 C300 R300 C450 R450 SEM

7.05 6.63 6.90 6.41 7.11 6.51 6.96 6.69 0.42

5.79ab 6.50b 5.81ab 5.94ab 5.77ab 5.74ab 5.16a 5.49ab 0.34

7.52c 7.22bc 7.16bc 7.12bc 6.15ab 6.62abc 5.91ab 5.68a 0.43

9.02c 8.63bc 8.96bc 8.68bc 8.13bc 7.92abc 7.84ab 6.99a 0.38

19.1 18.7 18.4 18.6 19.1 18.4 19.1 18.8 0.68

13.0b 15.0c 11.2a 13.1b 13.5bc 14.7c 14.1bc 14.3bc 0.58

10.1bc 11.0bcd 8.36a 9.80ab 9.68ab 11.5cd 11.0bcd 12.6d 0.58

9.77a 9.67a 9.38a 9.33a 9.10a 9.74a 9.35a 11.0b 0.42

5.77 5.78 5.81 5.78 5.83 5.78 5.79 5.78 0.04

5.82 5.82 5.80 5.79 5.80 5.80 5.76 5.77 0.03

5.75 5.71 5.74 5.72 5.72 5.73 5.69 5.68 0.03

5.81 5.79 5.80 5.78 5.79 5.79 5.80 5.77 0.04

P-SO2 P-DRE P-SO2 × DRE

N.S.

*

**

***

N.S.

***

***

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

***

***

*

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

DRE, dietary rosemary extract; lamb source: C (control) and R (supplemented with 400 mg DRE kg−1 ). SO2 addition levels: 0, 150, 300 and 450 mg kg−1 . All color coordinates expressed as average CIE units. SEM, standard error of mean; P, probability values. Means with different superscripts are different for P < 0.05 (same storage time). *** P < 0.001. ** P < 0.01. * P < 0.05. N.S. P > 0.05.

and C450 patties from day 4 onwards, meaning that SO2 could be reduced maintaining the same degree of protein oxidation in these patties. The most abundant VOC quantified in the headspace was hexanal followed at a distance by heptanal, 1-octen-3-ol, octanal, nonanal, E-2-octenal, 2pentylfuran, 1-hexanol, 2-octen-1-ol and hexanoate vinyl ester. VOCs were differently affected by both preservative treatments; DRE mainly inhibited (P < 0.05) heptanal, octanal and nonanal formation, while SO2 mainly reduced (P < 0.05) the concentration of hexanal and the rest of the VOCs. In general, VOC data did not provide clear information on possible antioxidant synergies between both preservative treatments. The oxidation pathways are complex in meat lipids, involving the formation of a large number of volatile compounds that are simultaneously interacting in sample headspace. Table 4 shows the effects of the combined treatments (DRE × SO2 ) on microbiological counts of lamb patties kept in retailing conditions. Freshly-made patties had initial loads of around 3.7, 2.4 and 3.0 CFU log for TVC, TCC and LAB, respectively. These microbial counts hardly increased during storage up to 12 days. DRE reduced (P < 0.05) TVC from day 4 onwards, reduced (P < 0.05) TCC throughout the whole retailing period and inhibited LAB

growth (P < 0.05) from day 8 onwards. The addition of SO2 also reduced (P < 0.05) TVC and LAB from day 8 onwards, while its inhibitory effect on total coliforms was relevant (P < 0.05) from day 4 onwards. The data suggest that microbial spoilage was controlled by the combination of 70/30 O2 /CO2 MAP and chilling (2 ◦ C) and therefore, both preservative treatments showed minor antimicrobial effects. Table 5 shows the effects of the preservative treatments (DRE × SO2 ) on the appearance and odor of lamb patties kept in retailing conditions. Raw lamb suffered gradual lean browning, fat graying, loss of its metallic-blood odor and incipient rancidity during storage. Both preservative treatments, SO2 and, to a lesser extent, DRE, contributed to color stabilization and rancidity prevention. In general, lean brownness and fat grayness were scored as less intense in R than in C patties at day 8 and 8–12, respectively. Lean color scored higher (P < 0.001) in sulphited patties from the beginning of the retailing period. Noticeable color stabilization was reached in R450 patties, as shown by the high score given to their lean color, fat color and freshness. SO2 addition also contributed (P < 0.01) to prevent odor deterioration (from day 4 onwards) and rancidity (from day 0 onwards). Rancid odor also scored lower (P < 0.01) in R450 than in other patties. Acid or putrid odors were

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273

Table 3 Effects of diet and SO2 addition level on the total carbonyls (POx), water holding capacity (WHC) and volatile oxidation compounds (VOCs) of lamb patties kept in retail conditions for up to 12 days. POx

WHC

2-Pentylfuran

Treatment/storage day

0

4

8

12

0

4

8

12

0

4

8

12

C0 R0 C150 R150 C300 R300 C450 R450 SEM

2.53b 2.08ab 2.57b 2.01ab 2.20ab 1.68a 2.49ab 1.74ab 0.29

3.70c 3.30abc 3.88c 3.46bc 3.31abc 3.02ab 3.23abc 2.76a 0.23

5.60de 4.51bcd 6.20e 4.44bcd 5.05cde 3.47ab 3.85abc 3.01a 0.44

7.33c 5.14ab 7.52c 5.14ab 6.57bc 4.14a 5.59b 4.04a 0.62

83.6 81.6 81.2 81.7 80.8 80.6 79.8 80.7 1.04

90.1 87.4 87.0 87.2 87.3 87.2 86.7 86.8 0.97

89.9 88.3 87.7 89.1 87.5 88.3 87.9 88.2 0.72

83.6 81.6 81.2 81.7 80.8 80.6 79.8 80.7 0.93

0.043b 0.033b 0.006a 0.005a 0.005a 0.004a 0.005a 0.003a 0.003

0.145c 0.098bc 0.112bc 0.079abc 0.099abc 0.062ab 0.094abc 0.056a 0.019

0.208d 0.176cd 0.170cd 0.145bc 0.102ab 0.093ab 0.091a 0.067a 0.018

0.261c 0.251c 0.203bc 0.196ab 0.227bc 0.162ab 0.159ab 0.118a 0.026

P-SO2 P-DRE P-SO2 × DRE

*

*

***

*

N.S.

N.S.

N.S.

N.S.

***

N.S.

***

***

N.S.

*

***

***

N.S.

N.S.

N.S.

N.S.

N.S.

**

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

Hexanal

Heptanal

Octanal

Treatment/storage day

0

4

8

12

0

4

8

12

0

4

8

12

C0 R0 C150 R150 C300 R300 C450 R450 SEM

2.03cd 2.94d 1.57bc 0.92ab 1.00abc 0.59ab 0.64ab 0.39a 0.32

43.1bc 39.2bc 47.8c 43.1bc 39.6bc 35.7b 26.7a 25.2a 2.92

41.5bc 45.7c 44.7c 45.5c 37.0ab 38.1abc 34.4ab 33.6a 2.72

45.6 50.5 47.5 50.8 50.9 45.6 49.5 43.8 3.42

0.35c 0.19abc 0.28bc 0.12ab 0.24bc 0.11ab 0.15ab 0.07a 0.05

1.23abc 0.88a 1.41abc 1.02ab 1.70c 1.40abc 1.52bc 1.04ab 0.21

1.22ab 0.93a 1.29ab 1.05ab 1.53ab 1.12ab 1.71b 1.26ab 0.22

1.36a 1.19a 1.50ab 1.39ab 1.96bc 1.32ab 2.22c 1.53abc 0.24

0.105b 0.061ab 0.060a 0.061a 0.053a 0.029a 0.033a 0.019a 0.013

0.285abc 0.193a 0.324abc 0.246ab 0.406c 0.318abc 0.356bc 0.246ab 0.049

0.277 0.240 0.312 0.252 0.298 0.289 0.355 0.316 0.049

0.345a 0.295a 0.370ab 0.384ab 0.529b 0.290a 0.523b 0.328a 0.063

P-SO2 P-DRE P-SO2 × DRE

***

***

***

N.S.

N.S.

N.S.

N.S.

N.S.

**

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

*

*

*

*

*

*

N.S.

**

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

Hexanoate vynil ester

1-Hexanol

Nonanal

Treatment/storage day

0

4

8

12

0

4

8

12

0

4

8

12

C0 R0 C150 R150 C300 R300 C450 R450 SEM

0.012b 0.016b 0.001a 0.001a 0.002a 0.001a 0.002a 0.001a 0.002

0.629d 0.484bcd 0.582cd 0.428abcd 0.376abc 0.305abc 0.326ab 0.287a 0.083

0.595bc 0.668c 0.635c 0.558bc 0.440ab 0.444ab 0.316a 0.353a 0.055

0.513 0.512 0.503 0.538 0.558 0.491 0.472 0.417 0.062

0.018b 0.014b 0.006a 0.003a 0.001a 0.004a 0.006a 0.003a 0.002

0.038c 0.031bc 0.025ab 0.021a 0.025ab 0.019a 0.022ab 0.019a 0.003

0.039d 0.029bcd 0.029cd 0.023abc 0.023abc 0.018ab 0.019abc 0.015a 0.004

0.072b 0.045ab 0.044ab 0.036a 0.044ab 0.027a 0.028a 0.021a 0.008

0.072c 0.038b 0.028ab 0.013ab 0.019ab 0.010a 0.013ab 0.007a 0.006

0.226abcd 0.131ab 0.237bcd 0.132ab 0.253d 0.142abc 0.242bc 0.135a 0.029

0.258c 0.192ab 0.270c 0.176a 0.247bc 0.150a 0.255bc 0.158a 0.022

0.301bcd 0.216ab 0.285abc 0.238ab 0.389d 0.192a 0.378cd 0.214ab 0.032

P-SO2 P-DRE P-SO2 × DRE

***

**

***

N.S.

***

***

***

*

***

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

*

*

*

N.S.

*

***

***

***

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

1-Octen-3-ol

(E)-2-Octenal

2-Octen-1-ol

Treatment/storage day

0

4

8

12

0

4

8

12

0

4

8

12

C0 R0 C150 R150 C300 R300 C450 R450 SEM

0.151b 0.150b 0.050a 0.042a 0.052a 0.043a 0.050a 0.034a 0.012

0.267c 0.251bc 0.215abc 0.193abc 0.182ab 0.159a 0.175a 0.153a 0.024

0.227b 0.231b 0.200ab 0.198ab 0.164a 0.166a 0.150a 0.154a 0.018

0.266 0.273 0.226 0.256 0.251 0.227 0.224 0.204 0.029

0.068b 0.091b 0.020a 0.014a 0.023a 0.018a 0.022a 0.015a 0.009

0.302b 0.230b 0.303b 0.203b 0.194ab 0.149ab 0.133a 0.128a 0.045

0.379b 0.416b 0.370b 0.370b 0.215a 0.229a 0.146a 0.160a 0.037

0.510c 0.546c 0.457bc 0.492bc 0.415abc 0.375ab 0.299a 0.318a 0.048

0.013b 0.012b 0.002a 0.001a 0.001a 0.001a 0.001a 0.001a 0.001

0.030d 0.022cd 0.019bc 0.013abc 0.013ab 0.009ab 0.010ab 0.008a 0.004

0.033c 0.033c 0.023b 0.023b 0.014a 0.013a 0.009a 0.009a 0.002

0.042c 0.045c 0.030b 0.034bc 0.028ab 0.023ab 0.018a 0.019a 0.004

P-SO2 P-DRE P-SO2 × DRE

***

**

***

N.S.

***

***

***

***

***

***

***

***

N.S.

N.S.

N.S.

N.S.

N.S.

*

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

DRE, dietary rosemary extract; lamb source: C (control) and R (supplemented with 400 mg DRE kg−1 ). SO2 addition levels: 0, 150, 300 and 450 mg kg−1 . POx, WHC and VOCs results expressed as average nmol g−1 , g 100 g−1 and mg kg−1 , respectively. SEM, standard error of mean; P, probability values. Means with different superscripts are different for P < 0.05 (same storage time). *** P < 0.001. ** P < 0.01. * P < 0.05. N.S. P > 0.05.

274

J. Ortu˜ no et al. / Small Ruminant Research 123 (2015) 269–277

Table 4 Effects of diet and SO2 addition level on the spoilage bacteria counts of lamb patties kept in retail conditions for up to 12 days. Total viable counts

Total coliforms

Lactic acid bacteria

Treatment/storage day

0

4

8

12

0

4

8

12

0

4

8

12

C0 R0 C150 R150 C300 R300 C450 R450 SEM

3.95 3.59 3.87 3.61 3.85 3.53 3.82 3.35 0.19

3.88b 3.49ab 3.86b 3.42ab 3.79ab 3.25a 3.48ab 3.36ab 0.17

3.99c 3.46abc 3.87bc 3.43abc 3.62abc 3.31ab 3.36ab 3.19a 0.18

4.15b 3.64ab 3.66ab 3.62ab 3.66ab 3.62ab 3.27a 3.14a 0.25

2.75bc 2.11a 2.83bc 2.15a 2.89c 2.10a 2.30ab 2.20a 0.22

2.72d 2.06bc 2.40cd 2.13bc 2.37cd 1.64a 1.83ab 1.96ab 0.18

2.83c 2.26ab 2.82c 2.17ab 2.59bc 1.94a 2.18ab 1.80a 0.19

2.95c 1.93ab 2.30b 1.79ab 2.27b 1.68ab 1.92ab 1.55a 0.25

3.18 2.88 3.07 3.06 3.21 2.81 3.14 2.53 0.21

3.13 2.89 3.19 2.75 3.13 2.60 2.89 2.66 0.24

3.69b 3.24ab 3.47ab 3.16ab 3.18ab 2.97ab 2.94ab 2.69a 0.26

3.87b 3.44ab 3.39ab 3.33ab 3.41ab 3.13ab 2.99a 2.85a 0.26

P-SO2 P-DRE P-SO2 × DRE

N.S.

N.S.

*

*

N.S.

**

*

*

N.S.

N.S.

*

*

N.S.

**

**

*

*

***

***

***

N.S.

N.S.

*

*

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

DRE, dietary rosemary extract; lamb source: C (control) and R (supplemented with 400 mg DRE kg−1 ). SO2 addition levels: 0, 150, 300 and 450 mg kg−1 . All results expressed as average Log CFU g−1 . SEM, standard error of mean; P, probability values. Means with different superscripts are different for P < 0.05 (same storage time). *** P < 0.001. ** P < 0.01. * P < 0.05. N.S. P > 0.05.

Table 5 Effects of diet and SO2 addition level on the appearance and odor of lamb patties kept in retail conditions for up to 12 days. Lean Color

Fat Color

Rancid odor

Treatment/storage day

0

4

8

12

0

4

8

12

0

4

8

12

C0 R0 C150 R150 C300 R300 C450 R450 SEM

4.36a 4.32a 4.90b 4.87b 4.90b 4.90b 4.93b 4.91b 0.07

3.43bc 3.84cd 2.50a 2.97ab 3.72cd 3.72cd 3.88cd 4.10d 0.21

2.05bc 2.34c 1.30a 1.73ab 2.19bc 2.65c 3.29d 3.73d 0.21

1.21a 1.48ab 1.15a 1.30a 1.50ab 1.89bc 2.20c 3.24d 0.14

4.85 4.85 4.93 4.96 4.97 4.94 4.90 4.97 0.05

4.05abc 4.34cd 3.61a 3.83ab 4.36bcd 4.27bcd 4.36cd 4.57d 0.17

3.08abc 3.18bc 2.64a 2.85ab 3.21bc 3.52c 4.05d 4.16d 0.16

2.46a 2.78abc 2.59ab 2.66ab 2.64ab 2.98bc 3.14c 3.91d 0.13

1.23bc 1.27bc 1.00a 1.01a 1.01a 1.03ab 1.01a 1.03ab 0.04

2.13bc 1.84ab 2.72d 2.36cd 1.84ab 1.96bc 1.58a 1.52a 0.14

3.07c 2.93c 3.07c 3.09c 2.93c 2.65bc 2.38ab 2.04a 0.16

3.23b 3.14b 3.19b 3.14b 3.36b 2.88b 2.92b 2.16a 0.16

P-SO2 P-DRE P-SO2 × DRE

***

***

***

***

N.S.

***

***

***

**

***

***

***

N.S.

N.S.

**

***

N.S.

N.S.

N.S.

***

N.S.

N.S.

N.S.

*

N.S.

N.S.

N.S.

*

N.S.

N.S.

N.S.

*

N.S.

N.S.

N.S.

N.S.

Meaty odor Treatment/storage day

0

Freshness 4

8 abc

12 ab

0 a

4 ab

8

12

C0 R0 C150 R150 C300 R300 C450 R450 SEM

4.52 4.48 4.85 4.75 4.75 4.82 4.78 4.78 0.08

2.98 3.29bcd 2.40a 2.83ab 3.33bcd 3.29bcd 3.49bc 3.69d 0.24

2.11 2.22ab 2.05a 2.10ab 2.24ab 2.64bc 3.01cd 3.28d 0.18

1.83 1.77a 1.87a 1.87a 1.68a 2.02a 2.22ab 2.78b 0.16

4.54 4.53a 4.95c 4.91c 4.95c 4.93c 4.96c 4.86bc 0.08

3.05 3.55bc 2.43a 2.96ab 3.58bc 3.48bc 3.76c 3.94c 0.23

1.99 2.24abc 1.66a 1.85ab 2.33bc 2.61cd 3.11de 3.60e 0.19

1.50a 1.56a 1.54a 1.67a 1.61a 1.91ab 2.10b 2.99c 0.14

P-SO2 P-DRE P-SO2 × DRE

N.S.

**

***

***

**

***

***

***

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

*

**

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

*

−1

b

ab

DRE, dietary rosemary extract; lamb source: C (control) and R (supplemented with 400 mg DRE kg ). SO2 addition levels: 0, 150, 300 and 450 mg kg−1 . All results expressed as average values of arbitrary units (1 minimum; 5 maximum). SEM, standard error of mean; P, probability values. Means with different superscripts are different for P < 0.05 (same storage time). *** P < 0.001. ** P < 0.01. * P < 0.05. N.S. P > 0.05.

J. Ortu˜ no et al. / Small Ruminant Research 123 (2015) 269–277 Table 6 Polynomial regression equations used to calculate the shelf-life of raw lamb patties. Treatment

R2

Equation

C0 R0 C150 R150 C300 R300 C450 R450

0.999 0.991 0.992 0.999 0.999 0.998 0.990 0.980

y = 0.016x2 − 0.442x + 4.547 y = 0.005x2 − 0.312x + 4.578 y = 0.038x2 − 0.725x + 4.895 y = 0.028x2 − 0.603x + 4.915 y = 0.010x2 − 0.404x + 4.971 y = 0.012x2 − 0.389x + 4.910 y = 0.003x2 − 0.266x + 4.915 y = 0.005x2 − 0.207x + 4.818

Lamb source: C (control) and R (supplemented with 400 mg DRE kg−1 ). SO2 addition levels: 0, 150, 300 and 450 mg kg−1 . R2 , regression coefficients; y, storage time; x, freshness score.

not detected in patties at any retailing time (data not shown). Thus, DRE was particularly efficient in preventing discoloration, and to a lesser extent, rancidity, in patties containing 450 mg SO2 kg−1 . Finally, the shelf life of the differently treated (DRE × SO2 ) patties was estimated by using polynomial regression equations between freshness average score and storage time (Table 6 and Fig. 1). Based on the loss of half of the initial freshness, the shelf life increased by 4.9 days in R450 compared with C450 patties; however, shelf life extension through DRE was not relevant for other combined preservative treatments. 4. Discussion Discolouration and microbial spoilage are often the factors limiting the shelf life of minced raw meat kept under refrigeration. Previous freezing–thawing and mincing may also trigger microbial and oxidative phenomena conducive to meat spoilage (Buckley et al., 1995). The use of 20–40% CO2 in MAP inhibits microbial growth in refrigerated meat (reviewed by McMillin, 2008). All the bacterial groups analyzed, total viable counts (TVC), total coliforms counts (TCC) and lactic acid bacteria (LAB), were clearly controlled by the MAP used. TVC remained below 7 log CFU g−1 , the limit of acceptability for minced meat (Borch et al., 1996), for 12 days of storage, even when the patties were not subjected to any preservative treatment, suggestion that 14

R450

Shelf Life time (d)

12 10

C450

8

C300

R0 6

C0 C150

R300

R150

4 2 0

SO2 addition level (mg kg-1) Fig. 1. Estimated time of raw lamb patties according to their loss of freshness.

275

antimicrobial agents are less needed to preserve the patties packed in high O2 /CO2 atmosphere. On the other hand, high-O2 atmospheres enhance meat reddening because the surface oxymyoglobin layer is increased, but also favor lipid and protein oxidizing reactions resulting in rapid odor deterioration and rancidity (McMillin, 2008). The addition of sulphite enhances the technological role of high-O2 /CO2 MAP in raw meat products. Sulphite is toxic for many microbes and is very active against Gram negative bacteria such as Enterobacteriaceae (Banks et al., 1985). In addition, sulphite reduces the heme groups contained in the myoglobin molecules, delaying its oxidation to sulphate, favoring the formation of oxymyoglobin and deoxymyoglobin, and thus providing a fresh appearance to red meat (Wedzicha and Mountfort, 1991). Sulphite can also transform hydroperoxides into non-radical forms, acting as a secondary lipid antioxidant (Günther et al., 1997). Carnosol and, to a lesser extent, carnosic acid, are bioavailable molecules for sheep that are deposited in mus˜ cle increasing the antioxidant status of lamb meat (Monino et al., 2008; Jordán et al., 2014). This was corroborated in a previous technological study, where the same DRE had antioxidant and antimicrobial effects, increasing the shelf life of raw lamb filets kept in similar retailing conditions ˜ et al., 2014). Carnosic acid from 9.3 to 13.4 days (Ortuno and carnosol are metal chelating agents and also act on free radicals since their benzene rings inhibit chain reactions during lipid oxidation (Frankel, 1998). Thus, both rosemary diterpenes would act as radical scavengers protecting SO2 against oxidation, and therefore, enabling sulphite to act for longer. In addition, both diterpenes severely affect lipid order and the packing of phospholipid model membranes (Pérez-Fons et al., 2010), and are capable of inhibiting the growth of Gram positive and Gram negative bacteria and yeast (Moreno et al., 2006). The shelf life was considerably extended by DRE in the patties with 450 mg SO2 kg−1 , while poor or contradictory results were found at lower doses. Indeed, 150 mg SO2 kg−1 might have had certain prooxidant effects on the patties. It is well-known that several molecules can act as antioxidants or pro-oxidants in food substrates, depending on their concentration, although previous studies found that 150 mg SO2 kg−1 or even lower ˜ doses inhibited the oxidation of raw minced pork (Banón et al., 2007; Roller et al., 2002). Most of the quality traits examined suggest that the use of DRE improved meat preservation in high-sulphited patties. The reflectance values and, in particular, the color scores pointed to increased color stabilization as a result of the DRE used in patties containing 450 mg SO2 kg−1 . Sensory evaluation often provides a more accurate discriminator of color than CIELab coordinates due to the variation in light reflection from samples containing fat ˜ grains and lean meat (Serrano and Banón, 2012). Color and fat stabilization were also reported in MAP (Kerry et al., 2000) and air-packed (Strohecker et al., 1997) refrigerated patties made from the meat of lambs fed a diet supplemented with alpha-tocopherol. DRE clearly inhibited myoprotein oxidation regardless of SO2 dosage. Changes in membrane permeability favor the exit of Fe+2 and other divalent cations involved in the initiation of oxidation reactions. Metal ion-catalysed oxidation is

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the main source of carbonyl formation in proteins. The protective effect exerted by rosemary diterpenes against POx might therefore be attributed to their capacity to act as (i) metal chelating agents through the inactivation of the pro-oxidant effect of non-heme iron and (ii) scavengers of hydroxyls and other radicals formed via the iron-mediated Fenton reaction (reviewed by Lund et al., 2011). The reduction of POx by DRE has been reported previously in fresh ˜ et al., lamb cuts kept in similar retailing conditions (Ortuno 2014) and in other meat products with added polyphenols (Vuorela et al., 2005; Rodríguez-Carpena et al., 2011). Flavor deterioration in raw meat, particularly rancidity, is mainly related with volatile lipid oxidation products. Hexanal, heptanal, 1-octen-3-ol, nonanal and 2-pentylfuran are among the major lipid oxidation products identified in meat (Boylston, 2012). Hexanal is the most abundant VOC present in rancid meat, where it is being considered as a sensitive and reliable indicator of the oxidative status and flavor acceptability (Shahidi and Pegg, 1993). Heptanal, octanal and nonanal are compounds mainly derived from oleate, while hexanal, 2-octenal, 2pentylfuran, 2-octen-1-ol and 1-octen-3-ol are mainly generated from linoleate autoxidation (Frankel, 1998; Grosch and Ullrich, 1987). All the VOCs detected in patties have previously been detected in raw lamb longissimus dorsi (Vasta et al., 2013). Rosemary diterpenes reduced VOC formation in previous studies of fresh lamb meat ˜ et al., 2014); however, no information on VOC inhi(Ortuno bition in raw meat products by SO2 is available. In general, each treatment (DRE and SO2 ) exerted selective antioxidant actions on different VOC formation pathways from the autoxidation of oleic and linoleic acids, which suggests certain differences in their lipid antioxidant activities. The shelf-life extension values observed suggest a possible biological synergistic interaction between high SO2 doses and the DRE diet. These findings could be corroborated by the interaction observed in other parameters (a*, H*, color and freshness). Therefore, the processing of lamb meat reinforced with rosemary diterpenes may enhance the antioxidant activity of 450 mg SO2 kg−1 , although this SO2 level could not be reduced without detrimental effects on meat preservation. In multi-component systems, different antioxidants can reinforce each other by cooperative effects, a phenomenon known as synergism. Rosemary diterpenes are generally considered metal chelating agents and free radical acceptors, whereas sulphites are described as secondary antioxidants, with an ability to decompose hydroperoxides, while acting as reducing agents (Günther et al., 1997). According to Frankel (1998), the most common mechanisms yielding significant synergism involve the combination either of chain-breaking antioxidants and preventive antioxidants or free radical acceptors with reducing agents. The results point to that there was synergistic effect of DRE × SO2 in the patties containing 450 mg kg−1 SO2 . For example, sodium ascorbate and similar reducing agents are widely used in meat products to stabilizer sulphites and nitrites, among other functions. Other authors mention the possibility of reducing the amount of sulphites required to preserve minced meat through combination with functional ingredients rich in polyphenols, such as green tea or grape seed extracts

˜ (Banón et al., 2007), or even non-phenolic antioxidants, such as chitosan (Roller et al., 2002; Serrano et al., 2012). 5. Conclusions The use of dietary bioavailable antioxidants, such as carnosic acid and carnosol from rosemary, is promising as an effective strategy to enhance the preservative effects of sulphite in minced meat. The preservative effects of sulphite might be improved by using meat from lambs fed a supplemented diet. Dineen et al. (2001) reached similar conclusions in low-nitrite meat products made from the meat of pigs fed a diet supplemented with vitamin E. However, according to our results, it seems more unlikely to lower SO2 addition by carnosic acid and carnosol supplementation. Future studies should explore the possibility of manufacturing both low-SO2 or even SO2 -free products, by processing meat reinforced with alternative dietary antioxidants. Conflict of interest None declared. References Baker, I.A., Alkass, J.E., Saleh, H.H., 2013. Reduction of oxidative rancidity and microbial activities of the Karadi lamb patties in freezing storage using natural antioxidant extracts of rosemary and ginger. Int. J. Agric. Food Res. 2, 31–42. Banks, J.G., Dalton, H.K., Nychas, G.J., Board, R.G., 1985. Sulfite, an elective agent in the microbiological and chemical changes occurring in uncooked comminuted meat products. J. Appl. Biochem. 7 (3), 161–179. ˜ Banón, S., Díaz, P., Rodríguez, M., Garrido, M.D., Price, A., 2007. Ascorbate, green tea and grape seed extracts increase the shelf life of low sulphite beef patties. Meat Sci. 77, 626–633. ˜ Banón, S., Méndez, L., Almela, E., 2012. Effects of dietary rosemary extract on lamb spoilage under retail display conditions. Meat Sci. 90, 979–983. Borch, E., Kant-Muermans, M.L., Blixt, Y., 1996. Bacterial spoilage of meat and cured meat products. Int. J. Food Microbiol. 33, 103–120. Botsoglou, N.A., Fletouris, D.J., Papageorgiou, G.E., Vassilopoulos, V.N., Mantis, A.J., Trakatellis, A.G., 1994. Rapid, sensitive, and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissue, food, and feedstuff samples. J. Agric. Food Chem. 42, 1931–1937. Boylston, T., 2012. Land animal product. In: Nollet, L.M.L. (Ed.), Handbook of Meat, Poultry and Seafood Quality. Wiley-Blackwell, Oxford, pp. 140–156. Buckley, D.J., Morrisey, P.A., Gray, J.I., 1995. Influence of dietary vitamin E on the oxidative stability and quality of pig meat. J. Anim. Sci. 73, 3122–3130. Dineen, N., Kerry, J.P., Buckley, D.J., Morrisey, P.A., Arendt, E.K., Lynch, P.B., 2001. Effect of dietary ␣-tocopheryl acetate supplementation on the shelf-life stability of reduced nitrite cooked ham products. Int. J. Food Sci. Technol. 36, 631–639. FAO/WHO. Food and Agriculture Organization/World Health Organization, 1986. Evaluation of Certain Additive and Polluting Agents in Foods. 291 Report of Mixed Committee FAO/WHO of Food Additive Experts. Series of Technical Information, N. 733, Geneva. Frankel, E.N., 1998. Lipid Oxidation. The Oily Press Ltd., Dundee. Grau, R., Hamm, R., 1953. Eine einfache methode zur bestimmung der wasserbindung im muskel. Naturwissenschafen 40, 29–30. Grosch, W., Ullrich, F., 1987. Identification of important volatile flavor compounds formed during autoxidation of linoleic and linolenic acids. J. Am. Oil Chem. Soc. 64, 624. Günther, A., König, T., Habicher, W.D., Schwetlick, K., 1997. Antioxidant action of organic sulphites-I. Esters of sulphurous acid as secondary antioxidants. Polym. Degrad. Stab. 55, 209–216.

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