Antioxidant and antimicrobial effects of dietary supplementation with rosemary diterpenes (carnosic acid and carnosol) vs vitamin E on lamb meat packed under protective atmosphere

Meat Science 110 (2015) 62–69

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Antioxidant and antimicrobial effects of dietary supplementation with rosemary diterpenes (carnosic acid and carnosol) vs vitamin E on lamb meat packed under protective atmosphere Jordi Ortuño, Rafael Serrano, Sancho Bañón ⁎ 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 13 November 2014 Received in revised form 8 July 2015 Accepted 9 July 2015 Available online 12 July 2015 Keywords: Polyphenols Vitamin E Lipid oxidation Protein oxidation Sensory evaluation Microbial quality

a b s t r a c t The antioxidant and antimicrobial effects on lamb meat of the dietary use of rosemary diterpenes and vitamin E were compared. Thirty fattening lambs were assigned to three diets: (C) control; (R) C plus 600 mg kg−1 carnosic acid and carnosol at 1:1 w:w; or (E) C plus 600 mg kg−1 α-tocopherol. The deposition of the dietary supplements in the muscle was determined. Microbial quality (total viable counts, Lactic Acid Bacteria, Enterobacteriaceae, Escherichia coli and Salmonella spp), oxidative stability (CIELab color, malondialdehyde and total carbonyls) and sensory attributes (appearance and odor) were determined in loin stored at 2 °C under 70% O2/30% CO2 atmosphere. Microbial quality was ensured by packaging and chilling. The E-diet was more effective (P ≤ 0.05) than the R-diet in preventing meat oxidation, although the latter had antimicrobial effects on meat. The shelf life of lamb (assessed as the loss of freshness) could be increased by 5 (R-diet) or 10 (E-diet) days. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction The extension of raw meat shelf life is a long-held aim of the meat industry as it would lead to great savings (Gill, 1996). Current strategies to preserve meat mainly involve the use of chilling systems, protective packaging and preservatives. High O2/CO2 modified atmosphere packaging (MAP) is commonly used to extend the shelf life of red meat, such as lamb, in retailing conditions: CO2 levels of 20–30% in atmosphere are often effective at inhibiting microbial spoilage (Gill & Tan, 1980; Stiles, 1991), whereas high O2 levels delay metmyoglobin formation prolonging meat redness (McMillin, 2008). However, MAP also promotes lipid oxidation due to the pro-oxidizing action of oxygen, which generates rancid off-odors. The shelf-life of chilled raw lamb loin kept in high O2/CO2 MAP would be around 8–9 days, by which time browning and/or rancidity reduces meat acceptance, even though the microbial quality is ensured (Bañón, Méndez, & Almela, 2012; Camo, Beltrán, & Roncalés, 2008; Ripoll, Joy, & Muñoz, 2011). Therefore, improving the antioxidant status of meat could help to increase its shelf life. Since 2008, the addition of antioxidants to meat cuts has not been permitted by the European Commission Regulation 1333/2008 on ⁎ Corresponding author at: Department of Food Science & Technology & Human Nutrition, Veterinary Faculty, University of Murcia, Campus Espinardo, 30071 Murcia, Spain. E-mail address: [email protected] (S. Bañón).

http://dx.doi.org/10.1016/j.meatsci.2015.07.011 0309-1740/© 2015 Elsevier Ltd. All rights reserved.

food additives. However, there is still the possibility of depositing antioxidants in the muscle by modifying the diet, thus increasing lipid and pigment stability during the meat retailing time. Vitamin E is the most widely used dietary antioxidant in animal feeding to prevent meat oxidation. The supplementation of sheep diet with vitamin E has been shown to offer protection against meat oxidation (Kasapidou et al., 2012; Kerry, Sullivan, Buckley, Lynch, & Morrissey, 2000; Lauzurica et al., 2005; López-Bote, Daza, Soares, & Berges, 2001; Turner, McClure, Weiss, Borton, & Foster, 2002; Wulf et al., 1995). The most biologically active form of vitamin E, α-tocopherol, is not degraded in the rumen but it is deposited in the muscle and fat tissues. The deposition of α-tocopherol in the muscle prevents lipid and pigment oxidation since it acts directly on the cell membranes (Higgins, Kerry, Buckley, & Morrissey, 1998). In order to achieve optimum protection in lamb meat effect, the minimum level of α-tocopherol for dietary inclusion has been established at around 500 mg kg−1 feed (López-Bote et al., 2001). In further studies, supplementation of the lamb diet with 250–1000 mg α-tocopherol kg−1 feed extended the shelf life of meat kept under MAP by up to 4 days due to its reduction of lipid and haem pigment oxidation, although the resultant sensory traits were not assessed. Moreover, vitamin E was seen to have no effect on microbial inhibition on meat (Álvarez et al., 2008; Lauzurica et al., 2005; Ripoll et al., 2011) In recent years, several alternative dietary strategies based on plant phenolic antioxidants have been successfully checked for improving lamb meat preservation (Andrés et al., 2014, 2013; Jerónimo et al.,

J. Ortuño et al. / Meat Science 110 (2015) 62–69

2012; Luciano et al., 2009; Rivas-Cañedo et al., 2013; Simitzis, Ilias-Dimopoulos, Charismiadou, Biniari, & Deligeorgis, 2013; Simitzis et al., 2008), and, in particular, by using rosemary and/or its derivatives (Bañón et al., 2012; Morán, Andrés, Bodas, Prieto, & Giráldez, 2012; Morán, Rodríguez-Calleja, et al., 2012; Morán et al., 2013; Nieto, Díaz, Bañón, & Garrido, 2010; Ortuño, Serrano, Jordán, & Bañón, 2014; Serrano, Ortuño, & Bañón, 2014). Carnosic acid and, in particular, carnosol, the main active diterpenes in rosemary, can be deposited in lamb muscle at sufficient levels to have antimicrobial and antioxidant effects on meat (Jordán, Castillo, Bañón, Martínez-Conesa, & Sotomayor, 2014; Moñino, Martínez, Sotomayor, Lafuente, & Jordán, 2008). Among the tested rosemary derivatives used in animal feeding, oil-free extracts provided effectual and steady results, probably due to their lack of heterogeneity and the possibility of adjusting the desired proportion of active compounds. For example, raw lamb shelf life was extended by up to 4 days when the lamb diet was supplemented with 200–600 mg diterpenes kg−1 feed (Bañón et al., 2012; Ortuño et al., 2014; Serrano et al., 2014). The supplementation of lambs with dietary rosemary extract (DRE) or vitamin E seems to yield similar benefits in terms of lamb preservation, although few comparative studies are available. When CaputiJambrenghi et al. (2005) used 500 and 1000 mg kg− 1 of a nonspecified DRE on air-packaged (AP) lamb, the results were poor compared with those obtained with 500 mg kg− 1 α-tocopherol. Neither the use of 600 and 1200 mg carnosic acid kg−1 feed did exert comparable antioxidant effects to 600 mg kg−1 vitamin E on lamb under MAP (Morán, Andrés, et al., 2012; Morán, Rodríguez-Calleja, et al., 2012). The lack of carnosol in the DRE used by these authors could have limited the transfer of rosemary diterpenes to lamb muscle (Jordán et al., 2014). Therefore, there are still issues to be clarified concerning the use of dietary treatments based on vitamin E or DRE, particularly as regards their antioxidant and antimicrobial effectiveness on meat. The aim of the present study was to compare the effects of DRE containing carnosic acid and carnosol and an equivalent dose of αtocopheryl acetate on the shelf life of lamb loin in retailing conditions (high O2/CO2 MAP, refrigeration and fluorescent lighting). 2. Material and methods 2.1. Dietary supplements Vitamin E (DL-α-tocopheryl acetate) and a DRE (containing carnosic acid plus carnosol) were used for the experimental lamb diets. Vitamin E (MicrovitTM E Promix 50) was provided by Lorca Alimentación Animal, S.A., Murcia, Spain. DL-α-tocopheryl acetate was obtained by adsorbing vitamin E oil on a silica support to obtain a powder with a vitamin E purity of 500 I.U. g−1. DRE was provided by Nutrafur-Furfural Español S.A., Murcia, Spain. The extract was obtained by successive extraction, drying and concentration stages using oil-free rosemary leaf and different solvents, including acetone and/or ethanol–water mixtures, as described by Del Baño et al. (2003). The resulting DRE was a dry (7.2 g water per kg extract) greenish-brown powder containing 0.31 kg rosemary diterpenes per kg extract (0.16 and 0.15 kg carnosic acid and carnosol, respectively). 2.2. Feed manufacturing The same dosage level (600 mg kg−1 feed) of rosemary diterpenes or vitamin E was incorporated, together with other additives (vitamins, minerals, etc.), to the respective experimental feeds for fattening lambs. The temperature and pressure during the whole pelleting process (17 min duration) were 70–75 °C and 2 bar, respectively. After pelleting, the remaining contents of active antioxidants in the feed were established in 515 mg kg− 1 diterpenes (252:263 carnosic acid:carnosol) and 525 mg kg−1 vitamin E. The HPLC methods for

63

determining rosemary diterpenes and vitamin E in feed will be described in Subsection 2.5. 2.3. Animals and diets Thirty weaned Segureña lambs (15 males and 15 females) with 13 ± 1 kg of live weight were selected from a collective feedlot. Lambs were individually identified and weighed, following which they were randomly assigned to one of three dietary treatments (10 lambs per treatment): lambs fed on a basal diet supplemented with 0.5 mg DRE kg−1 feed (R), 0.5 mg kg−1 α-tocopheryl acetate (E) or not supplemented (C) (Table 1). The lambs were fattened in individual pens located in an experimental farm from the period February to May. All handling practices followed the recommendations of the European Council Directive 86/609/EEC for the protection of animals used for experimental and other scientific purposes, and all of the animals were able to see and hear other lambs. All the animals were fed ad libitum with the corresponding fattening feed until they reached a live weight of 24 ± 1 kg. The fattening period lasted 50 ± 8 days. Finally, it was checked that the experimental diets given to lambs did not affect animal performance (average daily gain, conversion index or carcass weight) (data not shown). 2.4. Meat sampling The lambs were slaughtered in a local abattoir according to EC Regulations and the carcasses were chilled at 2 °C for 48 h. After chilling, a professional butcher removed the Longissimus thoracis et lumborum (LTL) muscle from both sides of the carcasses. Two batches composed of six loins (two loins from the same lamb carcass per each diet) were processed weekly until all thirty lamb carcasses had been analyzed. Meat sampling was carried out in a commercial butchery. The loins were filleted (1.5 cm thick) and packaged in polystyrene trays B5-37 Aerpack (Coopbox Hipania, Lorca, Murcia, Spain), in 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 2 per 24 h bar for oxygen, carbon dioxide and nitrogen, respectively (measured at 23 °C and 75% R.H.). The air in the packs was replaced by a modified atmosphere composed of 70% O2 and 30% CO2 (v:v) (EAP20, Carburos Metálicos, Barcelona, Spain) using a discontinuous INELVI VISC 500 packer (Industrial Eléctrica Vilar, Barcelona, Spain). Table 1 Ingredients and chemical composition of the experimental basal diet used in fattening lambs. Ingredients (g/kg fresh weight) Maize Barley Soybean meal DDGs Wheat bran Sugarcane molasses Calcium carbonate Crude soy oil Sodium chloride Sodium bicarbonate Vitamin D3 (E-671) (IU/kg DM) Vitamin A (E-672) (IU/kg DM) Vitamin E (mg/kg DM) Nutritional information (g 100 g−1) Crude protein Ether extract (crude lipids) Crude fiber Crude ash Calcium Phosphorus Sodium Magnesium

336.0 321.0 180.0 80.0 25.0 20.0 16.2 5.0 4.0 3.0 2000 10000 30.0

17.05 3.72 4.06 6.23 0.86 0.40 0.26 0.20

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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 CheckPoint II portable gas analyzer (PBI-Dansensor, Ringsted, Denmark). The proximate composition of fresh lamb meat from LTL was determined 48 h post-mortem to check that the samples from the different dietary trials had a similar proximate composition. The moisture (g 100 g− 1) was determined using a gravimetric method (ISO 1442:1997). Total protein (g 100 g−1) was determined following the Kjeldahl method (ISO 937:1981). Total fat (g 100 g−1) was determined using the Soxhlet method (ISO 1443:1973). For the shelf life study, the packed fillets from each lamb were stored at 2 ± 1 °C for 0, 7, 11, 14 or 18 days in a Climacell 707 display cabinet (MMM Medcenter Einrichtungen, München, Germany) continuously illuminated with white fluorescent light (800 lx). 2.5. Determination of α-tocopherol and rosemary diterpenes Meat samples (day 0) were frozen at −80 °C and then thawed at 4 °C for analyses. The α-tocopherol was extracted according to the method of Liu, Scheller, and Schaefer (1996) with modifications, and quantified by HPLC-UV according to the European Standard No EN-12822 (2000). Rosemary diterpenes were extracted and quantified using an HPLC system equipped with photodiode array UV/Vis (Moñino et al., 2008). The identification of diterpenic metabolite C19H22O3 (5,6-Dihydroxy-7isopropyl-1,1-dimethyl-2,3-dihydrophenanthren-9(1H)-one) was confirmed by means of HPLC-ESI-MS/TOF analysis. For more details, see Jordán et al. (2014). The concentrations of α-tocopherol and diterpenic metabolite C19H22O3 were expressed as mg kg−1 sample. 2.6. Microbiological analysis For microbiological assays, the bags containing samples were aseptically opened in a 131 Bio-II-A microbiology cabinet (Telstar, Tarrasa, Spain) before being weighed (10 g) with sterile tweezers into stomacher bags and blended with peptone water 0.1% w:w (Oxoid 133CM0087 Tryptone water, Basingstoke, Hampshire, United Kingdom) in a stomacher (IUL Instruments, GmBH, Köningswinter, Germany). 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 and 96 h, in an ST 6120 culture incubator (Heraeus S.A., Boadilla, Madrid, Spain). Enterobacteriaceae (ENB) were determined on Violet Red Bile Glucose Agar (CM04825, Oxoid) and incubated at 37 °C for 24 h (ISO 21528–2:2004). All the microbial counts were expressed as log CFU g−1. Escherichia coli (b10 CFU g−1) was determined using a chromogenic E. coli tryptone bile glucuronic agar (TBX) (Oxoid CM0945) after incubation at 44 °C for 24 h (ISO 16649–2: 2001). Salmonella spp. (absence in 25 g) was determined according to ISO 6579:2002. Samples were pre-enriched by incubating in peptone water at 37 °C for 18 h. A modified semi-solid Rappaport-Vassiliadis (MSRV) Agar (Oxoid CM1112) was used for the selective enrichment (at 41.5 °C for 24 h). Muller–Kauffmann Tetrathionate (MKT) Broth Base (Oxoid CM0343) was used as culture medium for the isolation of Salmonella spp and the suppression of Proteus species (at 37 °C for 24 h). The culture medium for the recovery of Salmonella was Xylose–Lysine–Desoxycholate AGAR XLD (Oxoid PO1132) (at 37 °C for 24 h). The selective and diagnostic culture medium was Brilliant Green Agar BGA (Oxoid: PO0171) (at 37 °C for 24 h). 2.7. Physical–chemical analysis Objective color was measured using a CR-200/08 Chroma Meter II (Minolta Ltd., Milton Keynes, United Kingdom) with a D65 illumination standard, 2° observer angle, and aperture size of 50 mm and calibrated against a standard white tile. Reflectance measurements

were taken directly on the meat surface after a blooming time of 30 min. The results were expressed as CIEL*a*b* values: Lightness (L*), Chroma (C*) and Hue angle (H*) (expressed as sexagesimal degrees), whose values were calculated as follows: C* = √ (a*2 + b*2); H* = tan− 1 (b*/a*). Nine 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 2,4dinitrophenylhydrazine (DNPH) to form protein hydrazones according the method of Oliver, Ahn, Moerman, Goldstein, and Stadtman (1987). The results were expressed as nmol DNPH fixed per milligram of protein. Lipid oxidation was assessed as thiobarbituric acid reactive substances (TBARS), as determined by (Botsoglou et al., 1994). The malondialdehyde (MDA) content (expressed as mg MDA kg− 1) was calculated as [MDA = (3D/3.769) × 10/W]; where: 3D was the value of the third derivate (Abs/532 nm) in the sample, and W was sample weight (g). Color measurements were made using a UV2 spectrophotometer (Pye Unicam, Cambridge, United Kingdom). All the chemicals were provided by Sigma Chemical CoAldrich (St. Louis, USA). 2.8. Sensory analysis A Quantitative Descriptive Analysis (ISO 4121:2003) was performed to assess lamb meat quality. Eight experienced panelists were specifically trained in six training sessions according to 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), exudation, odor (meaty, rancid acid and putrid) and freshness. Intensity scales graduated in one-point intervals (1: absent; 2: slight; 3: moderate; 4: intense; 5: very intense) were used to quantify these descriptors. Fresh and spoiled (stored at 4 °C under MAP for 21 days) lamb samples were used as extreme references for the scales of color, exudation and odor, except for putrid odor, for which samples stored at 4 °C for 7 days and kept under AP were used. No moldy odor was detected in meat kept under MAP. Finally, the panel scored the freshness using the same five-point numeric scale according to the loss of appearance and odor. The maximum score for freshness corresponded to fresh lamb, while the minimum score corresponded to the above mentioned spoiled lamb. Fresh lamb was characterized by lean redness, fat whiteness, slight serum-metallic odor and absence of exudate and off-flavors. Strongly spoiled lamb was characterized by lean browning, grayish fat, the presence of exudates, loss of the typical serum-metallic odor and intense off-flavors (rancid, putrid and/or acid). The panelists analyzed all samples in triplicate. 2.9. Statistical analysis An analysis of variance (Repeated Measures Model) was used to investigate the effect of the diet and storage time on the dependent variables. For the repeated-measures analysis, the degrees of freedom were adjusted for the correlation among the repeated measures using the correction by Greenhouse and Geisser. The LSD means test was used to compare the LSM, which were considered to be statistically different when P ≤ 0.05. MAP fillets from thirty lambs (10 lambs × 3 diets) were sampled at different storage times (5 control days). The data were analyzed using the IBM SPSS Statistics 19 software (IBM Software group, Chicago, IL, USA). The shelf life time of lamb was calculated by means

J. Ortuño et al. / Meat Science 110 (2015) 62–69

of polynomial regression equations between average freshness and storage time (Excell 2010, Microsoft Corporation, Redmond, WA, USA). The loss of half of the initial freshness was used to establish the maximum shelf life of the lamb (Serrano et al., 2014).

Table 3 Effects of diet and storage time on the microbial counts of lamb loin kept in retailing conditions for up to 18 days. Diet

Days 0

3. Results

65

TVC

C

4.31

Significance 7

a

11

4.49

ay

4.67

ab

14

4.79

ab

4.86

ab

18

Time

5.05

b

5.28

5.03

b

5.34c y *

Diet DxT

cy

y

No differences (P N 0.05) between treatments were found for the average contents of moisture, total lipids and total proteins (see Table 2). As regards muscle deposition of dietary supplements, metabolite C19H22O3 was the sole diterpene detected in the R-lamb at levels of 0.70 ± 0.48 mg kg−1 meat, whereas carnosic acid and carnosol were not detected in any sample. Vitamin E presented a higher accumulation level in lamb muscle than rosemary diterpenes. The mean concentration of vitamin E (expressed as mg α-tocopherol kg−1 meat) in loin differed (P ≤ 0.05) for the three diets, increasing in the following order: C-diet (0.71 ± 0.32) b R-diet (2.02 ± 0.63) b E-diet (3.95 ± 0.47). Compared with the concentration of vitamin E obtained with the basal diet, the muscle deposition of vitamin E strongly increased with the dietary supplementation of lambs with α-tocopheryl acetate. Table 3 shows the effects of diet and storage time on the microbial quality of lamb loin kept in retailing conditions. Regardless of the diet, lamb loin presented high microbial loads after processing in the butchery, although further bacterial growth was controlled by high O2/CO2 MAP and chilling. TVC and LAB counts did not reach 7 log CFU g−1 in any sample. Moreover, the R-diet was able to further inhibit bacterial growth in the lamb meat kept under protective atmosphere. There were significant (P ≤ 0.05) reductions in TVC and LAB in the R-lamb meat compared with meat from the other diets at different storage times. In contrast, ENB counts moderately increased (P b 0.05) during chilled storage and there was no diet effect on this bacterial group. As regards pathogenic bacteria, the absence of Salmonella spp in 25 g and E. coli counts below 1 log CFU g− 1 were verified in all the samples from all diets at all storage days. Thus, the supplementation of lamb diet with DRE reduced the bacterial growth in lamb loin, whereas the dietary use of vitamin E had no antibacterial effect on the meat. Table 4 shows the effects of diet and storage time on the physical– chemical traits of lamb loin kept in retailing conditions. Gradual browning, associated with a certain increase in brightness, was measured in lamb loin as the retailing time increased, which corresponded in terms of instrumental color with increases in H* angle and L*, respectively. The diet clearly (P b 0.001) modified the discoloration rate of meat, since H* increased to a much lesser extent in the E-lamb (from 13° to 20°) than in the R-lamb (from 13° to 52°) and C-lamb (from 15° to 62°) after the retailing period. When E- and R-lambs were compared, lower H* values were found in the former at several storage times. L* was also higher in the C-lamb and R-lamb than in the E-lamb from day 14 onwards, which could correspond to lower exudation in the Elamb. In contrast, the diet did not affect (P N 0.05) the pH or WHC, although the decrease in WHC during storage was more evident in the C-lamb, which would explain the differences in L* observed between diets. Possible deviations in color or exudation due to pH differences were discarded. On the other hand, there was a substantial (P ≤ 0.001) reduction in TBARS values in samples from the supplemented lambs. Table 2 Proximate composition of lamb loin from the different dietary trials. Diet

C E R SEM P

Loin composition Moisture

Total protein

Total fat

75.7 75.6 75.4 0.33

21.1 21.3 21.3 0.21

2.29 2.00 2.16 0.24

N.S.

N.S.

N.S.

All results are expressed as average g 100 g−1 and SEM (Standard Error of the Mean). P = levels of significance: ***(P ≤ 0.001); **(P ≤ 0.01); *(P ≤ 0.05); N.S (P N 0.05).

E R

LAB

4.17

a

y

y

3.65a

3.86ab

x

x

4.22ab

4.47b

4.67b

*

N.S.

***

**

N.S.

*

N.S.

N.S.

x

SEM 0.22 C 1.35a

0.22 2.86b y

0.28 3.49c y

0.31 4.19 c y

0.26 5.03d

1.42a

3.20b y

3.56bc

4.04c y

4.94d

y

E

y

R

0.88a

2.05b x

2.34b x

y

2.70bc

3.31c x

x

SEM ENB C E R SEM

0.24 1.41a 1.22a 0.57a 0.45

0.33 2.08ab 2.11ab 1.42b 0.50

0.39 2.24ab 2.29ab 1.59ab 0.41

0.35 2.41b 2.51b 1.82b 0.38

0.27 2.75b 2.96b 2.25b 0.30

TVC: Total viable counts; LAB: Lactic Acid Bacteria; ENB: Enterobacteriaceae. Diets: C (control); E (control plus α-tocopherol); R (control plus rosemary). All results are expressed as average log CFU g−1 and SEM (Standard Error of the Mean). a,b,c storage time effects in the same diet (P ≤ 0.05). x,y,z diet effects at the same storage time (P ≤ 0.05). Levels of significance: ***(P ≤ 0.001); **(P ≤ 0.01); *(P ≤ 0.05); N.S (P N 0.05).

The final MDA levels for C, R and E-lamb were 8.1, 5.5 and 1.0 mg kg−1 respectively, with the E-lamb having lower MDA levels than the R-lamb from day 7 onwards. In accordance with the lipid oxidation data, the increase in POx was also greater in the C-lamb (from 2.6 to 7.0 nmol carbonyl mg−1) than in the R-lamb (from 2.1 to 4.8) and E-lamb (from 2.2 to 3.2), with the E-lamb also having lower POx than R-lamb from days 7 onwards. The supplementation of the lamb diet with vitamin E or DRE delayed the oxidative deterioration of meat cuts kept under high O2/CO2 MAP, dietary vitamin E being more effective than DRE in inhibiting meat oxidation. Table 5 shows the effects of diet and storage time on the sensory quality of lamb kept in retailing conditions. Sensory changes began to be detected by the panel at day 7 as a result of the gradual lean browning, fat yellowing and the loss of serum odor due to rancidity and, to a lesser extent, acid odor, while putrid odors were not detected. In general, sensory deterioration was slower in samples from the supplemented lambs. Some sensory differences were found between the R-lamb and E-lamb. Lean color, meaty odor and rancid odor scored better (P ≤ 0.001) in the E-lamb than in the R-lamb from day 14 onwards. Fat color also scored higher (P ≤ 0.001) in the E-lamb than in R-lamb at day 18, whereas acid odor scored worse (P ≤ 0.05) in the E-lamb than in R-lamb at day 18, agreeing with LAB counts. Thus, both R- and Ediets maintained the sensory quality of lamb longer, although E supplementation was more effective in preventing discoloration and rancidity, corroborating previous CIELab and TBARS data. In contrast, the R-diet only seemed to be more effective than the E-diet in preventing acid off-odor. Having checked that chilling and MAP ensured the microbiological quality of meat for 18 days, the shelf life of lamb loin was estimated based on sensory criteria. According to the regression equations between freshness scores and storage time (Table 6), the initial score (day 0) of freshness had decreased by half after 7.5 (C lamb), 12.5 (R lamb) and 17.5 (E lamb) days of storage, meaning that the shelf life of lamb loin could be extended by 5 and 10 days, respectively, through the use of DRE and vitamin E. 4. Discussion Microbial standards must be observed in many countries for meat to be consumed. In our experiment, Enterobacteriaceae and the main

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Table 4 Effects of diet and storage time on the physical–chemical parameters measured in lamb loin kept in retailing conditions for up to 18 days. Diet

L*

C*

H*

pH

a

TBARS

POx

Days

C E R SEM C E R SEM C E R SEM C E R SEM C E R SEM C E R SEM C E R SEM

Significance

0

7

11

14

18

40.8 a 41.2 ab 41.2 a 0.81 14.9 a x 13.7 a y 14.2 a xy 0.36 15.1 a 12.5 a 13.5 a 1.95 5.70 5.71 5.69 0.01 80.8 a 81.1 a 79.9 a 1.16 0.05 a 0.04 a 0.04 a 0.01 2.58 a y 2.25 a x 2.09 a x 0.11

40.4 a 40.3 a 41.0 a 0.61 15.4 a 15.9 b 15.4 a 1.04 23.1 b x 19.1 b y 23.9 b x 1.06 5.72 5.72 5.70 0.02 73.0 b 74.0 b 71.8 b 0.93 4.56 b z 0.44 b x 3.07 b y 0.19 4.46 b z 2.54 ab x 3.59 b y 0.27

44.8 b x 41.6 bc y 43.0 b xy 0.71 11.5 b y 16.9 b x 15.1 a x 0.76 44.2 c x 21.2 b y 26.5 b y 3.08 5.71 5.72 5.70 0.02 71.0bc 72.7b 71.3b 0.98 6.22c z 0.56bc x 4.17c y 0.25 5.32bc z 2.71ab x 3.94b y 0.45

46.9 c x 42.2 bc z 44.3 b y 0.59 10.5 b z 16.2 ab x 13.5 ab y 0.90 56.0 c x 18.2 ab y 32.0 b y 5.31 5.70 5.71 5.68 0.01 69.6 c 72.0 b 70.6 b 1.03 7.45 d z 0.71 c x 5.02 d y 0.46 6.35 c x 2.98 b y 4.19 bc y 0.43

49.5 c x 43.8 c y 48.6 c x 1.09 11.1 b y 17.1 b x 11.9 b y 0.65 62.3 d x 19.7 ab y 52.3 c x 6.84 5.68 5.69 5.67 0.01 65.7d 68.2c 66.9c 0.96 8.15d z 1.03c x 5.51d y 0.31 6.96d z 3.16b x 4.76c y 0.33

Time

Diet

D×T

***

**

***

*

***

**

***

***

**

N.S.

N.S.

N.S.

***

N.S.

N.S.

***

***

***

***

***

***

L*: Lightness (CIE units); C*: Chroma (CIE units); H*: Hue angle (CIE units); WHC: water holding capacity (g 100 g−1); TBARS: thiobarbituric acid reactive substances (mg MDA kg−1 meat); POx: protein oxidation (nmol carbonyl g−1 protein). Diets: C (control); E (control plus α-tocopherol); R (control plus rosemary). All results are expressed as average values and SEM (Standard Error of the Mean). a,b,c storage time effects in the same diet (P ≤ 0.05). x,y,z diet effects at the same storage time (P ≤ 0.05). Levels of significance: ***(P ≤ 0.001); **(P ≤ 0.01); *(P ≤ 0.05); N.S (P N 0.05).

Table 5 Effects of diet and storage time on the appearance and odor of lamb loin kept in retailing conditions for up to 18 days. Diet

Lean color

Fat color

Meaty odor

Rancid odor

Acid odor

Freshness

C E R SEM C E R SEM C E R SEM C E R SEM C E R SEM C E R SEM

Days

Significance

0

7

11

14

18

4.96 a 5.00 a 4.98 a 0.03 4.93 a 4.97 a 4.95 a 0.04 5.00 a 5.00 a 5.00 a 0.00 1.00 a 1.00 a 1.00 a 0.00 1.00 a 1.00 a 1.00 a 0.00 4.98 a 5.00 a 5.00 a 0.01

4.36 b 4.53 b 4.53 a 0.10 4.43 b 4.58 b xy 4.68 a x 0.06 3.86 b y 4.88 a x 4.48 b x 0.14 1.98 b x 1.05 a y 1.32 b y 0.13 1.05 ab 1.05 ab 1.09 a 0.04 3.58 b y 4.20 b x 3.96 b x 0.13

2.54 c y 4.61 b x 4.14 b x 0.28 3.58 c y 4.64 b x 4.43 a x 0.17 2.31 c y 4.75 a x 3.97 b x 0.32 3.28 c x 1.13 a y 1.78 bc y 0.28 1.16 b 1.11 ab 1.04 a 0.05 1.95 c y 3.99 b x 3.38 b x 0.30

1.57 d z 4.45 b x 3.34 b y 0.24 3.03 d y 4.69 ab x 4.22 a x 0.17 1.59 cd z 4.53 b x 3.05 c y 0.27 3.85 cd x 1.19 a z 2.30 c y 0.20 1.45 c x 1.15 b y 1.21 b y 0.05 1.33cd z 3.83 b x 2.70 c y 0.24

1.50 d y 3.60 c x 2.07 c y 0.29 2.83 d y 4.03 c x 3.20 b y 0.20 1.27 d y 3.29 c x 1.61 d y 0.33 4.30 d x 1.80 b z 3.61 d y 0.22 1.44 c x 1.64 c y 1.39 c x 0.08 1.09 d y 2.83 c x 1.54 d y 0.32

Diets: C (control); E (control plus α-tocopherol); R (control plus rosemary). All results are expressed as average arbitrary units and SEM (Standard Error of the Mean). a,b,c Storage time effects in the same diet (P ≤ 0.05). x,y,z Diet effects at the same storage time (P ≤ 0.05). Levels of significance: ***(P ≤ 0.001); **(P ≤ 0.01); *(P ≤ 0.05); N.S (P N 0.05).

Time

Diet

D×T

***

***

***

***

***

***

***

***

***

***

***

***

***

*

N.S.

***

***

***

J. Ortuño et al. / Meat Science 110 (2015) 62–69 Table 6 Polynomial regression equations used to calculate the shelf-life of lamb loin. Diet

R2

Predictive equation

Shelf-life (days)

C E R

0.965 0.946 0.997

y = 0.004x2 − 0.310x + 5.095 y = −0.003x2 − 0.054x + 4.938 y = −0.005x2 − 0.091x + 4.972

7.5 17.5 12.5

Diets: C (control); E (control plus α-tocopherol); R (control plus rosemary). x: shelf life time; y: freshness score. The loss of half of the initial freshness was used to establish the maximum shelf life.

pathogenic bacteria that could colonize the meat were effectively inhibited by high O2/CO2 MAP and chilling, while mesophilic bacteria did not reach 7 log CFU g−1, the limiting value generally recognized as acceptable (Buys, Krüger, & Nortjé, 1994; Insausti et al., 2001). Among the different spoiling bacteria, LAB were more able to proliferate in meat under a high O2/CO2 atmosphere than aerobic putrefactive bacteria. The typically spoilage characteristics associated with LAB are sour and acid, due to the production of lactic and acetic acid (Dainty & Mackey, 1992). Unlike vitamin E, DRE showed antibacterial effects on lamb, even though microbial growth was strongly inhibited by MAP, which suggests that DRE might have been seen to be even more effective if MAP had not been used. Acidity assessment was consistent with the microbial data. The R-lamb scored lowest for acid odor and counted the lower LAB at the end of storage, while the acid odor became evident albeit slightly in the meat from the rest of dietary treatments, although rancidity could have partially masked it. Previous studies on lamb meat packed under high O2/CO2 MAP mention the potential of rosemary diterpenes to inhibit the growth of pathogenic and spoiling bacteria, molds and yeasts, where odor deterioration was not due to putrid or acid off-odors (Bañón et al., 2012; Ortuño et al., 2014). In contrast, other studies found no inhibitory effects on TVC, LAB or ENB in lamb reinforced with dietary vitamin E (Lauzurica et al., 2005). In a similar dietary study made in turkey, the use of rosemary inhibited TVC, LAB and ENB in breasts stored under refrigeration in darkness for 12 days, while dietary vitamin E had no antibacterial effects (Govaris et al., 2007). Meat shelf life can be estimated by reference to sensory criteria when its microbial quality is ensured by the packaging and storage conditions. Following this premise, the shelf life can be described as the length of time that the defined quality of meat remains acceptable under expected (or specified) conditions of distribution, storage and display (Gyesley, 1991). In the particular case of lamb raw meat packed under protective atmosphere, acceptance is mainly associated with freshness loss as a result of discoloration and flavor deterioration. Lean color strongly influences the acceptance of red meat. High O2 pressure favors oxymyoglobin formation, enhancing meat redness, although the myoglobin is gradually transformed to metmyoglobin under prooxidizing conditions, leading to gradual browning (Kerry et al., 2000). Color stabilization through high O2/CO2 MAP could be improved by the dietary use of vitamin E, and, to a lesser extent, of rosemary diterpenes. Other authors found similar results. For example, Morán, Andrés, et al. (2012); Morán, Rodríguez-Calleja, et al. (2012) compared loin cuts from lambs supplemented with 600 mg vitamin E kg− 1 or 1200 mg carnosic acid kg−1 stored under MAP (35 O2/35 CO2/30 N2) at 3 °C and fluorescent lighting for up to 14 days. Finding that vitamin E also was more effective than carnosic acid for stabilizing meat CIELab color values. Similar results were obtained by Caputi-Jambrenghi et al. (2005) when comparing loin cuts kept under AP for up to 7 days from lambs supplemented with 500 mg vitamin E kg−1 or 500–1000 kg−1 DRE, whose composition was not revealed. Lauzurica et al. (2005) reported that metmyoglobin was inhibited following 250–1000 mg dietary vitamin E kg− 1 supplementation in chilled-lamb loin packed under 70/30 O2/CO2 MAP for up to 28 days. In addition, color (assessed as CIELab color and by sensory descriptive analysis) was also stabilized using 200–600 mg of different DREs per kg feed in Segureño lamb loin

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processed under similar retailing conditions to those of the present study (Bañón et al., 2012; Ortuño et al., 2014; Serrano et al., 2014). Odor deterioration is another early sign of sensory spoilage in chilled-MAP lamb. The meaty odor quickly deteriorated after the first week of storage, when rancid odor began to be detected. Lipid autoxidation causes odor deterioration, in particular, rancid traits (Lillard, 1987). Hexanal and other volatile markers of lipid oxidation strongly increase in raw lamb meat during the first days of MAP storage, athough the formation of compounds responsible for rancid odor can be inhibited using dietary antioxidants (Olivares, Dryahina, Spanel, & Flores, 2012; Ortuño et al., 2014). Of particular note is the great potential of vitamin E as lipid antioxidant as indicated by the strong reduction in MDA levels and the absence of rancid odor in supplemented E-meat. Other studies agree that DREs are less effective than dietary vitamin E in preventing lipid oxidation, as assessed by TBARS (Caputi-Jambrenghi et al., 2005; Govaris et al., 2007; Morán, Andrés, et al., 2012), although no sensory tests were made. The antioxidant effects of diet were also reproduced in POx, with E-lamb having the lowest values. Little information is available on POx in lamb meat, although 2 nmol carbonyl mg−1 is considered a normal value for raw meat before retailing (Estévez, 2011). The amount of α-tocopherol has already been negatively correlated with carbonyl formation in lamb muscle (Santé-Lhoutellier, Engel, Aubry, & Gatellier, 2008) and POx formation was inhibited in lamb through DRE in a previous study (Ortuño et al., 2014). The effects of diet on oxidative stability may be explained by the deposition of dietary antioxidants in the muscle. Supplementing the lamb diet with vitamin E led to an approximately six-fold increase in the deposition of this vitamin in loin with respect to the basal diet. The dietary use of α-tocopherol at 500 mg kg−1 feed during the fattening stage was seen to provide vitamin E concentrations of between 1.8 and 5.9 mg kg−1 meat in different studies on light and heavy lambs (Álvarez et al., 2008; González-Calvo, Ripoll, Molino, Calvo & Joy, 2015; Kasapidou et al., 2012; Lauzurica et al., 2005; López-Bote et al., 2001; Wulf et al., 1995). Vitamin E levels of 2–5 mg kg−1 have been seen to be effective in protecting meat against oxidation in lamb (Álvarez et al., 2008; López-Bote et al., 2001) and beef (Liu et al., 1995; Arnold, Scheller, Arp, Williams, & Schaefer, 1993). In our study, the level of vitamin E in meat was within this range in the samples from E-lambs and R-lambs, but not in the samples from C-lambs. In addition, the same intake of α-tocopherol led to an almost three-fold increase in vitamin E concentration in the R-lamb compared with the Clamb, which suggests that muscle deposition of rosemary antioxidants might have contributed to preventing vitamin E degradation in the meat. As can been seen, rosemary diterpenes were also accumulated in lamb muscle. Diterpenic C19H22O3 is a secondary metabolite from carnosol oxidation which had already been identified by Jordán et al. (2014) in the muscle, kidney and liver of lambs supplemented with dietary carnosic acid and carnosol. In the above study, the supplementation of lamb diet with a similar DRE (at 640 mg kg−1) yielded 0.8–1.8 and 0.2–0.6 mg kg−1 of C19H22O3 and carnosol, respectively, in the deltoideus and abdominis muscles. This disparity in C19H22O3 deposition may be explained by the differences in the dose of DRE used and/or the muscle analyzed. The longissimus dorsi et thoracis muscle has a higher oxidative potential than the semitendinosus muscle in Segureño sheep (Peinado et al., 2004), which might explain both the absence of carnosol and the lower C19H22O3 levels found in the loin. In any case, rosemary diterpenes would be less bioavailable than vitamin E in lamb loin, since vitamin E is an endogenous molecule that can be deposited without transformation in the muscle, while carnosic acid and carnosol are exogenous molecules that are detoxified in liver and mainly deposited as secondary metabolites (Jordán et al., 2014). Dietary polyphenols might directly reduce free radicals resulting from the oxidation of lipids and proteins in meat and might exert an indirect antioxidant effect by reducing the oxidized form of vitamin E (Deckert et al., 2002; Gladine, Morand, Rock, Bauchart & Durand, 2007). Moreover, muscle deposition of C19H22O3 would be responsible for the slight

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antimicrobial effect found in the R-lamb, where diterpenes could have inhibited bacterial growth through membrane disruption mechanisms (Moreno, Scheyer, Romano, & Vojnov, 2006). The antioxidant and antimicrobial potential of rosemary diterpenes have been associated with their metal chelating activity, antiradical scavenging capacity, and capability to alter lipid order and the packing of phospholipid model membranes (Pérez-Fons, Garzón, & Micol, 2010). The doubt that emerges from our results is whether or not the antioxidant effects of rosemary diterpenes are linked to their intrinsic properties or to their indirect ability to protect vitamin E. The vitamin E integrated in phospholipid membranes is considered the primary lipid-soluble antioxidant in biological systems and the most powerful natural chain breaker in the muscle, since it is able to scavenge two peroxyradical molecules (Burton & Ingold, 1981; Descalzo & Sancho, 2008). Therefore, α-tocopherol is probably more efficient than rosemary diterpenes in terms of antioxidant activity which may explain the greater extension of lamb shelf-life achieved with vitamin E supplementation. Whatever the case, the use of rosemary products in sheep feed seems to be beneficial for meat production, although the exact supplementation protocol (active compounds, dose, feeding period, etc.) seems to be crucial to achieve positive results. As can be seen from different studies, supplementation of the ruminant diet with rosemary derivatives has produced dissimilar results. The dietary use of essential oils rich in terpenoids seems to have little preservative effect on lamb meat probably due to their low bioavailability and/or terpenoid degradation by ruminal bacteria (Vasta et al., 2013; Smeti, Atti, Mahouachi, & Muñoz, 2013), while oil-free rosemary leaves (Nieto et al., 2010) and their lipophilic derived extracts offer better results (Bañón et al., 2012; Morán, Andrés, et al., 2012; Morán, Rodríguez-Calleja, et al., 2012; Morán, Rodríguez-Calleja, et al., 2012; Ortuño et al., 2014). However, factors such as the dose and ratio of active molecules (O'Grady, Maher, Troy, Moloney, & Kerry, 2006; Morán, Andrés, et al., 2012; Morán, Rodríguez-Calleja, et al., 2012; Jordán et al., 2014), or the different animal response to dietary products (Mielnik, Aaby, & Skrede, 2003) may hinder standardisation of the supplementation method for livestock. The beneficial effects of dietary supplements from rosemary and other plants used in animal feeding to improve meat quality should be verified by studying the bioavailability of their active metabolites as well as their contribution to maintaining the levels of vitamin E, which appears to be the main contributor to the oxidative protection in the muscle. 5. Conclusion The dietary use of rosemary diterpenes or α-tocopherol in fattening lambs was seen to be effective in preventing meat oxidation, both showing clear synergism with the protective atmosphere used. Most of the physical–chemical and sensory traits determined in the meat revealed that dietary vitamin E was more effective as a meat antioxidant than rosemary diterpenes. This different antioxidant performance may be explained by differences in bioavailability and/or bioactivity. The strong pro-oxidizing and antimicrobial environment exerted by high O2/CO2 MAP conditioned the final outcome of both dietary supplements as meat preservatives. Under these conditions, α-tocopherol supplementation would be the most suitable dietary strategy to extend lamb meat shelf life. In contrast, rosemary diterpenes showed a microbial inhibitory potential, which could be effective when different storage conditions are applied, an issue that would be interesting to investigate in future studies. References Álvarez, I., de la Fuente, J., Díaz, M.T., Lauzurica, S., Pérez, C., & Cañeque, V. (2008). Estimation of α-tocopherol concentration necessary to optimise lamb meat quality stability during storage in high-oxygen modified atmosphere using broken-line regression analysis. Animal, 2(9), 1405–1411.

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