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Carnosic acid dietary supplementation at 0.12% rates slows
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down meat discolouration in gluteus medius of fattening lambs
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RUNNING TITLE:
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L. Morán1*, J. M. Rodríguez-Calleja2, R. Bodas1, N. Prieto1, F. J. Giráldez1
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and S. Andrés1
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24346 Grulleros, León, Spain.
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24071 León, Spain.
Instituto de Ganadería de Montaña (CSIC-Universidad de León). Finca Marzanas. E-
Departamento de Higiene y Tecnología de los Alimentos, Universidad de León, E-
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(Received - Accepted)
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CORRESPONDING AUTHOR: Lara Morán, Instituto de Ganadería de Montaña
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(CSIC-ULE). Finca Marzanas. E-24346 Grulleros, León, Spain. Tel. +34 987 307 054
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Fax +34 987 317 161 E-mail: laramoran@eae.csic.es
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Abstract
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Thirty-two Merino lambs fed barley straw and a concentrate alone (CONTROL) or
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enriched with vitamin E (VITE006) or carnosic acid (CARN006; CARN012) were used
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to assess the effect of these antioxidant compounds on meat quality attributes. The
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animals were slaughtered after being fed for at least 5 weeks with the experimental
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diets. The longissimus lumborum samples of VITE006, CARN006 and CARN012
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groups showed higher values (P<0.001) of L* (lightness) thorough the complete storage
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period under modified atmosphere when compared to the CONTROL group. Moreover,
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the VITE006 and CARN012 samples revealed lower discolouration when compared to
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the CONTROL group, these differences being more apparent in a less colour stable
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muscle such as gluteus medius (P<0.05 for hue after 14 days of refrigerated storage).
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Meat sensory traits were not significantly affected by carnosic acid and microbiological
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analyses were not conclusive at the doses administered.
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Keywords: Microbial spoilage; colour; sensory evaluation; water holding capacity;
carnosic acid; rosemary
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Introduction
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The shelf life of meat and meat products is seriously shortened by two main factors:
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microbial spoilage and colour stability, the last one being closely related to lipid
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peroxidation (Young & West, 2001). Therefore, any finding focused on delaying either
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of these processes is highly relevant for the meat industry. However, the high quality
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meat demanded by the consumer in developed countries (Boleman et al., 1997) must be
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free of chemically synthesized additives, so the addition of synthetic food stabilizers to
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meat in order to extend the shelf life of these products in the market does not seem to be
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the best choice. A suitable alternative is the inclusion of natural compounds (plant
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origin) in animal feedstuff, thus avoiding any further manipulation of the meat.
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In this context, especial attention has been paid to the antimicrobial and antioxidant
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effects promoted by rosemary (Rosmarinus officinalis L.), a herb commonly used as a
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flavouring agent. The bioactive properties of this herb have been attributed to the
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phenolic compounds in rosemary plants (Hernández-Hernández, Ponce-Alquicira,
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Jaramillo-Flores, & Legarreta, 2009). These compounds have demonstrated
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antimicrobial and antioxidant activities when added to food as additives (McBride,
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Hogan, & Kerry, 2007), but they have also shown beneficial effects on eggs, milk and
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meat products when rosemary is included in the diet of the animal (Botsoglou, Govaris,
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Giannenas, Botsoglou, & Papageorgiou, 2007; Galobart, Barroeta, Baucells, Codony, &
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Ternes, 2001; Jordán, Moñino, Martínez, Lafuente, & Sotomayor, 2010; Nieto, Díaz,
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Bañón, & Garrido, 2010). In this last sense, the main phenolic compound retained in
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animal tissues after the consumption of rosemary is carnosic acid (Moñino, Martínez,
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Sotomayor, Lafuente, & Jordán, 2008), so it can be hypothesized that the antimicrobial
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and antioxidant properties observed in meat quality are mainly due to the increment of
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this phenolic compound at this level. However, the amount of carnosic acid which must
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be fed to the animals to have beneficial effects on meat quality has not yet been
61
quantified. It must also be considered that the concentration of phenolic compounds in
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the plants varies depending on the maturity stage or climatic conditions, mainly
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conditions of drought (Munné-Bosch, Mueller, Schwarz, & Alegre, 2001). This is the
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reason why feeding rosemary extracts with a known richness of carnosic acid instead of
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intact plants will allow recommendations to be established about the amount of
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rosemary which should be fed to the animals according to its levels of carnosic acid.
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Therefore, the aim of the present study was to investigate the shelf-life extension of
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meat (antimicrobial properties and colour stabilization) when two different doses of
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carnosic acid (from rosemary extract) were included in the diet of lambs. Likewise,
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vitamin E (one of the most frequently used antioxidants in animal nutrition) was
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included in another group as a positive control.
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Material and Methods
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2.1 Animals and diets
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Two weeks before the commencement of the trial, 32 male Merino lambs were
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treated with Ivermectin (Ivomec, Merial Labs, Barcelona, Spain) and vaccinated against
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enterotoxaemia (Miloxan, Merial Labs, Barcelona, Spain).
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After random stratification on the basis of body weight (average body weight (BW),
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15.2 ± 0.749 kg), the lambs were allocated to four different groups: a control group
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(CONTROL), a group fed vitamin E (α- tocopheryl acetate) at a rate of 0.6 g kg-1 of
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feed concentrate (VITE006, equivalent to 900 IU of vitamin E kg-1 of feed concentrate),
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a third group fed a similar dose as the previous group (0.6 g carnosic acid kg-1 of feed
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concentrate, CARN006) of carnosic acid (Shaanxi Sciphar Biotechnology Co., Ltd,
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Xi'an, China) and the fourth group fed a double dose of carnosic acid (1.2 g carnosic
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acid kg-1 of feed concentrate, CARN012). The animals were then individually penned.
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All handling practises followed the recommendations of the European Council
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Directive 86/609/EEC for the protection of animals used for experimental and other
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scientific purposes, and all of the animals were able to see and hear other lambs.
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After 7 days of adaptation to the basal diet comprised of concentrate (50% barley,
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20% soybean meal, 15% maize, 8% wheat, 4% molasses and 3% mineral premix;
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chemical composition: dry matter (DM) 888 g kg-1, crude protein (CP) 178 g kg-1 DM,
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neutral detergent fibre (NDF) 134 g kg-1 DM, and ash 56 g kg-1 DM) and barley straw
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(DM 913 g kg-1, CP 35 g kg-1 DM, NDF 757 g kg-1 DM, ash 55 g kg-1 DM), all of the
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lambs were fed barley straw and the corresponding concentrate feed alone (CONTROL
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group) or supplemented with either vitamin E or carnosic acid. The concentrate (35 g
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kg-1 BW day-1) and forage (200 g day-1) were weighed and supplied in separate feeding
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troughs at 9:00 a.m. every day, and fresh drinking water was always available.
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2.2 Slaughter procedure, packaging, storage and sampling
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The animals were slaughtered on four different days, two lambs per group each day.
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The lambs were selected each day according to their weight (25 kg intended body
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weight) and slaughtered by stunning and exsanguination from the jugular vein; they
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were then eviscerated and skinned. The hot carcass of each lamb was weighed, chilled
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at 4° C for 24 h and weighed again. The pH value from the longissimus thoracis muscle
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at the sixth rib was determined in triplicate at 0 h, 45 min and at 24 h post-mortem
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before the muscle was removed from the carcass, using a pH meter equipped with a
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penetrating glass electrode (pH meter Metrohm® 704, Switzerland).
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The left hind leg was removed and stored at -30 ºC until sensory evaluation.
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Moreover, the longissimus thoracis (LT) et lumborum (LL) and gluteus medius (GM)
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muscles were removed from the right and left carcass sides. The LT samples were used
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for chemical analysis in accordance with the methods described by the Association of
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Official Analytical Chemists (AOAC, 2003), whereas the LL and GM muscles were cut
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into slices 2.5 cm thick, placed on impermeable polypropylene trays and wrapped with
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ML40-G bags (Krehalon; Proveedora Hispano Holandesa S.A., Barcelona, Spain),
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which were immediately modified-atmosphere packaged (MAP) using a tabletop
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Multivac
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Wolfertschwenden, Germany). The air in the bags was replaced by a commercial gas
A300
packaging
machine
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(Multivac
Verpackungsmaschinen,
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blend intended for red and poultry meats consisting of 35% CO2, 35% O2 and 30% N2,
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with a gas:meat volume ratio of about 2:5:1. The ML40-G bags had O2 and CO2
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transmission rates of 20 and 100 ml m-2 24 h-1, respectively, at 23 ºC and 80% relative
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humidity. All packages were stored under simulated retail display conditions [12 h daily
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fluorescent illumination (34 W) and 3±1 ºC] and the air temperature was monitored
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using a Testo175-T2 data logger (Instrumentos Testo S.A., Cabrils, Barcelona, Spain).
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The meat in these polypropylene trays was used to study the extract-release volume
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(ERV, on LT muscle), microbial spoilage (on LT muscle), the rate of discolouration (on
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LL and GM muscles) and the water holding capacity (WHC, on LL muscle).
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2.3 Colour, extract release volume (ERV) and water holding capacity (WHC)
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On each sampling day, the concentrations of CO2 and O2 inside each tray were
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determined using a CheckMate 9900 (PBI Dansensor, Denmark). After opening the
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packages, a slice of fresh meat from each muscle was measured for colour parameters
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on days 0, 3, 7, 9 and 14. The L* (lightness), a* (redness) and b* (yellowness) values
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(Centre Internationale de l'Eclairage, 1986) were used to determine the meat colour of
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the muscles using a chromameter (Minolta® Chroma Meter 2002, Germany). The hue
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angle (h*), which defines colour (0° is red; 90° is yellow), was calculated as arctangent
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(b*/a*), and the chroma (C*), a measure of colour intensity (0 is dull; 60 is vivid), was
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computed as
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(ERV) was measured (on days 0, 7 and 14) as previously described (Rodríguez-Calleja,
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Santos, Otero, & García-López, 2004). Briefly, minced lamb (15 g) was mixed with 60
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ml of the extraction reagent (0.2 M KH2PO4 and 0.2 M NaOH; pH 5.8) and
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homogenized for 2 min. The homogenate was filtered through Whatman no. 1 paper and
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the ERV was recorded as the volume collected in 15 min.
(a *2 b *2 ) (Young & West, 2001). Also, the extract release volume
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The water holding capacity (WHC) was measured on LL muscle via cooking losses,
according to Honikel (1998).
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2.4 Microbiological analysis
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Twenty-five grams of LL muscle from each tray (0, 7 and 14 days) was placed into
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sterile Stomacher bags, rinsed with peptone water (1:5 dilution) and the rinsate was then
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diluted tenfold. The numbers of total viable bacteria at 4.5ºC (TVB), Pseudomonas spp.,
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lactic acid bacteria (LAB) and Enterobacteriaceae (EC) were determined and
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confirmed as described elsewhere (Rodríguez-Calleja et al., 2004; Rodríguez-Calleja,
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García-López, Santos, & Otero, 2005). Briefly, TVB were determined by the pour plate
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technique on Plate Count Agar (PCA; Oxoid, Basingstoke, UK) incubated at 4.5 ºC for
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14 days. Pseudomonas spp. numbers were determined after 2 days incubation at 25 ºC
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on a Pseudomonas agar base (Oxoid) to which a CFC (cetrimide, fucidin, cephaloridine;
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Oxoid) supplement was added. The oxidase test (Oxidase Touch sticks, Oxoid) was
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performed on randomly selected colonies and only oxidase-positive colonies were
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counted as Pseudomonas spp. The LAB colonies were enumerated on overlaid plates of
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MRS (de Man, Rogosa and Sharpe; Oxoid) agar following 3 days incubation at 30 ºC.
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Brochothrix thermosphacta was enumerated on streptomycin sulphate cycloheximide
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thallous acetate agar (STAA, Oxoid) after incubation for 2 days at 25 ºC. Overlaid
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plates of Violet Red Bile Glucose agar (VRBGA, Oxoid) were used for EC counts after
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24 h incubation at 37 ºC.
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2.5 Sensory evaluation
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The muscle vastus intermedius (VI) of the left hind leg was chosen for sensory
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evaluation because it is considered a high value retail cut -lamb leg roast, chump-
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(Johansen, Aastveit, Egelandsdal, Kvaal, & Røe, 2006). Sensory analysis was carried
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out by 24 consumers. The left hind leg was defrosted at 4 ºC for 48 h and the VI was
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dissected and cut into steaks 20 mm thick. The steaks were cooked a pre-warmed clam-
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shell grill to an internal temperature of 74º C in the geometric centre of the steak
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(measured by a Digi-Sense thermocouple probe, Cole-Parmer Instrument Company,
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Illinois, USA), after which all fat and connective tissue was trimmed and the muscle cut
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into blocks of 2×1×1 cm. The blocks were wrapped in pre-labelled foil (the blocks from
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each animal were coded with the same alphabetical letter), placed in a heated incubator
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and then given to the assessors in a random order chosen by a random number
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generator. Two triangle tests (ISO 4120:1983) were carried out to discriminate between
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meat from the lambs fed (1) CONTROL vs. CARN012, and (2) CARN006 vs. VITE006
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diets. In the triangle test, the assessors were requested to identify which two of three
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products were most alike and hence to indicate which product was the most different
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from the other two. Additionally, all consumers participated in two blind preference
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tests in which they received two meat samples on a coded paper plate. Each consumer
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tasted and evaluated their preferences for the meat samples from lambs fed (1)
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CONTROL vs. CARN012, and (2) CARN006 vs. VITE006 diets, and provided the
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reasons for their choices.
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2.6 Statistical analysis
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Microbiological counts were transformed and expressed as log CFU g-1. The basic
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descriptive statistics for each parameter (mean and standard deviation) were calculated.
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The pH data from different times were analysed by a one-way analysis of variance. The
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ERV, WHC, microbial population and colour parameters data (L*, C* and h*) were
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subjected to a two-way analysis of variance, with treatment and day as the main factors.
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The Tukey’s honestly significant difference test was used to compare means where the
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main effects were significant (P<0.05). All analyses were performed using the GLM
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procedure of the SAS package (SAS, 1999). Data from the sensorial analysis were
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analysed using the chi-square test (Stone & Sidel, 1993) of the FREQ procedure in SAS
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(SAS, 1999).
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Results and discussion
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A previous study (López-Bote, Daza, Soares, & Berges, 2001) described a
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significant positive effect with low supplementation levels of vitamin E (270 mg kg-1
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diet) on lipid oxidation in meat; however, the protection effects at different storage
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times, and particularly the effect on meat surface redness, were optimized at a dietary
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inclusion of 550-625 mg α-tocopheryl acetate kg-1 diet. Consequently, in the present
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study, vitamin E was supplied at a rate of 0.6 g kg-1 of feed concentrate (VITE006
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group). The same dose of carnosic acid was supplied to another experimental group
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(CARN006) to compare the effectiveness of both components. The last group was fed a
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double dose of carnosic acid (CARN012) in order to clarify whether this substance
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could show a dose-dependent effect at the meat level.
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3.1 Chemical composition
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The mean and standard deviation (SD) of the chemical data corresponding to the
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lamb meat samples (LT) are summarized in Table 1.
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[INSERT TABLE 1 NEAR HERE, PLEASE]
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As can be observed, chemical composition of LT meat samples seemed to be
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affected by carnosic acid added to the diet, thus giving rise to higher levels of crude
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protein content when compared to the VITE006 group (Table 1).
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3.2 Colour parameters
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Table 2 summarizes the results of LL and GM muscles when the colour stability was
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studied after packaging in a controlled gas mixture for 14 days. These muscles have
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been described in bovine as high (LL) and medium (GM) colour-stable muscles
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(McKenna et al., 2005) due to the different values observed in their metmyoglobin
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reductase activity (MRA) and oxygen-consumption rate (OCR) parameters (McKenna
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et al., 2005; Young & West, 2001).
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[INSERT TABLE 2 AND FIGURE 1 NEAR HERE, PLEASE]
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As can be observed (Figure 1a), antioxidant treatments (CARN006, CARN012 and
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VITE006) showed higher L* values in the LL muscle throughout the complete storage
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period when compared to the CONTROL (P<0.05). However, no differences in L*
221
values were detected among CARN006, CARN012 and VITE006 groups (Figure 1a and
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Table 2). It has been previously demonstrated that phenolic-rich extracts are iron
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chelating agents (Saman et al., 2001) thus promoting either lower red blood cells counts
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or low haemoglobin levels. In fact, lower levels of haemoglobin content were measured
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in the blood of CARN006, CARN012 and VITE006 lambs when compared to the
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CONTROL group (data not shown). The inhibition of iron absorption caused by
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phenolic compounds in the diet of the lambs of the present experiment might have
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caused lower myoglobin contents and hence higher values of L* in CARN006 and
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CARN012 groups when compared to the CONTROL lambs. High doses of vitamin E
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have also demonstrated to be effective in reducing total iron concentrations to normal
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levels in the brain of mice (Lan & Jiang, 1997). The evidence observed by Lan & Jiang
232
(1997) would be in accordance with the low L*values in the LL of the VITE006 group.
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Moreover, oxygenation (which involves oxymyoglobin formation) reduces L* values
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while oxidation (which involves metmyoglobin formation) had an opposite effect,
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suggesting that lightness is also affected by the state of the myoglobin (Fernández-
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López, Pérez-Álvarez & Sayas-Barberá, 2000; Linares, Bórnez & Vergara, 2008). This
237
last argument might be the reason why L* was increased in the GM (medium colour10
238
stable muscles) of the CONTROL lambs after 7 days of refrigerated storage (Figure 1b),
239
whereas this parameter was quite stable in the GM of the lambs being fed antioxidants
240
(Figure 1b). Regarding LL muscle, L* was also very steady even in the CONTROL
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group (Figure 1a), probably due to the high colour stability of this muscle.
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As far as C* is concerned, the evolution of this parameter during refrigerated storage
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under MAP was similar in both muscles (Figure 2). Thus, C* evolution could be fitted
244
successfully to a second order polynomial function in all of the treatments either on LL
245
(R2= 0.77-0.95) or GM (R2= 0.77-0.98) muscles. Consequently, the first derivative was
246
calculated for each curve in order to know when the most intense colour (C*max) was
247
reached for each muscle in each group (Fernández-López et al., 2000). Thus, in LL
248
muscle C*max was reached after 6.8 days of refrigerated storage in the CONTROL
249
lambs, and slightly later in the antioxidant groups (7.3, 7.4 and 7.6 days for CARN006,
250
CARN012 and VITE006 groups, respectively). Thereafter, LL discolouration took
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place, with CARN006, CARN012 and VITE006 groups showing no differences in C*
252
when compared to the CONTROL lambs after 14 days of refrigerated storage (Figure
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2a).
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[INSERT FIGURE 2 NEAR HERE, PLEASE]
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The previous statement is in agreement with the higher values (although not
256
significant) of the h* parameter in the LL muscle after 14 days of refrigerated storage in
257
the CONTROL and CARN006 groups (Figure 3a), which also indicated that the meat
258
become dull faster in these lambs when compared to VITE006 and CARN012 samples
259
(Young & West, 2001). However, it must be stressed that none of these differences in
260
C* and h* values reached a level of significance in LL samples. This might have been
261
due to either a lack of effect of treatment or the high colour stability of LL muscle.
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[INSERT FIGURE 3 NEAR HERE, PLEASE]
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On the contrary, once again a medium colour-stable muscle such as GM showed
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more promising results when antioxidants were fed to the lambs. Thus, in GM muscle
265
C*max was reached after 5.5 days of refrigerated storage in the CONTROL lambs, and
266
after 5.9, 7.6 and 7.5 days in CARN006, CARN012 and VITE006 groups, respectively.
267
According to these data meat discolouration would be delayed at least two days in
268
medium-colour stable muscles from lambs being fed the highest antioxidant doses.
269
Thereafter, a more pronounced decrease in C* values was observed in CONTROL
270
samples, thus indicating a faster rate of metmyoglobin accumulation in this group when
271
compared to the others (Figure 2b). This is in agreement with the lower values in h*
272
(P<0.05) observed in VITE006 and CARN012 groups (more intense redness) when
273
compared to the CONTROL and CARN006 groups after 14 days of refrigerated storage
274
(Figure 3b). However it must be stated that at the same doses (CARN006 vs. VITE006),
275
carnosic acid showed a less potent effect than vitamin E on colour of GM muscle, and
276
only the highest dose of carnosic acid (CARN012) tended to show similar effects than
277
vitamin E.
278
3.3 pH, ERV and WHC
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The pH values of the meat samples are summarized in Table 3. As expected, the pH
280
values significantly decreased in all of the groups (P<0.001) after 45 minutes and 24 h
281
post-mortem; however, no significant differences (P>0.05) between the groups were
282
detected at any time. Accordingly, no differences in WHC among the groups due to pH
283
variations were expected.
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[INSERT TABLES 3 AND 4 NEAR HERE, PLEASE]
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Table 4 summarizes the ERV values throughout the refrigerated storage period. As
286
can be observed, the ERV values became significantly reduced during the storage
287
period (P<0.001) in all the groups, probably due to increases in the bacterial populations
288
(Jay, 1966). However, no differences among groups were detected at any time.
289
Regarding WHC (Figure 4), the lost of water in the CONTROL group (20.4%) was
290
not statistically different from that measured in the VITE006 (17.5%) and CARN012
291
(17.5%) groups after 7 days of refrigerated storage. However, the endogenous proteases
292
(µ-calpain and m-calpain) have been suggested to be protected by antioxidants against
293
post-mortem oxidation,thus allowing their activity (Huff-Lonergan & Lonergan, 2005),
294
so the water expelled from the intramyofibrillar spaces would be kept within the cells
295
thus reducing drip production (Huff-Lonergan & Lonergan, 2005). Consequently, the no
296
significant differences in the present study might have been due to either the lack of
297
treatment effect or the high intra-group variability observed in WHC values.
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[INSERT FIGURE 4 NEAR HERE, PLEASE]
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3.4 Microbiological analysis
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All of the counts of Brochothrix thermosphacta were under the detection level (<1.4
301
log ufc/g, data not shown). The means of Pseudomonas spp., EC, TVB (4.5ºC) and
302
LAB for each storage time in each treatment are shown in Table 5.
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[INSERT TABLE 5 NEAR HERE, PLEASE]
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In agreement with the lack of significant differences in ERV (Table 4),
305
microbiological growth for Pseudomonas spp., EC (Gram-negative), LAB (Gram-
306
positive) and TVB (4.5ºC) did not seem to be significantly affected by dietary carnosic
307
or vitamin E supplementation. It must be stressed that Pseudomonas spp. counts have
308
been found to decrease when rosemary extract is directly added to meat (Zhang, Kong,
13
309
Xiong, & Sun, 2009), thus indicating antimicrobial properties of rosemary extract
310
against one of the common meat spoilage bacteria at refrigerated temperatures, i.e.
311
Pseudomonas spp. However, it must be considered that Pseudomonas spp. is among the
312
most sensitive species to CO2 (Jay, Loessner, & Golden, 2005), so the MAP used in the
313
present study might have inhibited this microorganism, thus hindering a possible effect
314
of carnosic acid. In any case, the lack of significant differences in the counts of the rest
315
of microorganisms (EC, LAB and TVB, Table 5) also suggests that other compounds
316
(but not carnosic acid) might be responsible for the antimicrobial properties of rosemary
317
(Pintore, Usai, Bradesi, Juliano, Boatto, Tomi, Chessa, Cerri & Casanova, 2002).
318
3.5 Sensory evaluation
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When meat from the lambs fed the CONTROL and CARN012 diets were compared,
320
most of the consumers (17 out of 24, 70.8%, P<0.05, Table 6) could not distinguish
321
between the treatments. Moreover, 62.5% of the consumers preferred the CONTROL
322
samples describing them as more tender and juicy, but this percentage was not
323
statistically different from the percentage of consumers who preferred the CARN012
324
samples (P>0.05). When meat from lambs fed the CARN006 and VITE006 diets were
325
compared, only eight correct judgements (33.3%) were given. Therefore, the consumers
326
could not distinguish between these samples (P<0.10, Table 6). Likewise, the
327
CARN006 samples were preferred in 10 out of 24 cases (P>0.05), and the VITE006
328
samples were judged as more tender and tasty. Hence, in both cases, the meat samples
329
from lambs fed carnosic acid (CARN006 and CARN012) were neither clearly
330
distinguished nor preferred by the consumers when they were compared with those
331
from lambs fed either the CONTROL or VITE006 diets.
332
[INSERT TABLE 6 NEAR HERE, PLEASE]
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333
Conclusion
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At the doses used in the present study, it can be concluded that carnosic acid shows a
335
lower antioxidant activity when compared to vitamin E but it seems to be useful to
336
protect meat from discolouration after a long period of time under MAP at refrigerated
337
storage, particularly in low colour stable muscles such as gluteus medius. Moreover,
338
carnosic acid does not significantly affect meat sensory traits. The microbiological
339
analyses were not conclusive at the doses administered in the present study. Future
340
experiments should include higher rates of carnosic acid in the diet of fattening lambs to
341
check the potential of this compound in further delaying the microbial spoilage of meat.
342
Acknowledgements
343
Financial support received from ‘Consejería de Educación de la Junta de Castilla y
344
León’ (Project GR158) is gratefully acknowledged. Raúl Bodas and Nuria Prieto have a
345
JAE-Doc contract and Lara Morán was supported by a JAE-Predoc grant from the CSIC
346
under the programme ‘Junta para la Ampliación de Estudios’. We would also like to
347
thank M. J. Jordán from IMIDA (Murcia, Spain) for the carnosic acid quantification by
348
HPLC of the rosemary extract used in the study. M. Y., A. S. C., and D. D. G. designed
349
the research, M. Y. conducted the research, M. Y. and D. D. G. analysed the data and
350
wrote the paper. D. D. G. had primary responsibility for the final content.
351
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19
444
Table 1. Chemical composition of longissimus thoracis muscle (g kg-1 meat)
CONTROL
CARN006
CARN012
VITE006
P-value
Ash
11.3 ± 0.28
11.9 ± 0.65
11.9 ± 0.81
12.0 ± 0.52
0.080
CP
196.8ab ± 4.28
201.2a ± 7.54
201.7a ± 4.56
194.3b ± 3.27
0.020
EE
23.8 ± 6.26
26.3 ± 4.51
24.7 ± 7.86
26.6 ± 4.30
0.753
DM
233.5 ± 6.34
239.5 ± 7.67
238.4 ± 9.50
236.1 ± 4.46
0.373
445
446
447
448
449
450
Mean ± standard deviation; CONTROL group (no antioxidants); CARN006 (0.6 g
carnosic acid kg-1 of feed concentrate); CARN012 (1.2 g carnosic acid kg-1 of feed
concentrate); VITE006 (0.6 g α-tocopheryl acetate kg-1 of feed concentrate); CP, crude
protein; EE, ether extract; DM, dry matter; a, b, c: different superscripts in the same row
indicate statistical differences (P<0.05) among treatments
20
451
Table 2. Colour parameters for each treatment and storage day at 4oC under modified atmosphere package (35% CO2, 35% O2 and 30% N2)
452
21
longissimus lumborum
TREATMENT
DAY
P-value
gluteus medius
L*
a*
b*
L
a*
b*
CONTROL
39.3b ± 1.63
10.3 ± 1.41
9.9a ± 1.08
41.4 ± 1.65
9.8 ± 1.50
10.8ª ± 0.85
CARN006
40.6ª ± 2.19
10.2 ± 1.45
10.3b ± 1.21
41.7 ± 1.43
9.9 ± 1.45
11.0a ± 1.12
CARN012
40.3ª ± 1.50
10.2 ± 1.30
9.8a ± 1.18
41.4 ± 1.71
10.1 ± 1.28
10.6ª ± 0.94
VITE006
40.5ª ± 2.08
10.2 ± 0.85
9.7a ± 1.25
41.3 ± 1.48
10.1 ± 0.87
10.2b ± 1.09
0
39.9 ± 2.27
9.9b ± 1.07
8.6b ± 1.14
41.1 ± 1.70
10.3ª ± 1.31
9.6b ± 1.09
3
40.4 ± 1.83
10.7ª ± 1.17
10.2ª ± 0.56
41.8 ± 1.36
10.3ª ± 1.07
10.9ª ± 0.93
7
40.4 ± 1.55
10.8ª ± 0.85
10.5ª ± 0.60
41.4 ± 1.58
10.5ª ± 0.78
10.9ª ± 0.78
9
40.3 ± 1.89
10.4ab ± 0.96
10.2ª ± 0.71
41.5 ± 1.39
10.0a ± 0.82
11.0a ± 0.59
14
40.0 ± 2.07
9.2c ± 1.55
10.1a ± 1.60
41.5 ± 1.77
8.9b ± 1.67
10.9a ± 1.09
Treatment
0.014
0.956
0.021
0.720
0.542
0.001
Day
0.821
<0.0001
<0.0001
0.585
<0.0001
<0.0001
Treatment*Day
0.999
0.404
0.293
0.432
0.153
0.940
22
453
454
455
Mean ± standard deviation; CONTROL group (no antioxidants); CARN006 (0.6 g carnosic acid kg-1 of feed concentrate); CARN012 (1.2 g carnosic acid kg-1 of
feed concentrate); VITE006 (0.6 g α-tocopheryl acetate kg-1 of feed concentrate); a, b, c: different superscripts in the same column indicate statistical
differences (P<0.05) among treatments or days
456
457
23
458
Table 3. pH values at 0 h, 45 min and 24 h post- slaughter in longissimus thoracis muscle
459
460
461
462
463
pH 0 h
pH 45 minutes
pH 24 h
CONTROL
6.79a ± 0.198
6.43b ± 0.159
5.84c ± 0.040
CARN006
6.87a ± 0.178
6.49b ± 0.192
5.76c ± 0.061
CARN012
6.74a ± 0.371
6.47b ± 0.211
5.82c ± 0.088
VITE006
6.77a ± 0.308
6.55b ± 0.139
5.83c ± 0.060
464
465
466
467
468
Mean ± standard deviation; CONTROL group (no antioxidants); CARN006 (0.6 g carnosic acid kg-1 of feed concentrate); CARN012 (1.2 g
carnosic acid kg-1 of feed concentrate); VITE006 (0.6 g α-tocopheryl acetate kg-1 of feed concentrate); a, b, c: different superscripts in the same
row indicate statistical differences (P<0.05) measuring times
469
470
24
471
472
Table 4. Extract release volume (ERV, ml) of lamb meat samples from longissimus thoracis muscle stored at 4 ºC under modified atmosphere
package (35% CO2, 35% O2 and 30% N2) during 14 days.
0 days
7 days
CONTROL
36.5a ± 3.42
26.4b ± 7.65
CARN006
31.5a ± 5.21
23.4b ± 6.72
CARN012
35.6a ± 8.72
21.1b ± 4.49
VITE006
33.1a ± 10.20
21.9b ± 2.75
473
474
18.6c ± 3.66475
476
18.3b ± 3.99477
478
18.3b ± 4.17
479
14 days
16.6b ± 2.83
480
481
482
25
Mean ± standard deviation; CONTROL group (no
antioxidants); CARN006 (0.6 g carnosic acid kg-1 of feed
concentrate); CARN012 (1.2 g carnosic acid kg-1 of feed
concentrate); VITE006 (0.6 g α-tocopheryl acetate kg-1 of
feed concentrate); a, b, c: different superscripts in the same
row indicate statistical differences (P<0.05) among
measuring times
483
484
Table 5. Effects of carnosic acid and vitamin E on microbial populations in lamb meat samples from longissimus thoracis muscle stored at 4 ºC
under modified atmosphere package (35% CO2, 35% O2 and 30% N2) during 14 days
485
Day 0
Day 7
Day 14
486
CONTROL
CARN006
CARN012
VITE006
2.69 ± 0.905
2.02 ± 1.110
1.54a ± 0.660
1.29a ± 0.681
3.05 ± 1.053
2.97 ± 0.472
2.60ab ± 0.521
2.59b ± 0.683
4.40 ± 1.403 487
2.97 ± 1.417
3.66b ± 0.999488
3.64b ± 0.830
EC
CONTROL
CARN006
CARN012
VITE006
1.04 ± 0.614
1.18 ± 0.512
1.42ab ± 0.688
1.09a ± 0.269
1.35 ± 0.296
1.05 ± 0.287
1.01a ± 0.380
1.21a ± 0.387
2.06 ± 1.030 490
1.78 ± 0.409
491
2.10b ± 0.378
2.23b ± 0.440492
TVB at 4.5ºC
CONTROL
CARN006
CARN012
VITE006
2.20a ± 1.137
1.97 ± 0.959
1.81a ± 0.845
1.66 ± 0.893
2.54b ± 0.795
2.70 ± 0.477
2.53ab ± 0.611
2.17 ± 0.506
3.20b ± 1.797
3.47 ± 1.815 494
4.03b ± 1.446
495
3.60 ± 1.123
Pseudomonas
489
493
496
LAB
CONTROL
CARN006
CARN012
VITE006
2.74 ± 1.227
2.54 ± 0.900
1.89 ± 0.410
1.71 ± 0.279
2.74 ± 1.233
2.34 ± 0.707
2.39 ± 0.390
1.97 ± 0.839
2.99 ± 2.202
497
3.05 ± 1.261
3.27 ± 1.951 498
2.88 ± 2.212
26
499
500
501
502
503
Mean ± standard deviation; CONTROL group (no antioxidants); CARN006 (0.6 g carnosic acid kg-1 of feed concentrate); CARN012 (1.2 g
carnosic acid kg-1 of feed concentrate); VITE006 (0.6 g α-tocopheryl acetate kg-1 of feed concentrate); a, b, c: different superscripts in the same
row indicate statistical differences (P<0.05) among refrigerated storage days; Enterobacteriaceae (EC), total viable bacteria (TVB), lactic acid
bacteria (LAB)
504
27
505
506
507
508
509
Table 6. Results from sensory analysis carried out to discriminate between cooked meat samples from lambs fed 1) CONTROL and CARN012
and 2) CARN006 and VITE006
Triangle test
Correct judgements (n = 24)
P-value
CONTROL vs. CARN012
7
0.041
CARN006 vs. VITE006
8
0.100
Preference test
Preferences for B (n=24)
P-value
CONTROL (A) vs. CARN012 (B)
9
0.221
VITE006 (A) vs. CARN006 (B)
10
0.414
CONTROL group (no antioxidants); CARN006 (0.6 g carnosic acid kg-1 of feed concentrate); CARN012 (1.2 g carnosic acid kg-1 of feed
concentrate); VITE006 (0.6 g α-tocopheryl acetate kg-1 of feed concentrate)
510
28
511
512
Figure 1. Evolution of L* parameter in longissimus lumborum and gluteus medius muscles
stored at 4oC under modified atmosphere package (35% CO2, 35% O2 and 30% N2)
513
514
515
Figure 2. Evolution of chroma (C*) in longissimus lumborum and gluteus medius muscles
stored at 4oC under modified atmosphere package (35% CO2, 35% O2 and 30% N2)
516
517
518
Figure 3. Evolution of hue (h*) in longissimus lumborum and gluteus medius muscles stored at
4oC under modified atmosphere package (35% CO2, 35% O2 and 30% N2)
519
520
521
Figure 4. Evolution of water holding capacity (WHC) in longissimus lumborum muscle stored at
4ºC under modified atmosphere package (35% CO2, 35% O2 and 30% N2)
522
523
29
524
Figure 1.
525
526
(a) longissimus lumborum
527
528
529
530
531
532
533
534
535
536
537
(b) gluteus medius
538
539
540
541
542
543
544
545
CONTROL group (no antioxidants); CARN006 (0.6 g carnosic acid kg-1 of feed
concentrate); CARN012 (1.2 g carnosic acid kg-1 of feed concentrate); VITE006
30
(0.6 g α-tocopheryl acetate kg-1 of feed concentrate)
CONTROL group (no antioxidants); CARN006 (0.6 g carnosic acid kg-1 of feed
concentrate); CARN012 (1.2 g carnosic acid kg-1 of feed concentrate); VITE006
546
547
Figure 2.
(a) longissimus lumborum
548
549
550
551
552
553
554
555
556
557
558
(b) gluteus medius
559
560
561
562
563
564
565
566
567
568
569
570
571
572
31
573
574
Figure 3.
(a) longissimus lumborum
575
576
577
578
579
580
581
582
583
584
585
586
(b) gluteus medius
587
588
589
590
591
592
593
594
595
596
597
598
599
32
600
Figure 4.
601
602
603
604
605
606
607
608
609
33