Animal Science Journal (2017) ••, ••–••
doi:10.1111/asj.12768
ORIGINAL ARTICLE Incorporating rosemary diterpenes in lamb diet to improve microbial quality of meat packed in different environments Jordi ORTUÑO, Rafael SERRANO and Sancho BAÑÓN 1
Department of Food Science and Technology and Nutrition, Faculty of Veterinary Science, University of Murcia, Murcia, Spain
ABSTRACT The dietary use of phytochemicals may contribute to improving lamb meat preservation under different packing atmospheres. The objective was to test the preservative potential of a dietary rosemary extract (RE) containing carnosic acid and carnosol (at 1:1 w:w) in chilled lamb patties packed in air, vacuum and 70/30 O2/CO2 modified atmosphere. Three experimental diets, (C) control, (RE) C plus 600 mg RE/kg feed and (E) C plus 600 mg vitamin E/kg, were given to fattening lambs. Unlike the C- and E-diets, the RE-diet had a double antimicrobial and antioxidant effect on the lamb patties packed in all the environments studied. The RE-diet inhibited total viable and lactic acid bacteria and Enterobacteriaceae, but not Brochothrix thermosphacta and Pseudomonas spp. and also improved oxidative stability (measured as CIE Lab color and thiobarbituric reagent substances), appearance and odor. The E-diet had a better antioxidant effect than the RE-diet but had no antimicrobial effects. Escherichia coli and Salmonella spp. were not detected. The dietary use of RE was most suitable for preserving vacuum-packed meat, which is more exposed to spoilage by anaerobic bacteria, while the use of dietary vitamin E allowed better control of oxidation in the meat packed in a bacteriostatic and oxidizing environment.
Key words: carnosic acid, carnosol, dietary supplements, lamb meat, rosemary.
INTRODUCTION The sheep meat market is expanding due to increased demand from developing countries, such as China, India and Turkey, among others (FAO 2012). There is a growing need to preserve lamb meat because of the long distances between producer and consumer countries, as well the need of extending the shelf life of the meat (Mills et al. 2014). This will involve improvements in the technique used, such as packing, chilling and freezing. In the case of lamb, both vacuum packing (VP) and high O2/CO2 modified atmosphere packing (MAP) are often used to extend the shelf life of meat cuts and minced meat. Both packing methods are able to inhibit pathogenic and spoilage bacteria, although each has its advantages and disadvantages: high O2/CO2 MAP extends reddening but enhances rancidity, whereas VP avoids rancidity but might lead to meat discoloration and a higher risk of lactic acid bacteria (LAB) proliferation. On the other hand, the prospect of using preservatives in meat does not seem appropriate, especially in developed countries, where healthcare organizations and consumers are increasingly rejecting their use (for health reasons) and legal restrictions flourish. © 2017 Japanese Society of Animal Science
Thus, the development of alternative strategies for meat preservation should be an overriding aim for the future of the industry. One possible strategy that may be suitable for use with VP or MAP is to include phytochemicals in the diet of animals to preserve the resulting meat. Indeed, several diets based on plant phenolic compounds have been used in recent years to improve lamb meat preservation (Luciano et al. 2009; Jerónimo et al. 2012; Nieto et al. 2012; Rivas-Cañedo et al. 2013; Inserra et al. 2014), in particular, rosemary and/or its products (Nieto et al. 2011; Bañón et al. 2012; Morán et al. 2012a, 2012b; Smeti et al. 2013; Vasta et al. 2013; Ortuño et al. 2014, 2015; Serrano et al. 2014). Among the products tested in lambs, a dietary rosemary extract (RE) containing carnosic acid and carnosol has been seen to lead to the deposition in muscle of a diterpenic metabolite derived Correspondence: Sancho Bañón, Department of Food Science and Technology and Nutrition, Faculty of Veterinary Science. University of Murcia. Campus Espinardo, 30071. Murcia, Spain. (Email:
[email protected]) Received 21 July 2016; accepted for publication 14 November 2016.
J. ORTUÑO et al.
from the carnosol with antioxidant and antimicrobial potential effects (Jordán et al. 2014). Other studies suggest that the antimicrobial potential of RE would not be well used when lamb fillets are packed under a bacteriostatic atmosphere (Bañón et al. 2012; Serrano et al. 2014; Ortuño et al. 2015), hence the interest in knowing whether RE is able to delay microbial spoilage in meat products that present a high microbial risk, such as raw patties made from frozen–thawed pieces (Khalafalla et al. 1993). The efficacy of dietary antimicrobials will depend on their intrinsic properties and the type of bacteria present in the meat. Gram-negative spoilage microflora (Pseudomonas spp. and, to a lesser extent, Enterobacteriaceae) predominate in the meat stored in air, whereas the use of high-CO2 MAP or VP favors the dominance of facultative anaerobic populations, including LAB and Brochothrix thermosphacta (Skandamis & Nychas 2002). Depending on the dominant bacteria, fresh meat can suffer surface changes and develop off-odors when bacterial loads of 6–9 log colony-forming units (CFU)/g are reached (Sun & Holley 2012). Moreover, pathogenic bacteria such as E. coli and Salmonella spp. may also proliferate, potentially affecting meat safety. Besides using bacteriostatic environments, introducing preservatives in muscle through the diet may contribute to improving microbial quality in chilled packed meat products. Vitamin E, the dietary supplement most widely used in animal feeding to preserve meat, appears to be effective as an antioxidant but not as an antimicrobial agent (Macit et al. 2003; Lauzurica et al. 2005; Ripoll et al. 2011; Kasapidou et al. 2012). There seems to be a case therefore for using other dietary supplements, such as rosemary diterpenes, for their potential antimicrobial and antioxidant activities on meat. The objective was to test the ability of a RE (compared with dietary vitamin E) for inhibiting microbial spoilage in lamb patties packed in three different environments (air, vacuum and high O2/CO2 atmosphere). Other signs of spoilage (discoloration, pH drop and lipid oxidation) were also studied because both microbial and oxidative phenomena are important for meat quality.
MATERIALS AND METHODS Dietary supplements A RE containing carnosic acid plus carnosol and vitamin E (DL-α-tocopheryl acetate) were used for the experimental lamb diets. RE was provided by NutrafurFurfural Español S.A. (Murcia, Spain). The extract was obtained by successive stages of extraction, drying and concentration of oil-free rosemary leaf, using different solvents, including acetone and/or ethanol–water mixtures, as described by Del Baño et al. (2003). The resulting RE 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 © 2017 Japanese Society of Animal Science
and carnosol, respectively). Vitamin E (Microvit™ 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 creamy beige color and a vitamin E purity of 600 IU/g.
Animals and diet Twenty-four weaned Segureño lambs of 13 1 kg live weight were selected from a collective feedlot. The lambs were individually identified and weighed before being randomly assigned to one of the three dietary treatments (eight lambs per treatment): a basal or control diet (Cdiet), the basal diet supplemented with 0.5 mg RE/kg feed (RE-diet) and the basal diet supplemented with 0.5 mg/kg α-tocopheryl acetate (E-diet). The content of both RE and vitamin E was checked in the feed after pelletizing. For more details regarding the diets and feed characteristics, see Ortuño et al. (2015). The lambs were fattened from February to May in individual pens located on an experimental farm. All handling practices followed the recommendations of the European Council (RC) 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. The experiment was authorized by the Ethics Committee on Animal Research of the University of Murcia. All the animals were fed with the corresponding fattening pelletized feed supplied ad libitum until they reached a live weight of 24 1 kg. The fattening period lasted 50 8 days.
Meat sampling The lambs were slaughtered in a local abattoir according to European Union regulations and the carcasses were chilled at 2°C in a cooling room. Twenty-four hours post mortem, the legs were removed from the carcasses, boned by a professional butcher, vacuum packed, frozen and stored at 18°C in darkness for up to 6 months. For better monitoring of bacterial groups, lamb patties were made under unfavorable microbial conditions (frozen– thawed meat cuts processed in a traditional butchery). The frozen boned legs were vacuum thawed at 4°C for 48 h and minced in an atmospheric mincer using a 5 mm plate (Laska GMBH, WW1302; Nu-Meat Technology, Girona, Spain). Minced meat samples from the thigh (Gluteus, Quadriceps, Biceps femoris, Semimembranosus, Semitendinosus, Adductor and others minor muscles) were analyzed. The patties were formed using 20 g of minced meat with a manual burger former. Eight batches composed of three boned legs (one leg from each dietary treatment) were processed weekly until the meat from all 24 lambs had been analyzed. Around 20 patties were obtained from each leg. The patties made from each leg and diet were divided into three different groups for storage: (i) air packaging, whereby the patties were packaged in polystyrene Animal Science Journal (2017) ••, ••–••
DIETARY DITERPENES FOR LAMB PRESERVATION
Aerpack B5–37 trays, covered with an oxygenpermeable polypropylene film (650 cm3/2 m per 24 h) (Raelma Industries Madrid Ltd., Spain); (ii) vacuum packaging, in which the patties were vacuum packed using bags (170 × 250 mm) of plastic barrier film (three layers of coextruded polyamide/polyethylene 20/70, oxygen transmission rate 30–50 cm3/ 2 m per 24 h (Bolsera Murciana S.L., Murcia, Spain); (iii) high O2/ CO2 modified atmosphere packaging, where the patties were packaged in polystyrene B5–37 Aerpack trays (Coopbox Hipania, Lorca, Murcia, Spain) in BB4L bags (Cryovac Packaging, Barcelona, Spain) of low gas permeability (8–12 cm3/2 m per 24 h). All the values of oxygen permeability obtained from the suppliers were measured at 23°C, 75% relative humidity and 0.1 MPa). Four patties from the same dietary treatment were packed in each bag. The patties were arrayed for up to 7 days at 4°C under 12 h cycles of fluorescent light (800 lux) and darkness, and analyzed on days 1, 4 and 7 of storage.
Microbiological analysis For the microbiological assays, the bags containing samples were aseptically opened in a 131 Bio-II-A microbiology cabinet (Telstar, Tarrasa, Spain) before being weighed (20 g) with sterile tweezers into stomacher bags and homogenized with 180 mL buffered peptone water 0.1% w:w (Microkit BCD046, Madrid, Spain) in a stomacher (IUL Instruments, GmBH, Köningswinter, Germany). Serial decimal dilutions were prepared in peptone water (prepared at 0.1/100 w/w) and 1 mL of each diluent was poured in duplicate into agar media plates. Bacterial counts were determined as follows: total viable counts (TVC) on plate count agar (CultimedPanreac 413 799, Barcelona, Spain) incubated at 30°C for 72 h; LAB on deMan Rogosa Sharpe agar (Cultimed-Panreac 413 784) overlaid (double agar layer) with the same medium and incubated at 30°C for 96 h; Enterobacteriaceae (ENB) on violet red bile glucose agar (Microkit DMT136) overlaid with the same medium and incubated at 30°C for 48 h; Pseudomonas spp. on glutamate starch phenol agar (Microkit DMT148) incorporating sodium penicillin and incubated at 30°C for 48 h; and B. thermosphacta on tryptone soya agar incorporating a selective supplement (streptomycin sulphate, thallous acetate and cycloheximide) (Oxoid CM0881, Basingstoke, Hampshire, United Kingdom) and incubated at 25°C for 48 h. All the microbial counts were expressed as log CFU/g. E. coli ( 5 log CFU/g have been reported in other
Table 1 Average bacterial counts of lamb patties packed in different atmospheres (air, vacuum and high O2/CO2) and stored at 4°C for up to 7 days
Air Day
1
4
5.96†,¶ 5.58†,¶,†† 5.37†,†† 0.18 3.08†
Vacuum 7
1
6.81‡,†† 6.83‡,†† 6.23‡,¶ 0.18 3.50‡
8.14§ 8.03§ 7.96§ 0.10 4.58§
5.95† 5.61† 5.40† 0.19 2.93
2.93† 2.78† 0.31 2.57†,†† 2.40†,†† 1.99†,¶ 0.11 4.10†
3.63‡ 3.30‡ 0.29 3.32‡ 3.40‡ 2.91‡ 0.25 5.88‡
4.57§ 4.61§ 0.25 4.10§ 4.14§ 3.81§ 0.22 7.61§
3.89† 4.03† 0.15 4.26†,†† 3.88†,†† 3.40†,¶ 0.14
6.22‡ 5.95‡ 0.15 5.02‡,†† 4.87‡,†† 4.07‡,¶ 0.19
7.81§ 7.38§ 0.25 6.04§,†† 6.03§,†† 5.30§,¶ 0.18
4
High O2/CO2 7
1
4
7
6.57‡,†† 6.23‡,†† 5.71‡,¶ 0.20 3.02
7.22§ 7.36§ 6.76§ 0.16 2.90
5.79† 5.62† 5.38† 0.16 2.49†
6.60‡,†† 6.51‡,†† 5.67†,¶ 0.23 2.75‡
7.11§,†† 7.20§,†† 6.68‡,¶ 0.15 3.26§
2.75 2.77 0.24 2.41†,†† 2.36†,†† 1.89†,¶ 0.14 3.97†
2.85 2.81 0.28 3.23‡ 3.07‡ 2.61‡ 0.25 4.09†
2.81 2.80 0.22 3.39‡ 3.84§ 3.26§ 0.33 4.83‡
2.41† 2.58† 0.21 2.58†,†† 2.44†,†† 1.96†,¶ 0.15 3.81†
2.74‡ 2.73‡ 0.27 3.12‡ 2.99‡ 2.77‡ 0.30 5.12‡
3.02§ 2.90‡ 0.31 3.30‡ 3.35‡ 3.16‡ 0.32 6.13§
3.58† 3.76† 0.14 4.17†,†† 3.98†,†† 3.42†,¶ 0.18
4.20‡ 4.02‡ 0.14 4.98‡,†† 4.42‡,†† 3.62†,¶ 0.24
4.98§ 4.66§ 0.27 5.67§,†† 5.94§,†† 5.00§,¶ 0.33
3.75† 3.79† 0.15 4.02†,†† 3.91†,†† 3.35†,¶ 0.16
4.84‡ 4.91‡ 0.19 5.01‡,†† 4.88‡,†† 3.76‡,¶ 0.19
6.49§ 6.26§ 0.23 5.71§,†† 5.88§,†† 5.08§,¶ 0.25
Diet TVC
C E RE
SED Pseudomonas spp.
C E RE
SED ENB
C E RE
SED Brochothrix thermosphacta
C E RE
SED LAB
C E RE SED
Diets: C (control); E (control plus α-tocopheryl acetate); RE (control plus rosemary extract). †, ‡, §Time effects for the same diet (P ≤ 0.05). ¶, ††Diet effects at the same storage time (P ≤ 0.05). All results expressed as log CFU/g. TVC, total viable counts; ENB, Enterobacteriaceae; LAB, lactic acid bacteria; SED, standard error of the difference.
© 2017 Japanese Society of Animal Science
Animal Science Journal (2017) ••, ••–••
DIETARY DITERPENES FOR LAMB PRESERVATION
meat than psychrophilic anaerobic bacteria when the pieces are previously thawed inside vacuum bags (Borch et al. 1996; Tsigarida et al. 2000); this may help explain the lower counts observed for Pseudomonas in the AP patties compared with the counts of LAB or B. thermosphacta, once the meat was thawed at 4°C for 48 h, minced and re-packed under aerobiosis. Regardless of the packing atmosphere used, the RE diet decreased (P < 0.05) the counts of mesophilic bacteria (from day 4 onwards), LAB (whole storage period) and Enterobacteriaceae (day 1), although it did not affect (P > 0.05) the counts of other bacterial groups, such as Pseudomonas and B. thermosphacta. The inhibition of TVC and LAB by RE has previously been reported in chilled MAP lamb fillets (Bañón et al. 2012; Ortuño et al. 2014, 2015), as well as when other rosemary products were introduced into the diet of ewes (Nieto et al. 2010). However, in a similar study, the use of higher (600–1200 mg/kg) doses of dietary carnosic acid did not inhibit microbial spoilage in lamb fillets stored at 4°C in MAP (35% CO2, 35% O2 and 30% N2) (Morán et al. 2012b). Unlike in our study, B. thermosphacta was not detected, while Pseudomonas, psychrophilic bacteria and LAB reached less than 3 log CFU/g after 7 days of storage. Carnosic acid is an antimicrobial molecule (Moreno et al. 2006), which suggests that the lack of positive results obtained by Morán et al. (2012b) might have been due to the bacteriostatic atmosphere or to the low bioavailability of this acid in sheep muscle, as demonstrated by Jordán et al. (2014). The intake of RE by lambs leads to the deposition in muscle of a diterpenic metabolite derived from carnosol with antimicrobial potential (Jordán et al. 2014). A preliminary study confirmed that a supplementation level of 600 mg RE/kg led to the accumulation of 0.7 mg C19H22O3/kg meat, which resulted in a reduction of TVC from 4.5 to 3.9 log CFU/g, a reduction in LAB counts from 2.9 to 2.0 log CFU/g and in ENB counts from 4.5 to 3.9 log CFU/g, as determined in chilled MAP lamb loin fillets stored for 7 days (Ortuño et al. 2015). By contrast, other dietary studies observed no antimicrobial effects of vitamin E (provided at 500– 600 mg/kg) in MAP lamb meat (Lauzurica et al. 2005; Morán et al. 2012b; Ortuño et al. 2015). Whatever the case, using RE (antimicrobial and antioxidant) rather than vitamin E (antioxidant) would seem unnecessary when meat microbial spoilage can be controlled by a bacteriostatic atmosphere. As has been seen in patties, the dietary use of RE provided an additional inhibition of LAB but not of Pseudomonas spp. and/or B. thermosphacta. Gram-positive bacteria are more sensitive to the non-polar phenolic compounds present in rosemary extracts than Gram-negative bacteria (Davidson 1993). This was confirmed both in vitro (Moreno et al. 2006) and in meat (Zhang et al. 2009) by using a rosemary extract containing carnosic acid and carnosol at 2:1 w:w. As the de Man Rogosa Sharpe agar used for LAB enumeration in our study is not selective with Animal Science Journal (2017) ••, ••–••
respect to B. thermosphacta (Nowak et al. 2012), it is to be expected that LAB inhibition by RE will specifically affect species such as Lactobacillus, Leuconostoc and/or Carnobacterium, which are the most common LAB isolates from chilled meats (Tsigarida et al. 2000; Nychas & Skandamis 2005) The values of pH, CIELAB color and TBARS are shown in Table 2. The pH values of around 5.8 presented slight fluctuations during storage, probably as a consequence of bacterial metabolic activities. Regardless of the packing method used, the pH was slightly higher (P < 0.05) in the R patties on day 7, a result that seems be related with LAB inhibition by RE. Other microbial studies have shown that dietary treatments based on rosemary hardly affect the pH during the chilled storage of lamb meat, whether or not in a bacteriostatic environment (Morán et al. 2012b; Ortuño et al. 2015). In our study, the final counts of LAB reached in the patties (
