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Effect of Melissa officinalis supplementation on growth performance and meat quality characteristics in organically produced broilers a
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E. Kasapidou , I. Giannenas , P. Mitlianga , E. Sinapis , E. Bouloumpasi , K. Petrotos , A. f
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Manouras & I. Kyriazakis a
Department of Agricultural Technology, Division of Agricultural Products Quality Control, School of Agriculture Technology, Food Technology and Nutrition, Technological Educational Institution of Western Macedonia, Florina, Greece b
Laboratory of Animal Nutrition and Husbandry, Veterinary Faculty, University of Thessaly, Karditsa, Greece c
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Laboratory of Nutrition, Veterinary Faculty, Aristotle University of Thessaloniki, Thessaloniki, Greece d
School of Agriculture, Animal Production Department, Aristotle University of Thessaloniki, Thessaloniki, Greece e
School of Agriculture Technology, Food Technology and Nutrition, Department of Biosystems Engineering, Technological Educational Institute of Thessaly, Larisa, Greece f
School of Agriculture Technology, Food Technology and Nutrition, Department of Food Technology, Technological Educational Institute of Thessaly, Karditsa, Greece g
School of Agriculture, Food and Rural Development, Newcastle University, Newcastle, UK Accepted author version posted online: 09 Oct 2014.Published online: 18 Dec 2014.
To cite this article: E. Kasapidou, I. Giannenas, P. Mitlianga, E. Sinapis, E. Bouloumpasi, K. Petrotos, A. Manouras & I. Kyriazakis (2014) Effect of Melissa officinalis supplementation on growth performance and meat quality characteristics in organically produced broilers, British Poultry Science, 55:6, 774-784, DOI: 10.1080/00071668.2014.974140 To link to this article: http://dx.doi.org/10.1080/00071668.2014.974140
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British Poultry Science, 2014 Vol. 55, No. 6, 774–784, http://dx.doi.org/10.1080/00071668.2014.974140
Effect of Melissa officinalis supplementation on growth performance and meat quality characteristics in organically produced broilers
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E. KASAPIDOU, I. GIANNENAS1,2, P. MITLIANGA, E. SINAPIS3, E. BOULOUMPASI, K. PETROTOS4, A. MANOURAS5, AND I. KYRIAZAKIS6 Department of Agricultural Technology, Division of Agricultural Products Quality Control, School of Agriculture Technology, Food Technology and Nutrition, Technological Educational Institution of Western Macedonia, Florina, Greece, 1Laboratory of Animal Nutrition and Husbandry, Veterinary Faculty, University of Thessaly, Karditsa, Greece, 2 Laboratory of Nutrition, Veterinary Faculty, Aristotle University of Thessaloniki, Thessaloniki, Greece, 3School of Agriculture, Animal Production Department, Aristotle University of Thessaloniki, Thessaloniki, Greece, 4School of Agriculture Technology, Food Technology and Nutrition, Department of Biosystems Engineering, Technological Educational Institute of Thessaly, Larisa, Greece, 5School of Agriculture Technology, Food Technology and Nutrition, Department of Food Technology, Technological Educational Institute of Thessaly, Karditsa, Greece, and 6School of Agriculture, Food and Rural Development, Newcastle University, Newcastle, UK
Abstract 1. A trial was conducted to study the effect of Melissa officinalis supplementation on organic broiler performance and meat chemical, microbiological, sensory and nutritional quality. 2. Male and female day-old Ross 308 chicks were fed on a standard commercial diet containing 0, 2.5, 5 or 10 g/kg feed ground M. officinalis for 84 d before slaughter. 3. Weight gain and feed conversion ratio were significantly improved in the broilers receiving either 5 or 10 mg M. officinalis/kg feed. 4. Inclusion of M. officinalis did not affect muscle chemical and fatty acid composition. 5. On the basis of microbiological and sensory experimental data and subsequent extension of meat shelf life, M. officinalis did not reduce the microbial populations of the meat, but was effective in limiting lipid oxidation.
INTRODUCTION Herbs and spices are used as feed additives in poultry diets due to their antimicrobial, antiparasitic, antiviral and antioxidative properties that lead to improved animal performance and health, as well as better end product quality (Windisch et al., 2008; Leusink et al., 2010). With regard to broilers, herbs and/or their associated extracts such as thyme, rosemary, oregano, sage and clove have been extensively used in their diets as presented in review studies (Windisch et al., 2008; Bou et al., 2009; Brenes and Roura, 2010; Karre et al., 2013). Consumers are willing to pay premium prices for organically produced chickens
because they consider organic farming as environmentally friendly, promoting animal welfare and leading to the production of safer, healthier and high-quality animal products (Castellini et al., 2002; O’Donovan and McCarthy, 2002; Van Loo et al., 2010). In the USA, organic poultry has become popular with the consumers (Husak et al., 2008) and the demand is steadily increasing (O’Bryan et al., 2008). In the UK, the market for organic meat is also growing rapidly (McEachern and Willock, 2004). Chicken meat has a high content of unsaturated fatty acids, and it is notably abundant in polyunsaturated fatty acids (PUFA) (Valsta et al., 2005) and thus more susceptible to lipid oxidation and free radical production.
Correspondence to: E. Kasapidou, Department of Agricultural Technology, Technological Educational Institution of Western Macedonia, Terma Kontopoulou Street, 53100 Florina, Greece. E-mail: [email protected] Accepted for publication 16 August 2014.
© 2014 British Poultry Science Ltd
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MELISSA OFFICINALIS SUPPLEMENTATION AND ORGANIC BROILERS
Additionally, stability against lipid oxidation is lower in organic poultry meat due to the higher content of ferrous iron that catalyses oxidation (Castellini et al., 2002). Lipid oxidation is a major concern to the meat industry due to its adverse effects on flavour, odour, taste, texture, nutritional value and overall food quality (Morrissey et al., 1998; Ahn et al., 2007). Moreover, some of the compounds formed by lipid oxidation impose health risks as they are related to mutagenic and carcinogenic effects and cytotoxic properties (Jiménez-Colmenero et al., 2001). Furthermore, organic farming regulations, that is, raising animals outdoors, the use of slow-growing breeds and strict restrictions in the therapeutic use of antimicrobial agents may lead to potentially higher microbiological safety risks (Cui et al., 2005). In this respect, retardation of lipid oxidation and bacterial growth has a significant contribution to the extension of shelf life of poultry meat, particularly nowadays where meat is mainly sold via the supermarkets in a pre-packed form and consumers buy food in bulk. These facts highlight the need to explore new ways for the extension of the shelf life of meat, improvement in food safety as well as enhancing the performance and the health status of broilers. Melissa officinalis L. (lemon balm) belongs to the Laminaceae family and grows in the Mediterranean region, western Asia, south-western Siberia and northern Africa (Dastmalchi et al., 2008; Lara et al., 2011). M. officinalis has antioxidant and antimicrobial properties (Carnat et al., 1998; Tajkarimi et al., 2010). The antioxidant properties of M. officinalis are attributed to its polyphenolic compounds and particularly rosmarinic, caffeic and protocatechuic acids, whereas its antimicrobial properties have been associated with citrol and caryophyllene (Gutierrez et al., 2008). Plants (dehydrated leaves and/or other parts) of the Laminaceae family such as oregano, rosemary and thyme have been used in poultry diets due to their content in compounds with antibacterial, anticoccidial, antifungal, antioxidant and growth-promoting activities (Bou et al., 2009). On this basis, it was hypothesised that inclusion of M. officinalis into organic broiler diets would improve broiler growth performance and meat oxidative stability and fatty acid composition as well as reduce microbial growth in the meat.
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compliance with local and European laws and regulations, and in accordance with the principles and guidelines for the care of animals in experimentation (European Union, 2010; Directive 2010/63/EU of 22 September 2010).
Animals and experimental design A total of 320-d-old Ross 308 male and female broiler chickens were randomly allocated into 1 of 4 experimental treatments. Each treatment consisted of 4 replicates of 20 birds each. Each replicate was housed in a separate indoor pen (10 birds/m2), under controlled temperature and light conditions. All birds were reared on a commercial poultry farm, Karditsomagoula, Greece (latitude 39.23°, longitude 21.55°). The lighting programme was set at 60 watt/10 m2 during the first week with 23-h light per day for the first 3 d, and 10 watt/10 m2 with 14-h light per day thereafter. The temperature was set at 34ºC during the first day, was set at 33°C during the first week and was gradually reduced by 3°C per week to reach a minimum of 22°C at 21 d of age. Relative humidity was between 65% and 75%. After 21 d of age, each group (replicate) of chickens had free access to a separate yard during daytime and birds were confined indoors during the night. The experiment lasted for 84 d, and in order to meet the nutrient requirements of the chickens over this period, a complete basal diet was formulated for each of the three stages of growth: starter, grower and finisher. The feeds were formulated to meet National Research Council recommendations (National Research Council, 1994) and contained no antibacterial or anticoccidial supplements. Table 1 presents the ingredients and the composition of the basal diets that were in mash form. Proximate analysis of three batches of each of the basal diets showed no significant deviation from calculated values. The birds within the control group (CON) were given the basal diet for their respective growth stage. The other three groups were given experimental diets based on the basal diets, but containing an additional 2.5 g (MEL 2.5), 5 g (MEL 5) or 10 g/kg feed (MEL 10) ground dried M. officinalis at the expense of wheat bran. Access to feed and water was provided on an ad libitum basis.
MATERIALS AND METHODS This trial was carried out in accordance with the principles of regulations of the local Public Veterinary Service and the Institutional Committee for Animal Use and Ethics of The Veterinary Faculty of the University of Thessaly. Throughout the trial, the birds were handled in
Broiler performance measurements
Body weight and feed intake were monitored on a pen basis weekly, while weight gain and feed conversion ratio values were subsequently calculated. Mortality was also recorded on a daily basis in each pen.
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Table 1. Formulation (g/kg fresh weight), chemical composition (g/kg fresh weight) and fatty acid composition (as % of total fatty acids) of the basal diet
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Starter diet Grower diet Finisher diet (1–14 d) (15–35 d) (35–84 d) Wheat grains Maize grains Full fat soya bean Herring meal Sunflower cake Soya bean oil Wheat bran1 Calcium carbonate Dicalcium phosphate Vitamin, mineral and amino acids premix2 Chemical analysis3 Crude protein Fat Fibre Ash Calcium Phosphorus Lysine Methionine and cystine Metabolisable energy (MJ/kg DM)4 Fatty acids ∑saturated ∑Monounsaturated ∑Polyunsaturated ∑n-6 ∑n-3
394 200 180 50 90 15 25 14 12 20
412 130 180 20 140 25 50 12 11 20
432 120 150 10 170 25 50 12 11 20
212 42 38 51 9 6 13 10 12.6
202 51 45 49 9 6 12 10 13.0
194 61 49 47 8 6 11 09 13.2
36.32 29.26 32.59 30.51 2.08
Notes: 1Ground M. officinalis was added to replace an equal quantity of wheat bran. 2 Supplied per kg of diet: vitamin A, 12 000 IU; vitamin D3, 5000 IU; vitamin E (α-tocopheryl acetate), 30 IU; vitamin K3, 3 mg; vitamin B1, 1 mg; vitamin B2, 8 mg; vitamin B6, 3 mg; vitamin B12, 0.02 mg; nicotinic acid, 20 mg; Calcium pantothenate, 20 mg; folic acid, 2 mg; biotin, 0.2 mg; vitamin C, 10 mg; choline chloride, 480 mg; Zn, 125 mg; Mn, 100 mg; Fe, 62 mg; Cu, 7.5 mg; Co, 0.2 mg; I, 2 mg; Se, 0.2 mg; lysine, 200 g; methionine, 90 g; sodium chloride, 2.6 g. 3 According AOAC International (AOAC, 2003). 4 Calculated value.
M. officinalis preparation and analysis Cultivated M. officinalis consisted of flowered tops, leaves, stems and stalks of M. officinalis plants that had been dried and ground to pass through a 2mm screen. Extraction and GC-mass spectrometry analysis
A dried and ground M. officinalis sample was thoroughly mixed, and 25 g of the sample was mixed with 100 ml of ethanol (95% v/v). The mixture was placed for 24 h in a shaking water bath at 35°C, and following that, the mixture was filtered through a Whatman No. 1 filter paper. The filtrate was mixed, and an aliquot was collected in an airtight vial that was stored at 4°C before analysis. The extract was analysed by gas chromatography (GC)–mass spectrometry (MS) in an Agilent 7890 analyser with a Mass Spectrometer (MS) – detector type 5975 equipped with split/splitless
automatic injector and a capillary fused silica (100 m × 250 μm × 0.25 μm) column (J & W Scientific, Inc., Folsom, California, USA) using helium as carrier gas. The injected volume was 3 μl and the split ratio 1:100; injector and flame ionisation detector temperatures were 250°C and 300°C, respectively. The identification of the compounds was based on comparison of their retention times (RT) by using standards available in the laboratory and/or the National Institute of Standards and Technology (NIST) and Wiley libraries. Meat quality measurements At the end of the feeding period, 6 birds from each treatment were randomly selected for meat quality measurements and were killed by cervical dislocation. Skinless breast (M. pectoralis superficialis) and thigh (M. biceps femoris) samples were collected for colour assessment, lipid oxidation and microbiological analysis during storage. Samples were placed in plastic bags and stored at −40°C pending analysis. Prior to analyses, the stored samples were thawed at 4°C. The next day, they were placed in white plastic trays, overwrapped with transparent air-permeable polyethylene (cling) film as usual for retail sales and stored in a nonilluminated refrigerated cabinet at 4°C for 5 d. Additional skinless breast and thigh samples were prepared and stored at −40°C until analysed for proximate and fatty acid composition. Analytical methods Proximate analysis
Samples were homogenised and subjected to moisture, ash, crude protein and fat content analyses using AOAC (2003) methods 950.46, 920.153, 928.08 and 991.36, respectively. Moisture was determined by drying the homogenised sample in an air oven (model ED 115, Binder GmbH, Tuttlingen, Germany) at 105°C until a constant weight was obtained. Percentage of moisture content was calculated from pre- and post-drying weights. Ash was determined by incineration at 525°C for 12 h using a muffle furnace (model LM 412.07, Linn High Therm GmbH, Eschenfelden, Germany). Percentage of ash content was calculated by weight loss after incineration. Protein was estimated by the Kjeldahl method using nitrogen digestion (Turbotherm type TT/12 M) and distillation (Vapodest type 40) apparatuses (Gerhardt Apparate GmbH & Co. KG, Germany) and converted to crude protein by multiplying the nitrogen content determined by the Kjeldahl method by 6.25. Fat was extracted by petroleum ether using a Soxtherm/ Multistat type SE-416 macro automated system
MELISSA OFFICINALIS SUPPLEMENTATION AND ORGANIC BROILERS
(Gerhardt Apparate GmbH & Co. KG, Germany). Fat content was calculated as the proportional difference between weight of the sample before and after solvent extraction.
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Microbiological analyses
Microbiological analyses were carried out on thigh samples because leg meat is more perishable due to its higher pH than breast meat. On storage days 2 and 5, samples (25 g) were aseptically removed from each thigh and placed in individual stomacher bags. Maximum recovery diluent (MRD) (225 ml) was added, and the samples were homogenised for 2 min in a stomacher. Samples were examined for total counts of aerobic mesophilic microflora (APC), Staphylococcus aureus, Campylobacter jejuni, Clostridium (sulphite-reducing) spp. and lactic acid bacteria (LAB). Total aerobic mesophiles (APC) were incubated aerobically at 30°C for 48 h (ISO 4833, 2003). Staphylococcus aureus was grown aerobically at 37°C for 48 h in Baird Parker Agar supplemented with egg yolk tellurite (ISO 6888-1, 1999/ A1:2003). Campylobacter jejuni was grown in Campylobacter selective agar supplemented with Campylobacter selective supplement. The plates were incubated in anaerobic jars filled with a gas mixture of 5% O2, 10% CO2 and 85% N2 to create microaerophilic conditions at 37°C for 24–48 h. Sulphite-reducing Clostridium spp. were incubated at 37°C for 48 h in Tryptose Sulphite Cycloserine (TSC) agar supplemented with D-cycloserine. Plates were allowed to solidify, and an additional layer of the same culture medium layer was overlaid to create anaerobic conditions. LAB were cultivated in De Man–Rogosa–Sharpe (MRS) agar and incubated at 37°C for 72 h (De Man et al., 1960). All plates were visually inspected for typical colony types and morphology characteristics associated with each growth medium, and two replicates of appropriate dilutions were enumerated. Microbial counts are reported as log10 colony-forming units (CFU) per g of sample. Colour assessment
Breast samples (n = 4) instrumental colour measurements (L*, luminosity; a*, redness; and b*, yellowness) were taken daily at three different locations on the top side of each sample through the overwrap plastic film using a Minolta Chroma Meter (model CR-410, Minolta Camera Co, Osaka, Japan) with a 10-mm measuring area (aperture) and illuminant source C. The instrument was calibrated using the white calibration plate (Y = 93.66, x = 0.3150, y = 0.3217). The colorimeter-supplied optically inactive glass aperture cover was used to ensure a consistently flat sample
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surface (Bianchi et al., 2005). Sample thickness was similar, and colour measurements were taken following sample equilibration to room temperature (Bianchi and Fletcher, 2002). Colour changes over time were evaluated by colour difference (ΔE*) calculated as follows ΔE* = [(L1*−L2*)2 + (a1* −a2*)2 + (b1*−b2*)2]0.5 where L1*, a1* and b1* were measurements at day 1 and L2*, a2* and b2* were measurements at each subsequent time (Petracci and Baéza, 2011). Lipid oxidation
Lipid oxidation, on storage days 2 and 5, was determined on the basis of the formation of thiobarbituric acid reactive substances (TBARS) using a modification of the method of Vyncke (1975). Breast and thigh samples were blended in a domestic food processor, and subsamples were homogenised with aqueous trichloroacetic acid containing n-propyl gallate and ethylenediamine tetraacetic acid disodium salt. Samples were left for approximately 15–20 min to allow the extraction of the TBARS. The resulting slurry was filtered, and an aliquot of the filtrate was mixed with aqueous thiobarbituric acid. Samples were left overnight at room temperature in the dark, and the next day, absorbance was read at 532 nm against a blank sample using a UV–VIS spectrophotometer (U-2800 Double Beam Spectrophotometer, Hitachi, Tokyo, Japan). TBARS were calculated using 1,1,3,3-tetraethoxypropane as standard, and they were expressed as mg of malonaldehyde per kg of sample. Fatty acid composition
Fatty acid analysis was conducted at the Department of Animal Production of the School of Agriculture of Aristotle University of Thessaloniki. Total lipids (feed, breast and thigh muscle tissues) were extracted using a modification of the chloroform/methanol procedure of Folch et al. (1957). Fatty acids were transesterified with methanolic sulphuric acid, and the produced fatty acid methyl esters were extracted with hexane (Christie, 2003). Fatty acids were identified by using 37 component mixture (Supelco, Bellefonte, PA, USA) and PUFA No II animal source fatty acid methyl esters (Supelco, Bellefonte, PA, USA) as reference standards. Fatty acids were quantified by peak area measurement, and the results are expressed as percentage (%) of the total peak areas for all quantified acids. The average total area of unidentified peaks in muscle samples was < 7.8% of total peak area. The fatty acid methyl esters were analysed using an Agilent Technology (model 6890 N) gas chromatograph equipped with an autosampler
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(Agilent 7683), a split/splitless automatic injector (Agilent 7683B Series) and a DB-23 (60 m × 0.25 mm × 0.25 µm) column (J & W Scientific, Inc., Folsom, California, USA). The GC conditions were: carrier gas, He; split mode injection, 50:1 (3 μl); injector and flame ionisation detector temperatures, 250°C and 300°C, respectively; initial oven temperature, 110°C for 6 min, increased at 11°C per min to 165°C, increased at 15°C per min to 195°C, increased at 7°C per min to 230°C, held at 230°C for 7 min.
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Statistical analyses Experimental data were analysed as a randomised block design. Performance data were analysed by one-way analysis of variance (ANOVA) with initial body weight used as a covariate and the pen being the experimental unit. Individual broiler was the experimental unit for meat chemical, microbiological, sensory and nutritional quality data. One-way ANOVA was used to determine differences between treatments on the examined parameters. Levene’s test was performed to check homogeneity of variances, and if homogeneity was violated, the GamesHowell test was applied (Kinnear and Gray, 2000). Linear and quadratic responses to M. officinalis supplementation concentration for broiler performance and meat microbial growth and lipid oxidation were also tested. Post hoc analysis was undertaken using either Tukey’s or Games-Howell tests. All statistical tests were assessed at α = 0.05. Statistical software package SPSS version 13.0 (SPSS, 2004) for Windows (SPSS, Chicago, IL, USA) was used.
RESULTS AND DISCUSSION Chemical characterisation of M. officinalis extract The main components of the M. officinalis extract were citronellol and citrol followed by trans-caryophyllene. Linalool and caryophyllene were also present at significantly smaller concentrations. The composition of the M. officinalis extracts (oils) is variable and dependent on many Table 2.
parameters such as light intensity, nutrients, temperature, cultural practice, genotype, plant part age, harvesting time, region, plant morphological traits as well as harvesting, drying and distillation methods (Khalid et al., 2008, 2009; Moradkhani et al., 2010; Seidler-Łożykowska et al., 2013). Broiler performance Final live weight, carcass weight, feed intake, daily gain and feed conversion ratio values of the control and supplemented broilers are presented in Table 2. Slaughter weight and carcass weight were higher (P ≤ 0.05) in the broilers from the MEL 5 group. The MEL 10 group slaughter and carcass weight values did not differ statistically (P ≥ 0.05) from the CON, MEL 2.5 and MEL 5 groups, albeit they were numerically higher than the corresponding values in the CON and MEL 2.5 groups. Feed intake was not affected (P ≥ 0.05) by M. officinalis supplementation. Daily gain was higher (P ≤ 0.05) in all supplemented groups compared to CON, and similarly feed conversion ratio was lower (P ≤ 0.05) in the same groups. Mortality was also not affected (P ≥ 0.05) by the level of M. officinalis supplementation, although it was numerically higher in the CON group. A linear (P ≤ 0.05 or P ≤ 0.01) and a quadratic (P ≤ 0.01 or P ≤ 0.001) effect was observed with final body weight, carcass weight, daily weight gain and feed conversion ratio. Proximate analysis of breast and thigh tissues Inclusion of M. officinalis in the diet had a significant effect (P ≤ 0.01 and P ≤ 0.05) on the protein content from breast and thigh muscles, respectively. M. officinalis supplementation resulted in a significant (P ≤ 0.001) linear decrease in breast muscle protein content but not in thigh muscle protein content which increased as the supplementation concentration of M. officinalis increased (Table 3). The lowest ash content was observed in the thigh meat of broilers fed on the highest M. officinalis concentration (MEL 10). A linear response (P ≤ 0.01) was also observed in thigh ash content, which decreased as
Effect of M. officinalis supplementation on broiler performance at 84 d Treatment
Measurement Final body weight (g) Carcass weight (g) Feed intake (g/d) Daily gain (g) Feed conversion ratio Mortality (%)
Significance
CON
MEL 2.5
MEL 5
MEL 10
Treatment
Linear
Quadratic
2702a 2026.5a 83.8 31.6a 2.651b 2702a
2755.6a 2066.7a 76.1 32.3a 2.358a 2755.6a
2970.2b 2227.6b 82.5 34.8b 2.369a 2970.2b
2806.5a,b 2104.8a,b 77.7 32.9a,b 2.362a 2806.5a,b
0.011 0.012 0.154 0.015 0.011 0.338
0.016 0.014 0.180 0.016 0.012 0.444
0.001 0.001 0.240 0.001 0.001 0.454
Notes: MEL 2.5: 2.5 g Melissa officinalis per kg feed; MEL 5: 5.0 g Melissa officinalis per kg feed; MEL 10: 10.0 g Melissa officinalis per kg feed. a,b Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05. Four birds per replicate, 16 birds per group, n = 4.
MELISSA OFFICINALIS SUPPLEMENTATION AND ORGANIC BROILERS
period are presented in Table 4. There was a significant increase (P ≤ 0.001–0.01) in the populations of all microbial groups in the samples from the M. officinalis supplemented broilers in comparison with the CON. APC populations, which affect the shelf life of meat, were high (> 5 log10/CFU) on storage day 2 in all groups, whereas on storage day 5 they exceeded the maximum safety limit of 7 log10 CFU/g for fresh poultry meat (International Commission on Microbiological Specifications for Foods (ICMSF), 1986). M. officinalis did not affect the counts of Staphylococcus spp. that were within the reported range (< 3 and > 5 log10 CFU/g) for poultry meat in all treatments and both test days (Mead et al., 1993; Waldroup, 1996). Inclusion of M. officinalis resulted in significantly (P ≤ 0.001) lower counts of C. jejuni in both storage periods. Rosenquist et al. (2003) reported that even a 2 log10/CFU reduction in carcass contamination would reduce the human incidence of Campylobacteriosis by 30-fold in a quantitative risk assessment study. In this aspect, the reduction by 1 log10/CFU in the MEL 10 treatment in comparison with the CON samples on storage day 5 would have a significant impact on the health and safety of the produced meat. Populations of Clostridium sulphite-reducing spp. were low (< 2 log10 CFU/g) in all treatments with the exception of samples in the MEL 5 treatment where Clostridium sulphite-reducing spp. counts were significantly higher but did not exceed the value of 4 log10 CFU/g that is associated with potential serious food hygiene problem (Eisgruber and Reuter, 1995). Initial (day 2) LAB counts were low (ca 2 log10/CFU) in treatments CON and MEL 10. Counts increased as the storage period was extended in all treatments, and the highest counts, exceeding 5 log10/CFU, were observed in treatments MEL 2.5 and MEL 5. LAB are considered as the major spoilage micro-organisms in
Table 3. Effect of M. officinalis supplementation on the chemical composition of breast and thigh muscle Treatment1 Measurement
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Breast muscle Moisture (%) Ash (%) Protein (%) Lipids (%) Thigh muscle Moisture (%) Ash (%) Protein (%) Lipids (%)
CON
MEL 2.5 MEL 5 MEL 10 Significance
76.05 1.00 22.30b 0.76
75.66 1.03 22.26a 0.93
75.64 1.02 22.58a 0.75
76.62 1.00 21.50a 0.78
0.057 0.710 0.001 0.676
76.36 1.02b 19.60b 3.00
76.64 1.01b 19.56 2.68
77.27 0.96a,b 18.81a 2.81
76.91 0.92a 19.37 3.39
0.278 0.010 0.036 0.471
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Notes: MEL 2.5: 2.5 g Melissa officinalis per kg feed; MEL 5: 5.0 g Melissa officinalis per kg feed; MEL 10: 10.0 g Melissa officinalis per kg feed. 1 For each group, n = 6. a,b,c Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05.
the supplementation concentration of M. officinalis increased. Moisture and lipid contents were not significantly different (P ≥ 0.05) between the treatment groups in both thigh and breast muscles. Although there were statistically significant differences in the protein content of breast and thigh meat, and in the ash content of thigh meat, these differences may not be of practical significance, from both a production and a nutritional point of view. Marcinčáková et al. (2011) reported no statistical differences in breast and thigh muscle chemical composition (dry matter, fat and protein content) in conventionally produced broilers fed on a commercial diet supplemented with ground M. officinalis at a concentration of 20 g/kg feed. Microbiological analyses The results of the microbiological analyses of the air-packed thigh samples during the 5-d storage
Table 4. Effect of M. officinalis supplementation on the microbiological quality of thigh muscle during refrigerated storage for 5 d Treatment1 Bacteria (log10 CFU/g) Aerobic mesophilic microflora Staphylococcus aureus Campylobacter jejuni Clostridium (sulphite-reducing) Lactic acid bacteria (LAB) Aerobic mesophilic microflora Staphylococcus aureus Campylobacter jejuni Clostridium (sulphite-reducing) Lactic acid bacteria (LAB)
(APC)
Day
CON
MEL 2.5
MEL 5
MEL 10
Treatment
Linear
Quadratic
2
5.10a
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Effect of Melissa officinalis supplementation on growth performance and meat quality characteristics in organically produced broilers a
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a
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E. Kasapidou , I. Giannenas , P. Mitlianga , E. Sinapis , E. Bouloumpasi , K. Petrotos , A. f
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Manouras & I. Kyriazakis a
Department of Agricultural Technology, Division of Agricultural Products Quality Control, School of Agriculture Technology, Food Technology and Nutrition, Technological Educational Institution of Western Macedonia, Florina, Greece b
Laboratory of Animal Nutrition and Husbandry, Veterinary Faculty, University of Thessaly, Karditsa, Greece c
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Laboratory of Nutrition, Veterinary Faculty, Aristotle University of Thessaloniki, Thessaloniki, Greece d
School of Agriculture, Animal Production Department, Aristotle University of Thessaloniki, Thessaloniki, Greece e
School of Agriculture Technology, Food Technology and Nutrition, Department of Biosystems Engineering, Technological Educational Institute of Thessaly, Larisa, Greece f
School of Agriculture Technology, Food Technology and Nutrition, Department of Food Technology, Technological Educational Institute of Thessaly, Karditsa, Greece g
School of Agriculture, Food and Rural Development, Newcastle University, Newcastle, UK Accepted author version posted online: 09 Oct 2014.Published online: 18 Dec 2014.
To cite this article: E. Kasapidou, I. Giannenas, P. Mitlianga, E. Sinapis, E. Bouloumpasi, K. Petrotos, A. Manouras & I. Kyriazakis (2014) Effect of Melissa officinalis supplementation on growth performance and meat quality characteristics in organically produced broilers, British Poultry Science, 55:6, 774-784, DOI: 10.1080/00071668.2014.974140 To link to this article: http://dx.doi.org/10.1080/00071668.2014.974140
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British Poultry Science, 2014 Vol. 55, No. 6, 774–784, http://dx.doi.org/10.1080/00071668.2014.974140
Effect of Melissa officinalis supplementation on growth performance and meat quality characteristics in organically produced broilers
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E. KASAPIDOU, I. GIANNENAS1,2, P. MITLIANGA, E. SINAPIS3, E. BOULOUMPASI, K. PETROTOS4, A. MANOURAS5, AND I. KYRIAZAKIS6 Department of Agricultural Technology, Division of Agricultural Products Quality Control, School of Agriculture Technology, Food Technology and Nutrition, Technological Educational Institution of Western Macedonia, Florina, Greece, 1Laboratory of Animal Nutrition and Husbandry, Veterinary Faculty, University of Thessaly, Karditsa, Greece, 2 Laboratory of Nutrition, Veterinary Faculty, Aristotle University of Thessaloniki, Thessaloniki, Greece, 3School of Agriculture, Animal Production Department, Aristotle University of Thessaloniki, Thessaloniki, Greece, 4School of Agriculture Technology, Food Technology and Nutrition, Department of Biosystems Engineering, Technological Educational Institute of Thessaly, Larisa, Greece, 5School of Agriculture Technology, Food Technology and Nutrition, Department of Food Technology, Technological Educational Institute of Thessaly, Karditsa, Greece, and 6School of Agriculture, Food and Rural Development, Newcastle University, Newcastle, UK
Abstract 1. A trial was conducted to study the effect of Melissa officinalis supplementation on organic broiler performance and meat chemical, microbiological, sensory and nutritional quality. 2. Male and female day-old Ross 308 chicks were fed on a standard commercial diet containing 0, 2.5, 5 or 10 g/kg feed ground M. officinalis for 84 d before slaughter. 3. Weight gain and feed conversion ratio were significantly improved in the broilers receiving either 5 or 10 mg M. officinalis/kg feed. 4. Inclusion of M. officinalis did not affect muscle chemical and fatty acid composition. 5. On the basis of microbiological and sensory experimental data and subsequent extension of meat shelf life, M. officinalis did not reduce the microbial populations of the meat, but was effective in limiting lipid oxidation.
INTRODUCTION Herbs and spices are used as feed additives in poultry diets due to their antimicrobial, antiparasitic, antiviral and antioxidative properties that lead to improved animal performance and health, as well as better end product quality (Windisch et al., 2008; Leusink et al., 2010). With regard to broilers, herbs and/or their associated extracts such as thyme, rosemary, oregano, sage and clove have been extensively used in their diets as presented in review studies (Windisch et al., 2008; Bou et al., 2009; Brenes and Roura, 2010; Karre et al., 2013). Consumers are willing to pay premium prices for organically produced chickens
because they consider organic farming as environmentally friendly, promoting animal welfare and leading to the production of safer, healthier and high-quality animal products (Castellini et al., 2002; O’Donovan and McCarthy, 2002; Van Loo et al., 2010). In the USA, organic poultry has become popular with the consumers (Husak et al., 2008) and the demand is steadily increasing (O’Bryan et al., 2008). In the UK, the market for organic meat is also growing rapidly (McEachern and Willock, 2004). Chicken meat has a high content of unsaturated fatty acids, and it is notably abundant in polyunsaturated fatty acids (PUFA) (Valsta et al., 2005) and thus more susceptible to lipid oxidation and free radical production.
Correspondence to: E. Kasapidou, Department of Agricultural Technology, Technological Educational Institution of Western Macedonia, Terma Kontopoulou Street, 53100 Florina, Greece. E-mail: [email protected] Accepted for publication 16 August 2014.
© 2014 British Poultry Science Ltd
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MELISSA OFFICINALIS SUPPLEMENTATION AND ORGANIC BROILERS
Additionally, stability against lipid oxidation is lower in organic poultry meat due to the higher content of ferrous iron that catalyses oxidation (Castellini et al., 2002). Lipid oxidation is a major concern to the meat industry due to its adverse effects on flavour, odour, taste, texture, nutritional value and overall food quality (Morrissey et al., 1998; Ahn et al., 2007). Moreover, some of the compounds formed by lipid oxidation impose health risks as they are related to mutagenic and carcinogenic effects and cytotoxic properties (Jiménez-Colmenero et al., 2001). Furthermore, organic farming regulations, that is, raising animals outdoors, the use of slow-growing breeds and strict restrictions in the therapeutic use of antimicrobial agents may lead to potentially higher microbiological safety risks (Cui et al., 2005). In this respect, retardation of lipid oxidation and bacterial growth has a significant contribution to the extension of shelf life of poultry meat, particularly nowadays where meat is mainly sold via the supermarkets in a pre-packed form and consumers buy food in bulk. These facts highlight the need to explore new ways for the extension of the shelf life of meat, improvement in food safety as well as enhancing the performance and the health status of broilers. Melissa officinalis L. (lemon balm) belongs to the Laminaceae family and grows in the Mediterranean region, western Asia, south-western Siberia and northern Africa (Dastmalchi et al., 2008; Lara et al., 2011). M. officinalis has antioxidant and antimicrobial properties (Carnat et al., 1998; Tajkarimi et al., 2010). The antioxidant properties of M. officinalis are attributed to its polyphenolic compounds and particularly rosmarinic, caffeic and protocatechuic acids, whereas its antimicrobial properties have been associated with citrol and caryophyllene (Gutierrez et al., 2008). Plants (dehydrated leaves and/or other parts) of the Laminaceae family such as oregano, rosemary and thyme have been used in poultry diets due to their content in compounds with antibacterial, anticoccidial, antifungal, antioxidant and growth-promoting activities (Bou et al., 2009). On this basis, it was hypothesised that inclusion of M. officinalis into organic broiler diets would improve broiler growth performance and meat oxidative stability and fatty acid composition as well as reduce microbial growth in the meat.
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compliance with local and European laws and regulations, and in accordance with the principles and guidelines for the care of animals in experimentation (European Union, 2010; Directive 2010/63/EU of 22 September 2010).
Animals and experimental design A total of 320-d-old Ross 308 male and female broiler chickens were randomly allocated into 1 of 4 experimental treatments. Each treatment consisted of 4 replicates of 20 birds each. Each replicate was housed in a separate indoor pen (10 birds/m2), under controlled temperature and light conditions. All birds were reared on a commercial poultry farm, Karditsomagoula, Greece (latitude 39.23°, longitude 21.55°). The lighting programme was set at 60 watt/10 m2 during the first week with 23-h light per day for the first 3 d, and 10 watt/10 m2 with 14-h light per day thereafter. The temperature was set at 34ºC during the first day, was set at 33°C during the first week and was gradually reduced by 3°C per week to reach a minimum of 22°C at 21 d of age. Relative humidity was between 65% and 75%. After 21 d of age, each group (replicate) of chickens had free access to a separate yard during daytime and birds were confined indoors during the night. The experiment lasted for 84 d, and in order to meet the nutrient requirements of the chickens over this period, a complete basal diet was formulated for each of the three stages of growth: starter, grower and finisher. The feeds were formulated to meet National Research Council recommendations (National Research Council, 1994) and contained no antibacterial or anticoccidial supplements. Table 1 presents the ingredients and the composition of the basal diets that were in mash form. Proximate analysis of three batches of each of the basal diets showed no significant deviation from calculated values. The birds within the control group (CON) were given the basal diet for their respective growth stage. The other three groups were given experimental diets based on the basal diets, but containing an additional 2.5 g (MEL 2.5), 5 g (MEL 5) or 10 g/kg feed (MEL 10) ground dried M. officinalis at the expense of wheat bran. Access to feed and water was provided on an ad libitum basis.
MATERIALS AND METHODS This trial was carried out in accordance with the principles of regulations of the local Public Veterinary Service and the Institutional Committee for Animal Use and Ethics of The Veterinary Faculty of the University of Thessaly. Throughout the trial, the birds were handled in
Broiler performance measurements
Body weight and feed intake were monitored on a pen basis weekly, while weight gain and feed conversion ratio values were subsequently calculated. Mortality was also recorded on a daily basis in each pen.
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Table 1. Formulation (g/kg fresh weight), chemical composition (g/kg fresh weight) and fatty acid composition (as % of total fatty acids) of the basal diet
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Starter diet Grower diet Finisher diet (1–14 d) (15–35 d) (35–84 d) Wheat grains Maize grains Full fat soya bean Herring meal Sunflower cake Soya bean oil Wheat bran1 Calcium carbonate Dicalcium phosphate Vitamin, mineral and amino acids premix2 Chemical analysis3 Crude protein Fat Fibre Ash Calcium Phosphorus Lysine Methionine and cystine Metabolisable energy (MJ/kg DM)4 Fatty acids ∑saturated ∑Monounsaturated ∑Polyunsaturated ∑n-6 ∑n-3
394 200 180 50 90 15 25 14 12 20
412 130 180 20 140 25 50 12 11 20
432 120 150 10 170 25 50 12 11 20
212 42 38 51 9 6 13 10 12.6
202 51 45 49 9 6 12 10 13.0
194 61 49 47 8 6 11 09 13.2
36.32 29.26 32.59 30.51 2.08
Notes: 1Ground M. officinalis was added to replace an equal quantity of wheat bran. 2 Supplied per kg of diet: vitamin A, 12 000 IU; vitamin D3, 5000 IU; vitamin E (α-tocopheryl acetate), 30 IU; vitamin K3, 3 mg; vitamin B1, 1 mg; vitamin B2, 8 mg; vitamin B6, 3 mg; vitamin B12, 0.02 mg; nicotinic acid, 20 mg; Calcium pantothenate, 20 mg; folic acid, 2 mg; biotin, 0.2 mg; vitamin C, 10 mg; choline chloride, 480 mg; Zn, 125 mg; Mn, 100 mg; Fe, 62 mg; Cu, 7.5 mg; Co, 0.2 mg; I, 2 mg; Se, 0.2 mg; lysine, 200 g; methionine, 90 g; sodium chloride, 2.6 g. 3 According AOAC International (AOAC, 2003). 4 Calculated value.
M. officinalis preparation and analysis Cultivated M. officinalis consisted of flowered tops, leaves, stems and stalks of M. officinalis plants that had been dried and ground to pass through a 2mm screen. Extraction and GC-mass spectrometry analysis
A dried and ground M. officinalis sample was thoroughly mixed, and 25 g of the sample was mixed with 100 ml of ethanol (95% v/v). The mixture was placed for 24 h in a shaking water bath at 35°C, and following that, the mixture was filtered through a Whatman No. 1 filter paper. The filtrate was mixed, and an aliquot was collected in an airtight vial that was stored at 4°C before analysis. The extract was analysed by gas chromatography (GC)–mass spectrometry (MS) in an Agilent 7890 analyser with a Mass Spectrometer (MS) – detector type 5975 equipped with split/splitless
automatic injector and a capillary fused silica (100 m × 250 μm × 0.25 μm) column (J & W Scientific, Inc., Folsom, California, USA) using helium as carrier gas. The injected volume was 3 μl and the split ratio 1:100; injector and flame ionisation detector temperatures were 250°C and 300°C, respectively. The identification of the compounds was based on comparison of their retention times (RT) by using standards available in the laboratory and/or the National Institute of Standards and Technology (NIST) and Wiley libraries. Meat quality measurements At the end of the feeding period, 6 birds from each treatment were randomly selected for meat quality measurements and were killed by cervical dislocation. Skinless breast (M. pectoralis superficialis) and thigh (M. biceps femoris) samples were collected for colour assessment, lipid oxidation and microbiological analysis during storage. Samples were placed in plastic bags and stored at −40°C pending analysis. Prior to analyses, the stored samples were thawed at 4°C. The next day, they were placed in white plastic trays, overwrapped with transparent air-permeable polyethylene (cling) film as usual for retail sales and stored in a nonilluminated refrigerated cabinet at 4°C for 5 d. Additional skinless breast and thigh samples were prepared and stored at −40°C until analysed for proximate and fatty acid composition. Analytical methods Proximate analysis
Samples were homogenised and subjected to moisture, ash, crude protein and fat content analyses using AOAC (2003) methods 950.46, 920.153, 928.08 and 991.36, respectively. Moisture was determined by drying the homogenised sample in an air oven (model ED 115, Binder GmbH, Tuttlingen, Germany) at 105°C until a constant weight was obtained. Percentage of moisture content was calculated from pre- and post-drying weights. Ash was determined by incineration at 525°C for 12 h using a muffle furnace (model LM 412.07, Linn High Therm GmbH, Eschenfelden, Germany). Percentage of ash content was calculated by weight loss after incineration. Protein was estimated by the Kjeldahl method using nitrogen digestion (Turbotherm type TT/12 M) and distillation (Vapodest type 40) apparatuses (Gerhardt Apparate GmbH & Co. KG, Germany) and converted to crude protein by multiplying the nitrogen content determined by the Kjeldahl method by 6.25. Fat was extracted by petroleum ether using a Soxtherm/ Multistat type SE-416 macro automated system
MELISSA OFFICINALIS SUPPLEMENTATION AND ORGANIC BROILERS
(Gerhardt Apparate GmbH & Co. KG, Germany). Fat content was calculated as the proportional difference between weight of the sample before and after solvent extraction.
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Microbiological analyses
Microbiological analyses were carried out on thigh samples because leg meat is more perishable due to its higher pH than breast meat. On storage days 2 and 5, samples (25 g) were aseptically removed from each thigh and placed in individual stomacher bags. Maximum recovery diluent (MRD) (225 ml) was added, and the samples were homogenised for 2 min in a stomacher. Samples were examined for total counts of aerobic mesophilic microflora (APC), Staphylococcus aureus, Campylobacter jejuni, Clostridium (sulphite-reducing) spp. and lactic acid bacteria (LAB). Total aerobic mesophiles (APC) were incubated aerobically at 30°C for 48 h (ISO 4833, 2003). Staphylococcus aureus was grown aerobically at 37°C for 48 h in Baird Parker Agar supplemented with egg yolk tellurite (ISO 6888-1, 1999/ A1:2003). Campylobacter jejuni was grown in Campylobacter selective agar supplemented with Campylobacter selective supplement. The plates were incubated in anaerobic jars filled with a gas mixture of 5% O2, 10% CO2 and 85% N2 to create microaerophilic conditions at 37°C for 24–48 h. Sulphite-reducing Clostridium spp. were incubated at 37°C for 48 h in Tryptose Sulphite Cycloserine (TSC) agar supplemented with D-cycloserine. Plates were allowed to solidify, and an additional layer of the same culture medium layer was overlaid to create anaerobic conditions. LAB were cultivated in De Man–Rogosa–Sharpe (MRS) agar and incubated at 37°C for 72 h (De Man et al., 1960). All plates were visually inspected for typical colony types and morphology characteristics associated with each growth medium, and two replicates of appropriate dilutions were enumerated. Microbial counts are reported as log10 colony-forming units (CFU) per g of sample. Colour assessment
Breast samples (n = 4) instrumental colour measurements (L*, luminosity; a*, redness; and b*, yellowness) were taken daily at three different locations on the top side of each sample through the overwrap plastic film using a Minolta Chroma Meter (model CR-410, Minolta Camera Co, Osaka, Japan) with a 10-mm measuring area (aperture) and illuminant source C. The instrument was calibrated using the white calibration plate (Y = 93.66, x = 0.3150, y = 0.3217). The colorimeter-supplied optically inactive glass aperture cover was used to ensure a consistently flat sample
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surface (Bianchi et al., 2005). Sample thickness was similar, and colour measurements were taken following sample equilibration to room temperature (Bianchi and Fletcher, 2002). Colour changes over time were evaluated by colour difference (ΔE*) calculated as follows ΔE* = [(L1*−L2*)2 + (a1* −a2*)2 + (b1*−b2*)2]0.5 where L1*, a1* and b1* were measurements at day 1 and L2*, a2* and b2* were measurements at each subsequent time (Petracci and Baéza, 2011). Lipid oxidation
Lipid oxidation, on storage days 2 and 5, was determined on the basis of the formation of thiobarbituric acid reactive substances (TBARS) using a modification of the method of Vyncke (1975). Breast and thigh samples were blended in a domestic food processor, and subsamples were homogenised with aqueous trichloroacetic acid containing n-propyl gallate and ethylenediamine tetraacetic acid disodium salt. Samples were left for approximately 15–20 min to allow the extraction of the TBARS. The resulting slurry was filtered, and an aliquot of the filtrate was mixed with aqueous thiobarbituric acid. Samples were left overnight at room temperature in the dark, and the next day, absorbance was read at 532 nm against a blank sample using a UV–VIS spectrophotometer (U-2800 Double Beam Spectrophotometer, Hitachi, Tokyo, Japan). TBARS were calculated using 1,1,3,3-tetraethoxypropane as standard, and they were expressed as mg of malonaldehyde per kg of sample. Fatty acid composition
Fatty acid analysis was conducted at the Department of Animal Production of the School of Agriculture of Aristotle University of Thessaloniki. Total lipids (feed, breast and thigh muscle tissues) were extracted using a modification of the chloroform/methanol procedure of Folch et al. (1957). Fatty acids were transesterified with methanolic sulphuric acid, and the produced fatty acid methyl esters were extracted with hexane (Christie, 2003). Fatty acids were identified by using 37 component mixture (Supelco, Bellefonte, PA, USA) and PUFA No II animal source fatty acid methyl esters (Supelco, Bellefonte, PA, USA) as reference standards. Fatty acids were quantified by peak area measurement, and the results are expressed as percentage (%) of the total peak areas for all quantified acids. The average total area of unidentified peaks in muscle samples was < 7.8% of total peak area. The fatty acid methyl esters were analysed using an Agilent Technology (model 6890 N) gas chromatograph equipped with an autosampler
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(Agilent 7683), a split/splitless automatic injector (Agilent 7683B Series) and a DB-23 (60 m × 0.25 mm × 0.25 µm) column (J & W Scientific, Inc., Folsom, California, USA). The GC conditions were: carrier gas, He; split mode injection, 50:1 (3 μl); injector and flame ionisation detector temperatures, 250°C and 300°C, respectively; initial oven temperature, 110°C for 6 min, increased at 11°C per min to 165°C, increased at 15°C per min to 195°C, increased at 7°C per min to 230°C, held at 230°C for 7 min.
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Statistical analyses Experimental data were analysed as a randomised block design. Performance data were analysed by one-way analysis of variance (ANOVA) with initial body weight used as a covariate and the pen being the experimental unit. Individual broiler was the experimental unit for meat chemical, microbiological, sensory and nutritional quality data. One-way ANOVA was used to determine differences between treatments on the examined parameters. Levene’s test was performed to check homogeneity of variances, and if homogeneity was violated, the GamesHowell test was applied (Kinnear and Gray, 2000). Linear and quadratic responses to M. officinalis supplementation concentration for broiler performance and meat microbial growth and lipid oxidation were also tested. Post hoc analysis was undertaken using either Tukey’s or Games-Howell tests. All statistical tests were assessed at α = 0.05. Statistical software package SPSS version 13.0 (SPSS, 2004) for Windows (SPSS, Chicago, IL, USA) was used.
RESULTS AND DISCUSSION Chemical characterisation of M. officinalis extract The main components of the M. officinalis extract were citronellol and citrol followed by trans-caryophyllene. Linalool and caryophyllene were also present at significantly smaller concentrations. The composition of the M. officinalis extracts (oils) is variable and dependent on many Table 2.
parameters such as light intensity, nutrients, temperature, cultural practice, genotype, plant part age, harvesting time, region, plant morphological traits as well as harvesting, drying and distillation methods (Khalid et al., 2008, 2009; Moradkhani et al., 2010; Seidler-Łożykowska et al., 2013). Broiler performance Final live weight, carcass weight, feed intake, daily gain and feed conversion ratio values of the control and supplemented broilers are presented in Table 2. Slaughter weight and carcass weight were higher (P ≤ 0.05) in the broilers from the MEL 5 group. The MEL 10 group slaughter and carcass weight values did not differ statistically (P ≥ 0.05) from the CON, MEL 2.5 and MEL 5 groups, albeit they were numerically higher than the corresponding values in the CON and MEL 2.5 groups. Feed intake was not affected (P ≥ 0.05) by M. officinalis supplementation. Daily gain was higher (P ≤ 0.05) in all supplemented groups compared to CON, and similarly feed conversion ratio was lower (P ≤ 0.05) in the same groups. Mortality was also not affected (P ≥ 0.05) by the level of M. officinalis supplementation, although it was numerically higher in the CON group. A linear (P ≤ 0.05 or P ≤ 0.01) and a quadratic (P ≤ 0.01 or P ≤ 0.001) effect was observed with final body weight, carcass weight, daily weight gain and feed conversion ratio. Proximate analysis of breast and thigh tissues Inclusion of M. officinalis in the diet had a significant effect (P ≤ 0.01 and P ≤ 0.05) on the protein content from breast and thigh muscles, respectively. M. officinalis supplementation resulted in a significant (P ≤ 0.001) linear decrease in breast muscle protein content but not in thigh muscle protein content which increased as the supplementation concentration of M. officinalis increased (Table 3). The lowest ash content was observed in the thigh meat of broilers fed on the highest M. officinalis concentration (MEL 10). A linear response (P ≤ 0.01) was also observed in thigh ash content, which decreased as
Effect of M. officinalis supplementation on broiler performance at 84 d Treatment
Measurement Final body weight (g) Carcass weight (g) Feed intake (g/d) Daily gain (g) Feed conversion ratio Mortality (%)
Significance
CON
MEL 2.5
MEL 5
MEL 10
Treatment
Linear
Quadratic
2702a 2026.5a 83.8 31.6a 2.651b 2702a
2755.6a 2066.7a 76.1 32.3a 2.358a 2755.6a
2970.2b 2227.6b 82.5 34.8b 2.369a 2970.2b
2806.5a,b 2104.8a,b 77.7 32.9a,b 2.362a 2806.5a,b
0.011 0.012 0.154 0.015 0.011 0.338
0.016 0.014 0.180 0.016 0.012 0.444
0.001 0.001 0.240 0.001 0.001 0.454
Notes: MEL 2.5: 2.5 g Melissa officinalis per kg feed; MEL 5: 5.0 g Melissa officinalis per kg feed; MEL 10: 10.0 g Melissa officinalis per kg feed. a,b Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05. Four birds per replicate, 16 birds per group, n = 4.
MELISSA OFFICINALIS SUPPLEMENTATION AND ORGANIC BROILERS
period are presented in Table 4. There was a significant increase (P ≤ 0.001–0.01) in the populations of all microbial groups in the samples from the M. officinalis supplemented broilers in comparison with the CON. APC populations, which affect the shelf life of meat, were high (> 5 log10/CFU) on storage day 2 in all groups, whereas on storage day 5 they exceeded the maximum safety limit of 7 log10 CFU/g for fresh poultry meat (International Commission on Microbiological Specifications for Foods (ICMSF), 1986). M. officinalis did not affect the counts of Staphylococcus spp. that were within the reported range (< 3 and > 5 log10 CFU/g) for poultry meat in all treatments and both test days (Mead et al., 1993; Waldroup, 1996). Inclusion of M. officinalis resulted in significantly (P ≤ 0.001) lower counts of C. jejuni in both storage periods. Rosenquist et al. (2003) reported that even a 2 log10/CFU reduction in carcass contamination would reduce the human incidence of Campylobacteriosis by 30-fold in a quantitative risk assessment study. In this aspect, the reduction by 1 log10/CFU in the MEL 10 treatment in comparison with the CON samples on storage day 5 would have a significant impact on the health and safety of the produced meat. Populations of Clostridium sulphite-reducing spp. were low (< 2 log10 CFU/g) in all treatments with the exception of samples in the MEL 5 treatment where Clostridium sulphite-reducing spp. counts were significantly higher but did not exceed the value of 4 log10 CFU/g that is associated with potential serious food hygiene problem (Eisgruber and Reuter, 1995). Initial (day 2) LAB counts were low (ca 2 log10/CFU) in treatments CON and MEL 10. Counts increased as the storage period was extended in all treatments, and the highest counts, exceeding 5 log10/CFU, were observed in treatments MEL 2.5 and MEL 5. LAB are considered as the major spoilage micro-organisms in
Table 3. Effect of M. officinalis supplementation on the chemical composition of breast and thigh muscle Treatment1 Measurement
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Breast muscle Moisture (%) Ash (%) Protein (%) Lipids (%) Thigh muscle Moisture (%) Ash (%) Protein (%) Lipids (%)
CON
MEL 2.5 MEL 5 MEL 10 Significance
76.05 1.00 22.30b 0.76
75.66 1.03 22.26a 0.93
75.64 1.02 22.58a 0.75
76.62 1.00 21.50a 0.78
0.057 0.710 0.001 0.676
76.36 1.02b 19.60b 3.00
76.64 1.01b 19.56 2.68
77.27 0.96a,b 18.81a 2.81
76.91 0.92a 19.37 3.39
0.278 0.010 0.036 0.471
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Notes: MEL 2.5: 2.5 g Melissa officinalis per kg feed; MEL 5: 5.0 g Melissa officinalis per kg feed; MEL 10: 10.0 g Melissa officinalis per kg feed. 1 For each group, n = 6. a,b,c Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05.
the supplementation concentration of M. officinalis increased. Moisture and lipid contents were not significantly different (P ≥ 0.05) between the treatment groups in both thigh and breast muscles. Although there were statistically significant differences in the protein content of breast and thigh meat, and in the ash content of thigh meat, these differences may not be of practical significance, from both a production and a nutritional point of view. Marcinčáková et al. (2011) reported no statistical differences in breast and thigh muscle chemical composition (dry matter, fat and protein content) in conventionally produced broilers fed on a commercial diet supplemented with ground M. officinalis at a concentration of 20 g/kg feed. Microbiological analyses The results of the microbiological analyses of the air-packed thigh samples during the 5-d storage
Table 4. Effect of M. officinalis supplementation on the microbiological quality of thigh muscle during refrigerated storage for 5 d Treatment1 Bacteria (log10 CFU/g) Aerobic mesophilic microflora Staphylococcus aureus Campylobacter jejuni Clostridium (sulphite-reducing) Lactic acid bacteria (LAB) Aerobic mesophilic microflora Staphylococcus aureus Campylobacter jejuni Clostridium (sulphite-reducing) Lactic acid bacteria (LAB)
(APC)
Day
CON
MEL 2.5
MEL 5
MEL 10
Treatment
Linear
Quadratic
2
5.10a
