effect of ganoderma lucidum (reishi mushroom) or olea europaea (olive) leaves on oxidative stability...
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Meat Science 96 (2014) 1275–1280
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Effect of Ganoderma lucidum (Reishi mushroom) or Olea europaea (olive)leaves on oxidative stability of rabbit meat fortified with n-3 fatty acids
Tina Trebušak, Alenka Levart, Janez Salobir, Tatjana Pirman ⁎Department of Animal Science, Biotechnical Faculty, University of Ljubljana, 1230 Domžale, Slovenia
⁎ Corresponding author. Tel.: +386 1 320 38 95; fax: +E-mail address: [email protected] (T. Pirman
0309-1740/$ – see front matter © 2013 Elsevier Ltd. All rihttp://dx.doi.org/10.1016/j.meatsci.2013.11.018
a b s t r a c t
a r t i c l e i n f oArticle history:Received 6 May 2013Received in revised form 10 October 2013Accepted 17 November 2013
Keywords:Rabbit meatNatural antioxidantsFatty acid compositionOxidative status
The objective of the present study was to evaluate the effect of Ganoderma lucidum (Reishi mushroom) or Oleaeuropaea (olive tree) leaves on oxidative stability of rabbit meat fortified with n-3 fatty acids. Forty-eightslovenska kunka (SIKA) rabbits were divided into four homogeneous groups. The control group (CONT−) re-ceived diet with 6% palm fat; other groups received diet with 6% linseed oil and were either unsupplemented(CONT+) or supplemented with 1% of G. lucidum (REISHI) or O. europaea leaves (OLIVE). Rabbits wereslaughtered and fatty acid composition, concentration of vitamin E and malondialdehyde (MDA) in back musclewere analyzed. The results showed that linseed oil addition improved fatty acid composition by increasing poly-unsaturated fatty acid (PUFA) proportion, decreasing proportion of saturated fatty acid (SFA) and reducing n-6/n-3 ratio in rabbit meat. Groups that were supplementedwith linseed oil had lower content ofα-tocopherol andhigher content of γ-tocopherol, compared to the CONT− group. The addition of potential antioxidants did noteffectively prevent oxidation of rabbit meat.
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1. Introduction
Meat and meat products have always been important for healthygrowth and development because they are good sources of quality pro-teins, essential amino acids, minerals, vitamins as well as fats (Biesalski,2005). This reputation has recently decreased, because of the opinionthat meat contains a lot of fat with unfavorable composition. Fats havean impact on nutritional and sensory value of food including taste andflavor. Moreover, they are an important source of the fat-solublevitamins (vitamin A, D, E and K) and polyunsaturated fatty acids(PUFA) (Marcinčák, Popelka, Bystrický, Hussein, & Hudecková, 2005).
Nowadays people aremore aware of the importance of healthy dietsand the beneficial effects of PUFA, especially n-3 on human health arewell known and have been documented by numerous studies(Williams, 2000; Riediger, Othman, Suh, & Moghadasian, 2009). Thepopulations in the developedworld do not consume enough dietary es-sential nutrients, including n-3 PUFA and these deficiency increases therisk of developing various diseases, especially cardiovascular diseases.On the other hand, Western diets have excessive amounts of n-6 PUFA.This led to a very high n-6/n-3 ratio ranging from 15/1 to 16.7/1. The op-timal n-6/n-3 ratio supposed to be less than 4/1 (Simopoulos, 2002).Therefore, the interest in obtaining animal products with a highercontent of n-3 fatty acids has increased.
Rabbit meat contains low amount of fat and cholesterol and it hashigh content of PUFA, compared to other sources of meat (Dalle Zotte,
386 1 714 10 05.).
ghts reserved.
2002). This is the reason it is often recommended by nutritionists.Fatty acid composition and n-6/n-3 ratio of rabbit meat are better, com-pared to other sources of meat. A rabbit's diet usually contains alfalfawith high n-3 PUFA content (Kouba et al., 2008). It is also the speciescharacteristic of the PUFA digestion and absorption. Ruminants hydro-genated dietary fats in rumen before intestinal absorption so thatabsorbed fatty acids are more saturated than dietary fatty acids, whilein non-ruminants intestinal fatty acids digestibility depends on thelevel of saturation of dietary fatty acids (Doreau & Chilliard, 1997).The fatty acid composition of meat and also other animal products(eggs, milk) can be improved by different strategies (Leskanich &Noble, 1997; Raes, De Smet, & Demeyer, 2004; Wood et al., 2003) themost effective of which is the addition of n-3 fatty acids in animaldiets (Dalle Zotte, 2002). Rabbit dietmanipulation is very effective in in-creasing level, not only of fatty acids, but also of vitamin E, selenium andother and in that way rabbit meat could be considered as a functionalfood (Dalle Zotte & Szendro, 2011). Linseed or oil is a suitable and fre-quently used plant source of n-3 PUFA, because of its high α-linolenicacid (C18:3 n-3) content. Unfortunately PUFA easily undergoesperoxidative damage, so the higher content of PUFA could lead to ahigher susceptibility to lipid oxidation (formation of free radicals, lipidperoxides, aldehydes (e.g. malondialdehyde (MDA)) during storageand cookingmeat, and consequently has an impact on flavor, color, tex-ture and nutritional value of meat (Gray, Gomaa, & Buckley, 1996).Therefore, the incorporation of antioxidants in the diet in order to pre-vent lipid oxidation is very important. Themost frequently used antiox-idant is vitamin E, and its positive role in protecting meat fromperoxidation was demonstrated in previous research on rabbits
Table 1In vitro antioxidant potential determined by photochemiluminiscence, vitamin E concen-tration and proximate composition of used supplements.
G. lucidum (Galimmun®) O. europaea leaves
ACW (mg/kg) 521.0 110,981.0ACL (mg/kg) 15.4 720.0
Vitamin E (mg/kg)α-Tocopherol – 195.1γ-Tocopherol 2.1 2.8
Proximate composition (g/kg)Dry matter 979.1 938.8Crude protein 179.0 112.0Crude fat 12.1 28.8Crude fiber 197.2 225.4Crude ash 42.6 69.4Carbohydrates 548.2 503.2
ACW—water soluble antioxidants; mg/kg of gallic acid equivalents; ACL—lipid solubleantioxidants; mg/kg of trolox equivalents.
Table 2Ingredients and proximate composition of the diets (g/kg).
CONT– CONT+ REISHI OLIVE
IngredientsAlfalfa 458.3 458.3 448.3 448.3Barley 130.0 130.0 130.0 130.0Sunflower meal 210.0 210.0 210.0 210.0Hay meal 100.0 100.0 100.0 100.0Rapeseed oil 10.0 10.0 10.0 10.0Palm fat 60.0Linseed oil 60.0 60.0 60.0G. lucidum 10.0O. europaea leaves 10.0Methionine 0.5 0.5 0.5 0.5Lysine 2.0 2.0 2.0 2.0Vitamin–mineral mixa 5.0 5.0 5.0 5.0Lignobondb 20.0 20.0 20.0 20.0Salt 4.2 4.2 4.2 4.2
Proximate compositionDry matter 932.89 912.37 924.34 921.70Crude protein 178.39 178.50 181.21 177.43Crude fat 108.10 86.35 85.05 86.08Crude fiber 227.43 228.21 228.16 227.04Crude ash 69.17 69.68 71.45 69.85Carbohydrates 349.80 349.64 358.48 361.30
CONT− 6%palm fat in a diet; CONT+6% linseed oil in a diet; REISHI 6% linseed oil in a dietwith addition of 1% G. lucidum; OLIVE 6% linseed oil in a diet with addition of 1%O. europaea leaves.
a Vitamin–mineral mix (0.5 %) (mg/kg diet): vitamin A (10 mg), vitamin D3 (2 mg), vi-tamin E (60 mg), vitamin K3 (4 mg), vitamin B1 (2 mg), vitamin B2 (7.5 mg), vitamin B3(50 mg), vitamin B5 (20 mg), vitamin B6 (2 mg), vitamin B12 (10 mg), folic acid (5 mg),biotin (5 mg), choline (333 mg), Fe (156 mg), Cu (32 mg), Mn (161 mg), Co (100 mg),I (2.95 mg), Se (15 mg), Zn (125 mg). It also contained 3.9 g wheat bran in 5 g ofmixtureadded to diet.
b Pellet binder.
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(Castellini, Dal Bosco, Bernardini, & Cyril, 1998; Corino, Pastorelli,Pantaleo, Oriani, & Salvatori, 1999; Dal Bosco, Castellini, Bianchi, &Mugnai, 2004; Tres, Bou, Codony, & Guardiola, 2009) and other animals(Zhang, Xiao, Samaraweera, Lee, & Ahn, 2010; Voljč, Frankič, Levart,Nemec, & Salobir, 2011). Recently, the interest in using non-vitamin,natural antioxidants of plant origin such as polyphenols and flavonoids,has increased (Qwele et al., 2013; Moyo, Oyedemi, Masika, &Muchenje,2012).
In Europe, particularly in theMediterranean, O. europaea tree and itsproducts have always been important in folk medicine and recognizedas important components of a healthy diet (Visioli, Poli, & Galli, 2002).O. europaea leaves and their extracts are a rich source of phenoliccompounds such as oleuropein, which upon hydrolysis can produceother bioactive substance, namely elenolic acid and hydroxytyrosol,and tocopherol (Savournin et al., 2001). They are also known for theirantioxidant activity, because of their high content of phenolic com-pounds (Benavente-García, Castillo, Lorente, Ortuño, & Del Rio, 2000;Botsoglou, Govaris, Christaki, & Botsoglou, 2010). In Asia, medicinalmushrooms were proven to have antioxidant effects and have beenused as a supplement to reduce the risk of oxidation (Khatua et al.,2013). They contain a number of potential natural antioxidants(Lindequist, Niedermeyer, & Julich, 2005; Cheung, Cheung, & Ooi,2003; Cheung & Cheung, 2005) such as phenols, flavonoids, carotenesand ascorbic acid (Froufe, Abreu, & Ferreira, 2009).
The objectives of this study were to evaluate the influence of addingfat high in polyunsaturated fatty acids to a rabbit's diet on fatty acidcomposition of back muscle and the susceptibility of meat to oxidation,and to assess the effectiveness of dietary Ganoderma lucidum (Reishimushroom) or Olea europaea (olive tree) leaves supplementation onthe oxidative stability of rabbit meat under various storage and cookingconditions.
2. Materials and methods
2.1. Animals and diets
All procedures were performed according to current legislation onanimal experimentation in Slovenia. Animals used in this experimentwere reared and slaughtered at the Department of Animal Science,Biotechnical Faculty (Ljubljana, Slovenia).
Forty-eight SIKA rabbits (80 days old, average initial body weight2580 ± 299 g), were randomly stratified by gender and weight intofour groups and assigned into different dietary treatments for 22 days.The CONT− group received diet with 6% palm fat; other groups re-ceived diet with 6% linseed oil and were either unsupplemented(CONT+) or supplemented with 1% of G. lucidum (REISHI) or 1% ofO. europaea leaves (OLIVE). The ingredients of the diets and the proxi-mate composition are presented in Table 2. G. lucidum or Reishi mush-room was added to the feed as the product Galimmun® (Institute ofNatural Sciences, Ljubljana, Slovenia) which contains milled fruitingbodies and mycelium of organically produced mushrooms. O. europaealeaves were picked at local olive oil-press (Oljarna Babič, Marezige,Slovenia), dried, milled and stored in a dry and dark place. Theproximate composition, vitamin E and in vitro antioxidant potential ofboth supplements are presented in Table 1. During the experiment an-imals had free access to pelleted diet and water (nipple drinkers).They were individually housed in two-floor wire cages. During theexperiment, two animals (females from the CONT+ and OLIVE group)had health problems (feet blister) and were eliminated from theexperiment.
Diet samples were taken during the experiment for the purpose ofproximate analysis and for the determination of fatty acid compositionand vitamin E content. Proximate analysis of the supplements(Table 1) and diets (Table 2) were determined by standard procedures(AOAC, 2000), dry matter on oven drying at 95–100 °C (AOAC Officialmethod 934.01), crude protein by copper catalyst Kjeldahl method
(AOAC official method 984.13), crude fat or ether extract (AOAC offi-cial method 920.39), crude fiber by fritted glass crucible method(AOAC official method 978.10) and crude ash (AOAC official method942.05).
2.2. Experimental procedure and sample collection
Each day animals received weighed daily meal and the residue fromthedaybeforewasweighed anddiscarded. Bodyweightswere recordedweekly during the experimental period and just before slaughter.Rabbits were slaughtered at 102 days of age, by electric stunning(70 V, 50 Hz) and killed by cutting the carotid arteries and jugularveins and exsanguinated in accordancewith the Slovenian national reg-ulations for commercial slaughtering. Animals did not undergo trans-port. Carcasses were prepared by removing blood, skin, the distal part
Table 3Fatty acid composition and vitamin E content of the diets.
CONT– CONT+ REISHI OLIVE
Main fatty acids (% of the total fatty acids)C12:0 0.20 0.04 0.04 0.04C14:0 0.89 0.12 0.12 0.12C16:0 35.81 8.00 8.06 8.07C18:0 41.73 4.00 3.98 3.99∑ C18:1 7.75 23.48 23.70 23.56C18:2 n-6 9.01 22.01 22.20 21.92C18:3 n-3 2.78 40.23 39.77 40.21∑ SFA 80.08 13.48 13.53 13.56∑ MUFA 8.05 24.14 24.36 24.22∑ PUFA 11.86 62.38 62.11 62.22∑ n-3 PUFA 2.85 40.33 39.86 40.31∑ n-6 PUFA 9.01 22.05 22.25 21.92n-6/n-3 PUFA 3.16 0.55 0.56 0.54
Vitamin E (mg/kg)α-Tocopherol 24.27 21.35 21.71 29.75γ-Tocopherol 28.69 27.96 27.82 15.73
CONT− 6%palm fat in a diet; CONT+6% linseed oil in a diet; REISHI 6% linseed oil in a dietwith addition of 1% G. lucidum; OLIVE 6% linseed oil in a diet with addition of 1%O. europaea leaves.SFA—saturated fatty acids; MUFA—monounsaturated fatty acids; PUFA—polyunsaturatedfatty acids.
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of the tail, the distal portions of legs, head, gastrointestinal and urogen-ital tract according to Blasco and Ouhayoun (1993). After 24 h of chill-ing at 4 °C the back muscle (M. longissimus dorsi) was taken, dividedinto slices and treated using six different methods: fresh raw andcooked meat, refrigerated raw and cooked meat, and frozen raw andcooked meat. Rawmeat samples were packed in bags and stored eitherat −70 °C until analysis (fresh meat), at 4 °C for 6 days (refrigeratedmeat) or at−20 °C for 3 months (frozen meat). Cooked meat sampleswere packed in plastic centrifuge tubes with stopper, stored under thesame condition as raw meat and were then cooked at 85 °C for60 min. All samples were homogenized (Grindomix homogenizer,RetschGmbh&Co, Haan, Germany) and stored at−70 °C until analysis.
2.3. Determination of vitamin E (α- and γ-tocopherol) and antioxidantcapacity
Concentrations of vitamin E in supplements, diets and back musclewere measured according to the methodology of Abidi and Mounts(1997) and Rupérez, Martín, Herrera, and Barbas (2001). The sampleswere analyzed by reverse-phase HPLC, using a Luna 5u PFP(2) column(100A250 × 4.6 mm; Phenomenex Inc., Torrance, CA, USA) andWaters474 scanning fluorescence detector. The mobile phase consisted of 95%methanol and 5% water and the mobile phase rate was 1.2 ml/min. Theresults of the analysis were evaluated using the Millenium32 Chroma-tography Manager (Waters, Milford, MA, USA) program.
The antioxidant capacity of the lipid soluble (ACL) and watersoluble (ACW) compounds in supplements was measured with thephotochemiluminiscence method by PhotoChem® (Analytik Jena,Jena, Germany) and presented as Trolox equivalents (ACL) or gallicacid equivalents (ACW). Samples were extracted with methanoland the gained extracts were analyzed according to the ACL-Kit orACW-Kit protocol (Analytik Jena, Jena, Germany).
2.4. Determination of fatty acid composition
The fatty acid composition of diets and muscle samples wasanalyzed using a gas chromatographic method after the in situtransesterification of lipids. Each sample was analyzed in duplicate.Methyl esters of fatty acids were prepared according to the procedureof Park and Goins (1994). A brief summary of the procedure is as fol-lows: approximately 0.5–0.7 g of sample was weighed directly in atube with stopper and mixed with 3 ml 0.5 M sodium hydroxide inmethanol and 0.3 ml methylene chloride. In situ transesterificationwas performed by heating samples at 90 °C for 10 min in the closedtube. After cooling, 3 ml of 14% boron trifluoride in methanol wasadded and heating at 90 °C was continued for 10 min in the closedtube. Samples were cooled, and the fatty acid methyl esters (FAMEs)were extracted into 1 ml hexane. Analysis of FAMEs was performedby gas chromatographyusing an Agilent 6890 series gas chromatograph(Agilent Technologies, Wilmington, DE, USA) equipped with an Agilent7683 Automatic Liquid Sampler, a split injector, a flame ionizationdetector and a fused silica capillary column Omegawax 320 (Supelco,USA). The injection volumewas 1 μl. The chromatogramswere evaluat-ed by the Agilent GC Chem Station software. Separated FAMEs wereidentified by retention time.
2.5. Malondialdehyde (MDA) determination
The MDA concentration in meat samples was determined followingthemethod of Vila, Jaradat, Marquardt, and Frohlich (2002) with minormodifications. Briefly, approximately 0.3 g of the homogenized samplewas mixed with 1.5 ml of 2.5% trichloroacetic acid in Eppendorfmicrocentrifuge tubes, left for 10 min and then centrifuged (15,000 g,for 15 min at 4 °C). One milliliter of supernatant was mixed with1.5 ml of 0.6% thiobarbituric acid and 1 ml of Milli Q deionized waterin a tubewith stopper and heated at 90 °C for 60 min. After the samples
were cooled, they were filtered through Millipore filters (pore size0.22 μm) into autosampler vials. A Waters Alliance 2690 (Waters,Milford,MA) equippedwithWaters 474 scanning fluorescence detectorwas used to determine MDA concentration. For the purpose of separa-tion a reversed-phase HPLC chromatography column (HyperClone 5uODS (C18) 120 A, 4.6 × 150 mm 5 μm; Phenomenex Inc., USA) and aC18 ODS guard column (4 mm × 30 mm; Phenomenex Inc., USA)were used. The mobile phase consisted of 65% 50 mmol/l KH2PO4
buffer (pH 6.9) and 35% methanol. The mobile phase flow rate was1.0 ml/min. The results of the analysis were evaluated using theMillenium32 Chromatography Manager (Waters, Milford, MA, USA)program.
2.6. Statistical analysis
Data were analyzed by the general linear models (GLM) proce-dure of the SAS/STAT module (SAS 8e, 2000; SAS Inc., Cary, NC,USA), taking into consideration the diet as the only main effect. Dif-ferences between groups were determined on the basis of Tukey'smultiple comparisons test. The results in the tables are presentedas least square means ± SEM with P-values. If not stated otherwise,a least significant difference of 0.05 was used to separate treatmentmeans.
3. Results and discussion
3.1. Fatty acid composition and vitamin E content of the diets
As expected, the fatty acid composition of the diets differed accord-ing to the ingredients (Table 3). The linseed oil addition led to a higherproportion of n-3 and n-6 PUFA, total PUFA and monounsaturated fattyacids (MUFA), due to reduction of saturated fatty acids (SFA), comparedto the diet with palm fat (CONT−). The linseed diets (CONT+, REISHI,OLIVE) also had lower n-6/n-3 ratio (0.55 vs. 3.16) because of abetter proportion of linoleic acid (C18:2 n-6) and α-linolenic acid(C18:3 n-3). Diet OLIVE had the highest content of α-tocopherol;on the other hand diets CONT+ and REISHI have the lowest level ofα-tocopherol. The OLIVE diet had the lowest level of γ-tocopherol, butthis form of vitamin E is not effective in the prevention of oxidation(Tomažin, Frankič, Levart, & Salobir, 2012).
Table 4Body weight (BW), weight gain and diet intake.
CONT– CONT+ REISHI OLIVE SEM a P
Initial BW (g) 2574 2589 2563 2595 93.3 0.994Final BW (g) 3235 3362 3303 3396 102.7 0.689Weight gain (g/day) 28.8 33.6 32.2 34.8 2.19 0.225Feed intake (g/day) 168.3 178.5 168.7 174.7 5.46 0.477FCR (g/g) 6.09 5.66 5.39 5.18 0.37 0.322
CONT− 6% palm fat in a diet; CONT+6% linseed oil in a diet; REISHI 6% linseed oil in a dietwith addition of 1% G. lucidum; OLIVE 6% linseed oil in a diet with addition of 1%O. europaea leaves.FCR—feed conversion rate.
a n = 46.
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3.2. Productive performance
Table 4 represents the changes in body weights, daily growth andfeed intake. Results shows that diet had no effect (P N 0.05) on animalgrowth and feed consumption, which was also shown in previousstudies on rabbits (Bernardini, Dal Bosco, & Castellini, 1999; Dal Boscoet al., 2004; Kouba, Benatmane, Blochet, & Mourot, 2008; Bianchi,Petracci, & Cavani, 2009) where diets high in n-3 PUFA did not signifi-cantly influence productive performances. Our results of weight gainand feed conversion rate show trend of better values in the groupswith linseed oil addition (Table 4), but the differences were notsignificant.
3.3. Fatty acid and tocopherol content of backmuscle and oxidative stabilityof meat
Dietary linseed oil addition led to a higher proportion of total PUFAand lower proportion of total SFA in back muscle, while the level ofMUFA was not affected. Linseed oil supplemented diets significantly in-creased (P b 0.001) the proportion of theα-linolenic acid and also other
Table 5Fatty acid composition and vitamin E content of back muscle.
CONT– CONT+ REISHI OLIVE SEM 1 P
Main fatty acids (% of the total fatty acids)C12:0 0.15 0.09 0.14 0.11 0.02 0.057C14:0 1.86a 1.44b 1.28b 1.40b 0.10 0.001C16:0 23.21a 17.31b 17.66b 17.62b 0.34 b0.001C16:1 n-7 3.48 2.64 2.41 2.55 0.45 0.319C18:0 10.48a 6.02b 6.84b 6.49b 0.33 b0.001∑ C18:1 23.38 23.50 22.76 23.12 0.30 0.286C18:2 n-6 23.91 24.25 24.30 24.17 0.50 0.940C18:3 n-3 3.15b 14.68a 12.50a 13.48a 0.74 b0.001C20:4 n-6 4.12 3.43 4.44 3.91 0.39 0.309C20:5 n-3 0.14b 0.39a 0.45a 0.41a 0.04 b0.001C22:5 n-3 0.63b 1.11a 1.36a 1.23a 0.11 b0.001C22:6 n-3 0.11b 0.15ab 0.19a 0.17ab 0.02 0.005∑ SFA 38.52a 27.50b 28.98b 28.43b 0.53 b0.001∑ MUFA 27.80 27.06 26.11 26.60 0.74 0.397∑ PUFA 33.68b 45.44a 44.91a 44.97a 0.85 b0.001∑ n-3 PUFA 4.07b 16.52a 14.68a 15.50a 0.68 b0.001∑ n-6 PUFA 29.59 28.90 30.21 29.45 0.65 0.546n-6/n-3 PUFA 7.28a 1.81b 2.11b 1.96b 0.13 b0.001
Vitamin E (mg/kg)α-Tocopherol 3.99a 3.27ab 3.08b 3.11b 0.21 0.007γ-Tocopherol 0.46b 0.77a 0.71a 0.67a 0.03 b0.001
CONT− 6% palm fat in a diet; CONT+6% linseed oil in a diet; REISHI 6% linseed oil in a dietwith addition of 1% G. lucidum; OLIVE 6% linseed oil in a diet with addition of 1%O. europaea leaves.SFA—saturated fatty acids; MUFA—monounsaturated fatty acids; PUFA—polyunsaturatedfatty acids.a, bValues with different superscripts are significantly different (P b 0.05).
1 n = 46.
long chain n-3 PUFA. However, there was no effect of diet on n-6 PUFAlevel, but it reduced the n-6/n-3 ratio (Table 5). Similar results werefound with dietary inclusion of 160 g ground linseed/kg diet(Bernardini et al., 1999) or 30 g of extruded linseed/kg diet (Koubaet al., 2008); both significantly increased the proportion of α-linolenicacid and consequently decreased the n-6/n-3 ratio. The addition ofG. lucidum or O. europaea leaves did not have major effect on fatty acidcomposition of back muscle. The tocopherols content in back musclediffers between groups; the level of α-tocopherol was the highest inthe CONT− group, while in the REISHI (P = 0.012) and OLIVE(P = 0.018) groups it was significantly lower (Table 5). On the con-trary, the level of γ-tocopherol was significantly higher (P b 0.001) ingroups that were supplemented with linseed oil. These results werenot expected because they do not reflect the vitamin E composition ofthe diet, but the same ratio among the control and experimentalgroup and α- and γ-tocopherol were found in plasma, liver in leg mus-cle (unpublished data). According to previous research on pigs, the in-clusion of O. europaea leaves in diet significantly increased back fatand intramuscular α-tocopherol content (Paiva-Martins, Barbosa,Pinheiro, Mourão, & Outor-Monteiro, 2009). These authors have longerexperimental period and used higher quantities of O. europaea leaves(5% and 10%), which may explain the differences among the results.
The extent of lipid oxidation in meat samples, presented as MDAconcentration in back muscle, differed between groups (Table 6).Groups supplemented with linseed oil showed higher levels of MDAin meat than the CONT− group. Higher levels of MDA were alsofound when samples were heat processed or stored at 4 °C for 6 days,compared to the fresh or frozen raw meat samples. The inclusion ofG. lucidum or O. europaea leaves in the diet slightly reducedMDA valuesin all stored and heat treated conditions, but not significantly and theO. europaea leaves were more effective in heated (cooked) samples;on the other hand G. lucidum, on the raw samples (except fresh raw).Both those supplements have in vitro antioxidant potential, but itseems that it is not the same at in vivo conditions, which is in accordancewith the results of Dal Bosco et al. (2014) on Spirulina and Thyme. Sev-eral studies have revealed that increasing the content of n-3 PUFA hadbeneficial effect on fatty acid composition but had negative effect onlipid oxidation in rabbit meat which could be improved by addition ofantioxidants (Bielanski & Kowalska, 2008; Zsédely et al., 2008). To ourknowledge, there are no studies dealing with the effect of G. lucidumon the oxidative stability of meat and only few about the antioxidantproperties of G. lucidum. Jones and Janardhanan (2000) found out thatmethanol extracts of G. lucidum possess in vitro antioxidant activity,which was also obtained by Lakshmi, Ajith, Sheena, Gunapalan, andJanardhanan (2003)with ethanol extracts of themyceliumofG. lucidumthat inhibit Fe2+-induced peroxidation of lipid in rat liver (50% inhibi-tion) and maximally inhibit (37%) croton oil-induced peroxidation onthe mouse skin. Similarly Lee et al. (2001) reported that amino-polysaccharide fraction from G. lucidum significantly inhibited iron- or
Table 6MDA concentration (nmol/g) in fresh, refrigerator-stored (4 °C, 6 days), frozen-stored (−20 °C, 3 months) back muscle either raw or heat-processed (85 °C, 60 min).
CONT– CONT+ REISHI OLIVE SEM 1 P
Raw-fresh 0.67b 3.69a 2.61a 2.53a 0.45 0.001Heat-fresh 1.72b 11.47a 9.62a 7.43a 1.23 b0.001Raw-6 days 0.93b 13.99a 8.28ab 10.13a 2.46 0.002Heat-6 days 2.18b 12.13a 9.95a 7.92a 1.20 b0.001Raw-3 months 0.86b 3.32a 2.38ab 2.56ab 0.50 0.008Heat-3 months 2.32b 10.84a 8.25a 7.04a 1.20 b0.001
CONT− 6% palm fat in a diet; CONT+ 6% linseed oil in a diet; REISHI 6% linseed oil in adiet with addition of 1% G. lucidum; OLIVE 6% linseed oil in a diet with addition of 1%O. europaea leaves.a, bValues with different superscripts are significantly different (P b 0.05).
1 n = 46.
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iron plus ascorbic acid-induced lipid peroxidation in rat brain homoge-nates 64.2% at the highest concentration (2 mg/ml) and showed a dose-dependent inactivation of hydroxyl radicals and superoxide anions.Antioxidant activity of mushrooms is mainly related to their contentof phenolic compounds (Froufe et al., 2009), which is also typical forO. europaea fruit, oil and leaves (Benavente-García et al., 2000). Theinfluence of O. europaea leaves supplementation on oxidative stabilityof meat was investigated in several studies. Incorporation of only 5 gO. europaea leaves/kg in turkey diets significantly increased the oxida-tive stability of raw breast fillets during refrigerated storage and wasequal to 150 mg/kg α-tocopherol acetate supplementation (Botsoglouet al., 2010). Similar results were obtained by Paiva-Martins et al.(2009) on pork meat where chops from pigs fed the leaves diet hadlower peroxide and conjugated diene contents than chops from pigsfed conventional diets. They explained differences in oxidative stabilitywith higher α-tocopherol concentration in meat when O. europaealeaves were fed, which also could partly explain our results. Oleuropeinfrom trees and also leaves has been shown to be a potent antioxidant instudies using in vivo methods. It inhibits hyperglycemia and oxidativestress induced by diabetes in rabbits (Al-Azzawie & Alhamdani, 2006)with decreasing the level of MDA along with blood glucose to the levelof normal control rabbits.
Our results showed a certain trend in increasing oxidative stability,but in the future itwill be better to use different extracts fromG. lucidumor O. europaea leaves or higher amounts of supplements.
4. Conclusions
The results of the present study clearly demonstrate that theenrichment of the rabbit diet with linseed oil led to production of rabbitmeat with a high content of n-3 PUFA and better n-6/n-3 PUFA ratio.However, the higher content of PUFA inmeat causes higher susceptibil-ity to lipid oxidation, which is undesirable from a nutritional point ofview. Addition of G. lucidum or O. europaea leaves in rabbit diet showeda small reduction of oxidative processes, particularly in stored raw rab-bitmeat, but those differenceswere not significant. Nevertheless, fromascientific point of view, a higher inclusion of extracts of those (potential)natural antioxidants is worthy of further investigation.
Acknowledgments
This work was supported by a grant from the Slovenian ResearchAgency (Ljubljana, Slovenia).
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