Meat Science 106 (2015) 31–37

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The effect of dietary alfalfa and flax sprouts on rabbit meat antioxidant content, lipid oxidation and fatty acid composition A. Dal Bosco ⁎, C. Castellini, M. Martino, S. Mattioli, O. Marconi, V. Sileoni, S. Ruggeri, F. Tei, P. Benincasa Department of Agricultural, Food and Environmental Sciences, University of Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy

a r t i c l e

i n f o

Article history: Received 23 October 2014 Received in revised form 24 March 2015 Accepted 26 March 2015 Available online 3 April 2015 Keywords: Rabbit Meat Sprouts n-3 Fatty acid Tocopherols Phytochemicals

a b s t r a c t The aim of this study was to determine the effect of dietary supplementation with flax and alfalfa sprouts on fatty acid, tocopherol and phytochemical contents of rabbit meat. Ninety weaned New Zealand White rabbits were assigned to three dietary groups: standard diet (S); standard diet + 20 g/d of alfalfa sprouts (A); and standard diet + 20 g/d of flax sprouts (F). In the F rabbits the Longissimus dorsi muscle showed a higher thio-barbituric acid-reactive value and at the same time significantly higher values of alpha-linolenic acid, total polyunsaturated and n-3 fatty acids. Additionally n-3/n-6 ratio and thrombogenic indices were improved. The meat of A rabbits showed intermediate values of the previously reported examined parameters. Dietary supplementation with sprouts produced meat with a higher total phytoestrogen content. The addition of fresh alfalfa and flax sprouts to commercial feed modified the fat content, fatty acid and phytochemical profile of the meat, but the flax ones worsened the oxidative status of meat. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Sprouts, i.e., the young seedlings obtained from seed germination, are deemed to be healthy foods because they contain high levels of nutrients that may have positive effects on human health such as preventing cardiovascular diseases and cancer (Finley, 2005; Marton, Mandoki, Csapò-Kiss & Csapç, 2010; Schenker, 2002; Webb, 2006). Compared to sound seeds, sprouts are low in antinutritive compounds (e.g., trypsin inhibitor, phytic acid, tannins; Marton et al., 2010) and high in oligo- and monosaccharides (Koehler, Hartmann, Wieser & Rychlik, 2007), free fatty acids (Kim, Kim & Park, 2004), oligopeptides, amino acids (Urbano et al. 2005), vitamins and phytochemicals such as polyphenols (flavonoids, phenolic acids, lignans, phytoestrogens), glucosinolates and carotenoids (Amici, Bonli, Spina, Cecarini, Calzuola & Marsili, 2008; Fernandez-Orozco et al., 2006 and Krishna, Paridhavi & Patel, 2008). Flax sprouts have been found to be a better source of protein, crude fiber, simple sugars and essential micronutrients than sound seeds (Narina, Hamama & Bhardwaj, 2012). Flax sprouts are characterized by higher levels of water soluble proteins and free amino acids (Wanasundara, Shahidi & Brosnan, 1999a) higher levels of free fatty acids, glycolipid fractions, lysophosphatidylcholine, phosphatidic acid (from negligible amounts to 46% of the total) and similar phospholipid levels (Wanasundara, Wanasundara & Shahidi, 1999b).

∗ Corresponding author. Tel.: +390755857110; fax: +390755857122. E-mail address: [email protected] (A. Dal Bosco).

http://dx.doi.org/10.1016/j.meatsci.2015.03.021 0309-1740/© 2015 Elsevier Ltd. All rights reserved.

Alfalfa sprouts contain high amounts of vitamins, phytoestrogens and saponins. Sprouts have higher levels of vitamin A and C (1250- and 10fold increase with respect to the sound seed, respectively; Plaza, De Ancos & Cano, 2003), coumestrol, liquiritigenin, isoliquiritigenin, loliolide (Hong et al. 2011 and Horn-Ross et al. 2000) and saponins than raw seeds (Oleszek, 1998). Despite the increasing popularity of sprouts as a ‘healthy food’ in western countries, the risk of bacterial contamination (e.g., Escherichia coli, Salmonella enteritidis, Vibrio cholerae) represents a public health concern, as sprouts are normally home grown and used as components of salads and therefore undergo no thermal or other sanitation treatment (Taormina, Beuchat & Slutsker, 1999). Dried sprout powder has been proposed as dietary supplement that could be blended at proportions similar to those used in other conventional foods (e.g., wheat sprout powder in wheat bakery flours) to increase the nutritional value of foods without changing dietary behavior (Koehler et al., 2007); however, certain phytochemicals may be lost during processing. An alternative could be the use of sprouts in animal feeding: bioactive compounds from sprouts could be transferred to livestock products, which, in turn, would be transferred to humans. This scheme represents an attractive way of improving the quality and safety of food destined for human consumption and animal health. Some studies examined the effect of sprouts on the performance and health of animals (Jegede et al. 2008; Peer & Leeson, 1985) with controversial result; however, to the best of our knowledge, no specific data on the possibility of transferring bioactive compounds from sprouts to animal products are available. Evidence of bioactive transfer from sprouts to animal tissues could be presumed by some papers. Winarso,

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A. Dal Bosco et al. / Meat Science 106 (2015) 31–37

Purwo & Kusuma (2011) assessed that the improvement in spermatozoa quality of goat was due to the transfer of high vitamin E amount from legumes sprouts. Thus, given the lack of scientific data, the aim of the present study was to evaluate the effect of dietary alfalfa and flax sprouts on rabbit meat antioxidant content, lipid oxidation and fatty acid composition. 2. Materials and methods 2.1. Animals and diets Ninety New Zealand White mixed-sex rabbits were weaned at 30 days of age, allocated into three homogeneous groups (weight, sex) and subjected to the following dietary treatments until they were 80 days old: • Standard (S) diet; • Standard diet + 20 g/d of alfalfa sprouts (A); • Standard diet + 20 g/d of flax sprouts (F).

Fresh sprouts were placed daily near the feeders. The experimental protocol was devised according to the Italian directives (Gazzetta Ufficiale, 1992) on animal welfare for experimental and other scientific purposes, and the research was carried out at the experimental farm of the Department of Agricultural, Food and Environmental Sciences of the University of Perugia (Italy). All of the rabbits were housed individually in flat-deck cages (600 × 250 × 330 mm). The feeding program was adjusted according to previous studies (Martens & Villamide, 1998). Water was supplied ad libitum. The applied temperature and lighting schedules in the rabbit house were 15–18 °C and 16 L:8 D, respectively. 2.2. Production of alfalfa and flax sprouts Alfalfa (Medicago sativa L.) and flax (Linum usitatissimum L.) seeds were germinated on a substrate consisting of moistened tissue paper lying on a layer of silica sand sterilized at 105 °C in aluminum trays (22 cm × 30 cm for alfalfa and 30 cm × 36 cm for flax). In each tray, the sand (600 g for the alfalfa and 1 kg for the flax) was distributed to create a uniform layer on the bottom of the tray and moistened with demineralized water. The trays were placed in a temperature-controlled room at 20 °C in the dark and kept in these conditions for three days. Water was added periodically to compensate for sand water loss due to evaporation. In contrast to the usual sprouting procedure used for alfalfa in which sand water content is restored once a day, flaxseed requires several separate additions of water, as the seeds tend to produce a glue-like mucilaginous exudate in the presence of high water content that would hamper seedling development. For each species, the sprouts obtained on the third day from different trays were combined to prevent a possible tray effect and stored at 4 °C in plastic bags until use (i.e., within three days). 2.3. Sampling and analytical determination At 80 days of age, 20 rabbits per group with weights close to the average of the group (± 10%) were selected and slaughtered in the departmental processing plant 12 h after feed withdrawal under the supervision of veterinarians from the University of Perugia; none of the animals underwent any form of transportation. The rabbits were sacrificed by severing the carotid arteries and jugular veins following electro-stunning and the carcasses were prepared according to the methods described by Blasco and Ouhayoun (1993). Following carcass chilling (24 h at + 4 °C), the two Longissimus lumborum muscles were removed and carefully freed from connective and adipose tissues.

2.3.1. Chemical composition of the feed and sprouts The chemical composition of the feed and sprouts was determined according to the method of the AOAC (1995). 2.3.2. Fatty acid profile of the feed, sprouts and meat The fatty acid profile of the feed, sprouts and meat was determined by gas chromatography following lipid extraction according to the method described by Folch, Lees & Sloane-Stanley (1957). In particular, 1 mL of lipid extract was evaporated under a stream of nitrogen and the residue was derived by adding 3 mL of sulfuric acid (3% in methanol). Following incubation at 80 °C for 1 h, the methyl esters were extracted with petroleum ether, and 1 μL was injected into a gas chromatograph (Mega 2 — model HRGC; Carlo Erba, Milan, Italy) equipped with a flame ionization detector. The fatty acid methyl esters (FAMEs) were separated with an Agilent (J&W) capillary column (30 m × 0.25 mm I.D; CPS Analitica, Milan, Italy) coated with a DB-wax stationary phase (film thickness of 0.25 mm). The operating conditions used during the column injection of the 1 mL sample volume were as follows: the temperatures of the injector and detector were set at 270 °C and 280 °C, respectively, and the detector gas flows were H2 at 50 mL min−1 and air at 100 mL min−1. The oven temperature was programmed to provide a good peak separation as follows: the initial oven temperature was set at 130 °C; this temperature increased at a rate of 4.0 °C min−1 to 180 °C and was held for 5 min; the temperature was then increased at a rate of 5.0 °C min−1 to 230 °C; the final temperature was held for 5 min. Helium was used as a carrier gas at a constant flow rate of 1.1 mL min−1. Individual fatty acid methyl esters were identified by referring to the retention time of FAME authentic standards. The average amount of each fatty acid was used to calculate the sum of the total saturated (SFA), total monounsaturated (MUFA) and total polyunsaturated (PUFA) fatty acids. 2.3.3. Nutritional indexes The peroxidability index (PI) was calculated according to the equation proposed by Arakawa and Sagai (1986): PI = (% monoenoic × 0.025) + (% dienoic × 1) + (% trienoic × 2) + (% tetraenoic × 4) + (% pentaenoic × 6) + (% hexaenoic × 8). The amount of each fatty acid was used to calculate the indices of atherogenicity (AI) and thrombogenicity (TI) as proposed by Ulbricht and Southgate (1991) and the hypocholesterolemic/ hypercholesterolemic ratio (HH) as suggested by Santos-Silva, Bessa & Santos-Silva (2002). 2.3.4. Oxidative status of meat The extent of muscle lipid oxidation was evaluated by a spectrophotometer set at 532 nm (Shimadzu Corporation UV-2550, Kyoto, Japan), according to the modified method of Ke, Ackman, Linke, and Nash (1977), which measured the absorbance of thio-barbituric acid-reactive substances (TBARS). Briefly 5 g of meat, was homogenized in a 75 g/L trichloroacetic acid solution. After centrifugation 5 mL of extract was reacted with 2.88 g/L of fresh thio-barbituric acid (TBA), with a ratio of 1:2 (v/v). Oxidation products were quantified as malondialdehyde equivalents (mg MDA/kg muscle) through a 1,1,3,3-tetraethoxypropane calibration curve. The tocopherol (α-tocopherol and its isomers β + γ and δ) and retinol contents of the feed, sprouts and meat were quantified by HPLC according to the method described by Hewavitharana, Lanari & Becu (2004). In particular, 5 ml of distilled water and 4 ml of ethanol were added to 2 g of sample and vortexed for 10 s. After mixing, 4 ml of hexane containing BHT (200 mg/l) was added and the mixture was carefully shaken and centrifuged. An aliquot of supernatant (3 mL) was dried under a stream of nitrogen and dissolved in 300 μL of acetonitrile; 50 μL were injected into the HPLC (pump model Perkin Elmer series 200, equipped with an autosampler system, model AS 950-10, Tokyo, Japan) on an Ultrasphere ODS column (250 × 4.6 mm internal diameter, 5 μm particle size; CPS analitica, Milan, Italy). Tocopherols were identified using a FD detector (model Jasco, FP-1525 — excitation and emission

A. Dal Bosco et al. / Meat Science 106 (2015) 31–37 Table 1 Ingredients and chemical composition of feed and sprouts (% wet weight). Basal diet Ingredient Alfalfa Corn meal Soybean meal Barley Wheat bran Ca-Phosphate Vitamin mineral premixa Molasses Salt Ca-carbonate DL-Methionine Chemical composition Dry matter Crude protein Ether extract Ash Fiber NDF ADF ADL Hemicellulose Digestible energy Mj kg−1 f.m.

Sprouts Alfalfa

Flax

10.2 6.82 0.52 2.04 3.05 37.8 13.8 1.81 7.42 2.04

11.3 6.08 0.63 1.98 3.56 34.2 12.4 1.72 6.54 2.12

30.00 10.00 18.00 25.00 12.00 1.35 1.20 1.00 0.70 0.70 0.05 89.0 17.3 2.04 7.28 12.8 24.0 14.1 2.90 9.93 10.5

a Added per kg: vit. A U.I. 11.000; vit. D3 U.I. 2.000; vit. B1 mg 2.5; vit. B2 mg 4; vit. B6 mg 1.25; vit. B12 mg 0.01; vit. E mg 25; biotin mg 0.06; vit. K mg 2.5; niacin mg 15; folic. ac mg 0.30; D-panthotenic. ac. mg 10; choline mg 600; Mn mg 60; Cu mg 3; Fe mg 50; Zn mg 15; I mg 0.5; Co mg 0.5; lysine mg 50; methionine mg 40.

wavelengths of 295 nm and 328 nm, respectively) and quantified using external calibration curves prepared with increasing amounts of pure tocopherols in ethanol. The carotenoid contents were extracted with the same procedure of tocopherols and quantitatively determined by HPLC (Jasco, pump model PU-1580, equipped with an autosampler system, model AS 950–10, Tokyo, Japan) and a Ultrasphere ODS column (250 × 4.6 mm internal diameter, 5 μm particle size; CPS analitica, Milan, Italy). The solvent system consisted of solution A (methanol–water–acetonitrile, 10:20:70) and solution B (methanol–ethyl acetate, 70:30). The flow rate was 1 mL min−1, and the elution program was a gradient starting from 90% A in a 20 min step to 100% B and then a second isocratic step of 10 min. The detector was a UV–visible spectrophotometer (JascoUV2075 Plus) set at a wavelength of 450 nm for lutein, zeaxanthin and β-carotene, and at 325 nm for retinol. The different carotenoids were identified and quantified by comparing the sample with pure commercial standards in chloroform (Sigma-Aldrich, Steinheim, Germany; and Extrasynthese, Genay, France) and in ethanol for the retinol (Sigma-Aldrich, Steinheim, Germany; and Extrasynthese, Genay, France).

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sample was subjected to the enzymatic extraction proposed by Kuhnle, Dell'Aquila, Low, Kussmaul & Bingham (2007). The extracts were analyzed for the phytochemical determination by HPLC following the method proposed by Peñalvo et al. (2004) with slight modifications. The following equipment was used for the HPLC analysis: two Jasco PU-1580 (L. i. Service, S. r. l. Roma, Italy) pumps connected to a gradient solvent system, a HT310 L (HTA s.r.l., Brescia, Italy) autosampler with a 20 μl loop, an Inetsil ODS-3 (C18 150 mm × 4.6 mm i.d.; particle size = 5 μm) (GL Sciences, Japan) protected with a guard column, an Inetsil ODS-3 (C18 4.0 mm × 10 mm i.d.; particle size = 5 μm) (GL Sciences, Japan), a CoulArray (ESA, Inc., Chelmsford, MA) detector consisting of two cell packs in series, each pack containing four porous graphite working electrode channels with an associated palladium reference electrode and a platinum counter electrode, and CoulArray Software for Windows for the acquisition and analysis of data. The phytoestrogens in the extracts were identified by comparing their retention times with those of the standards; the compounds were then quantified using their calibration curves in the concentration range of 0.05, 0.25, 0.50, 1.0, 1.5 and 2.5 μg/mL. For the binary gradient elution, mobile phase A was 50 mM sodium acetate buffer, pH 5 /MeOH (80:20 v/v) and mobile phase B was 50 mM sodium acetate buffer, pH 5/ MeOH/ACN (40:40:20 v/v/v). The gradient cycle was as follows: 70% of phase A at the starting point followed by a decrease to 40% in 25 min, held for 10 min, decreased to 25% in 5 min, held for 10 min, brought back to the initial conditions in 5 min and finally held to 70% of phase A for 5 min. The variation in gradient solvent was linear and the eight electrode potentials were set at 150, 230, 300, 420, 470, 550, 600 and 650 vs. palladium reference electrodes at room temperature. The sample injection volume was 20 μl and the total run time for each sample was 60 min. The flow was 0.9 mL min−1. 2.4. Statistical analysis Data were analyzed with a linear model of STATA package (2005) with the diet as a fixed effect. Least square means and planned comparisons were used for mean separation when the model was significant (P b 0.05). To simultaneously analyze the global trend in the nutritional traits of the meat (fatty acids, antioxidants, TBARS), multivariate analysis was performed (proc. Factor). 3. Results and discussion 3.1. Chemical characteristics of feed and sprouts Table 1 shows the reported formulation, chemical composition and energetic value (MJ kg−1) of solid feed and sprouts. 3.2. Fatty acid profile of basal diet and sprouts

2.3.5. Phytochemical content of sprouts and meat Phytoestrogen (daidzein and coumestrol) and lignan (secoisolariciresinol diglucoside, isolariciresinol, hydroxymatairesinol, secoisolariciresinol and matairesinol) levels in the alfalfa and flaxseed sprouts and in the Longissimus lumborum muscle were quantified by high performance liquid chromatography equipped with a column array detector (HPLC-ECD) following extraction. The alfalfa and flax sprouts were subjected to acid and alkaline extractions. The extraction was performed using a combination of the methods proposed by Plaza et al. (2003) and Milder et al. (2004). The acid extraction proposed by Plaza et al. (2003) was found to be optimal for the extraction of phytoestrogens, especially from alfalfa sprouts, whereas the alkaline extraction proposed by Milder et al. (2004) is a standard procedure used for the extraction of lignans. The sprouts were frozen by immersion in liquid nitrogen, pulverized in a domestic grinder and used for basic extraction; the resulting pellet was subjected to acid extraction. Approximately 100 mg of freeze-dried Longissimus lumborum

Differences in the fatty acid profiles of basal diet and sprouts are shown in Table 2. The alfalfa and flax sprouts had higher PUFA concentrations than the conventional feed; in particular, linoleic acid (38.46%)

Table 2 Main fatty acid of control diet and sprouts (mean + s.d.). Control

C14:0 C16:0 C18:0 C18:1n-9 C18:2n-6 C18:3n-3

% " " " " "

1.24 ± 0.21 20.12 ± 1.25 23.20 ± 2.07 17.44 ± 1.84 21.83 ± 1.43 16.17 ± 1.36

Sprouts Alfalfa

Flax

0.47 ± 0.11 17.62 ± 1.11 3.27 ± 0.69 10.14 ± 1.20 38.46 ± 2.74 28.68 ± 2.47

0.29 ± 0.08 6.10 ± 0.87 2.36 ± 0.42 6.09 ± 0.78 15.76 ± 1.58 70.05 ± 2.91

Each value represents the mean of three replications.

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Table 3 Antioxidant compounds in control diet and sprouts (mean + s.d.).

α-Tocopherol α-Tocopherol acetate γ-Tocopherol δ-Tocopherol α-Tocotrienol γ-Tocotrienol Retinol Lutein + Zeaxanthin β-Carotene

μg g−1 f.m. " " " " " " " "

Control

Sprouts Alfalfa

Flax

121.4 ± 12.0 66.2 ± 1.25 n.d. 2.98 ± 0.26 4.30 ± 0.35 5.47 ± 0.62 59.9 ± 4.28 21.3 ± 3.81 13.6 ± 2.82

47.0 ± 3.08 n.d. 5.36 ± 1.01 n.d. n.d. n.d. 11.6 ± 2.14 114.1 ± 20.1 19.6 ± 3.12

42.0 ± 4.21 n.d. 4.48 ± 1.20 n.d. n.d. n.d. 10.9 ± 1.59 13.3 ± 2.00 n.d.

n.d = not detectable. Each value represents the mean of three replications.

and linolenic acid (70.05%) levels were highest in alfalfa and flax sprouts, respectively. Palmitic and stearic acid levels were higher in the conventional feed than in the sprouts. 3.3. Bioactive compounds in basal diet and sprouts The profiles of bioactive compounds in the feed and sprouts are shown in Table 3. The amounts of α-tocopherol in the feed, alfalfa and flax sprout groups were 121.48, 47.07 and 42.01 μg g−1, respectively. Alpha-tocopherol acetate, one the most commonly used antioxidants in animal feed, was detected only in the standard diet, confirming the high resistance of synthetic compounds to feed processing (Castellini, Dal Bosco, Bernardini & Cyril, 1998). Higher levels of antioxidants were found in the feed than in the sprouts due to the different water content of the feed and sprouts (12 vs 90%, respectively), with the only exception represented by γ-tocopherol. Levels of the carotenoids lutein and zeaxantin were much higher in the alfalfa sprouts than in the control feed and flax sprouts (114.12 vs 21.37 and 13.36 μg g−1, respectively). Beta-carotene content was higher in the alfalfa sprouts than in the control diet and not detected in flax sprouts. 3.4. Phytochemical compounds in sprouts and basal diet The phytochemical compounds found in alfalfa and flaxseed sprouts and in the basal diet are shown in Table 4. The flax sprouts contained very high amounts of secoisolariciresinol diglucoside (SDG). The other lignans (isolariciresinol, hydroxymatairesinol, secoisolariciresinol and matairesinol) were also present in significantly greater quantities in the sprouts. The alfalfa sprouts had a coumestrol content of approximately 8 mg 100 g−1 d.m. The content of coumestrol in alfalfa can be highly variable and is affected by a number of factors, including the age of the plant, climatic conditions, presence of pathogens and the processing

of the crop (Franke, Custer, Cerna & Narala, 1994; Moravcová et al., 2004). Moreover, alfalfa sprouts contained high amounts of other important isoflavones such as daidzein (23 mg 100 g−1 d.m.) whereas, as expected, the levels of lignans (except SDG, which was six times higher) were low. Even if the study of the performance of the rabbits was not the objective of this trial, we observed that all the sprouts provided were eaten and that the average daily gain and the final live weight were similar in all groups (data not shown). Jegede, Fafiolu, Oni, Faleye & Oduguwa (2006) and Jegede, Fafiolu, Falaye & Oduguwa (2008) observed a reduction in feed intake, weight gain and slaughter weight in growing rabbits fed malted sorghum sprouts. In particular, as the level of sprouts increased (10, 20 and 30%) the productive performance decreased, most likely due to anti-nutritional factors found in sorghum. The available information on the effect of sprouts on rabbit feed intake is limited. 3.5. Meat fatty acid profile The fatty acid profile of the Longissimus lumborum muscles is presented in Table 5. Effect of sprout supplementation was more profound in the flax group: the meat of this group had significantly lower SFA and linoleic acid contents and higher levels of linolenic acid (approximately two times more), PUFA and total n-3 fatty acids. This situation led to an improvement in the n-6/n-3 ratio and thrombogenic index, and a slight improvement in the atherogenic index, accordingly, the peroxidability index increased. Alfalfa had an intermediate effect on meat fatty acid profile in relation to the control and flax groups. As previously mentioned, it is very difficult to compare our results with those of other studies, as few papers are available on this specific topic. In our previous study (Dal Bosco et al., 2014), adding fresh alfalfa grass to the diet resulted in significant reductions in C14:0, C16:1, C18:1 and C18:2n-6 levels; in contrast, the C18:0 and all fatty acids of the n-3 series increased. In particular, the meat of the alfalfa group had higher PUFA concentrations and total n-3 fatty acid contents (35.9% vs. 31.9% and 12.4% vs. 7.1%, respectively) and lower MUFA concentrations (22.6% vs. 26.0%). The linolenic acid content of the alfalfa group was three times higher than that of the control group; in contrast, in this trial, the decrease of linolenic in the alfalfa group was less pronounced (+27.3%). The lipid contents of the meat were significantly different, with higher values in the control meat, most likely due to the lower feed consumption, digestible energy intake and lipid content of the feed (commercial feed + sprouts). 3.6. Oxidative status of meat The Longissimus lumborum muscle from the rabbits fed the flax sprouts had higher TBARS values (Table 6). The enhanced susceptibility of meat to lipid peroxidation is most likely due to the different fatty acid profile of the feed and the produced meat, as well as the vitamin E content of the muscle. Flax sprouts were characterized by a high proportion

Table 4 Phytochemical contents in control diet and sprouts (mean + s.d.). Sprouts

Secoisolariciresinol diglucoside Isolariciresinol Hydroxymatairesinol Secoisolariciresinol Matairesinol Daidzein Cumestrol

mg 100 g−1 d.m " " " " " "

n.d = not detectable. Each value represent the mean of three replications.

Control

Alfalfa

Flax

58.1 + 2.06 13.3 + 1.25 n.d. n.d. n.d. 12.0 + 1.65 2.31 + 0.28

29.3 + 1.06 4.03 + 0.28 2.08 + 0.27 6.14 + 0.42 n.d. 23.3 + 2.42 8.2 + 0.53

4799.1 + 201 106.1 + 9.06 74.2 + 6.36 137.4 + 9.06 73.2 + 4.18 20.5 + 1.58 154.2 + 11.1

A. Dal Bosco et al. / Meat Science 106 (2015) 31–37

of linolenic acid; however, the amount of antioxidants may not have been adequate to compensate for the pro-oxidant thrust induced by higher PUFA contents. The lower solid feed intake of these animals resulted in a reduction in α-tocopherol acetate and α-tocopherol levels (approximately − 6% and 4%, respectively) and a greater ingestion of PUFAs, which led to an imbalanced oxidative status. Our hypothesis is that the higher degree of unsaturation of the meat of the rabbits fed flax sprouts led to an increased consumption of antioxidants in the muscle cell membrane (Dalle Zotte and Szendrő, 2011). The meat from the alfalfa group, despite having higher α-tocopherol levels, had similar TBARS values as the meat of the control group. It is likely that the previously mentioned hypothesis could also apply to the alfalfa group. The factorial analysis clearly confirms such assumption: TBARS values were on the same order of magnitude as the levels of PUFA n-3 (Fig. 1), whereas α-tocopherol and α-tocotrienol levels were two-fold higher. The enrichment of meat with significant amounts of n-3 (+32% and +73% for alfalfa and flax sprouts, respectively) renders the meat more unstable (TBARS and n-3 are closely characterized in Factor 1), even if levels of the main antioxidants concomitantly increased (Factor 2; 52% and 15% for alfalfa and flax sprouts, respectively). Lipid oxidation in meat is associated with the development of rancid flavor, odor, drip loss, and a concomitant reduction in the acceptability and nutritional quality of it (Buckley, Morrissey & Gray, 1995).

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Table 6 Oxidative status of Longissimus dorsi muscle.

TBARS α-Tocopherol δ-Tocopherol γ-Tocopherol α-Tocotrienol γ-Tocotrienol Retinol

mg MDA kg−1 ng g−1 " " " " "

Control

Alfalfa

Flax

Pooled SE

0.07a 1349.1a 40.3b 57.6 230.7a 32.6 89.7

0.10a 2058.4b 40.6b 45.8 459.8b 28.3 85.2

0.21b 1552.9a 36.0a 56.3 304.9a 36.4 101.9

0.05 234.2 3.2 6.2 43.6 3.1 10.2

N = 20 per group; a..b: P b 0,05.

The present findings were in agreement with our previous results obtained with rabbits fed fresh grass (Cardinali, Rebollar, Mugnai, Dal Bosco & Castellini, 2008; Mugnai, Finzi, Zamparini, Dal Bosco & Castellini, 2008) and are consistent with those of other authors (Forrester-Anderson, Mc Nitt, Way & Way, 2006) who suggested that grass-based diets alter the fatty acid profiles and enhance the n-3 fatty acid contents of meat. It is important to note that the n-6/n-3 values in the control group were lower than the recommended dietary amounts for humans: Williamson, Foster, Stanner & Buttriss (2005) emphasized the need to eat foods with n-6/n-3 ratios lower than or equal to 6, whereas Simopoulos (2002) indicated that 4:1 is the optimal ratio. 3.7. Phytochemical content of meat

Table 5 Lipid content, fatty acid profile and nutritional indexes of longissmus dorsi muscle.

Lipid content (%) Fatty acid (%) C14:0 C16:0 C18:0 Others ∑ SFA C16:1Δ9 C18:1Δ9c C20:1Δ11c Others ∑ MUFA C18:2Δ9c.12c [n-6] C20:2Δ11.14 [n-6] C20:3Δ8c.11c.14c [n-6] C20:4Δ5c.8c.11c.14c [n-6] Others n-6 C18:3Δ9c.12c.15c [n-3] C20:3Δ11c.14c.17c [n-3] C20:5Δ8c.11c.14c.17c [n-3] C21:5Δ6c.9c.12c.15c.18c [n-3] C22:5Δ7c.10c.13c.16c.19c [n-3] C22:6Δ4c.7c.10c.13c.16c.19c [n-3] Others n-3 ∑ PUFA PUFA/SFA ∑ n-6 ∑ n-3 n-6/n-3 Atherogenicity index Thrombogenicity index Peroxidability index Hypochol./hyperchol. fatty acid ratio

Control

Alfalfa

2.15b

1.82a

Flax 1.94a

0.22

2.19 28.9 7.08 0.52 38.6b 3.59 24.1 0.42 0.62 28.7 22.5b 0.32 0.34 4.06 0.26 2.39a 0.32a 0.55 0.25 0.85 0.57 0.15 32.5a 0.84 27.5 5.08a 5.41c 0.62 0.88c 57.0a 1.77

2.33 27.7 7.88 0.48 38.3b 3.41 23.9 0.45 0.48 28.2 21.5ab 0.28 0.37 4.18 0.30 3.29b 0.51b 0.78 0.30 0.91 0.69 0.22 33.3a 0.87 26.6 6.70b 3.98b 0.61 0.79b 60.3a 1.84

2.18 27.1 7.06 0.43 36.7a 3.66 23.5 0.40 0.57 28.1 20.4a 0.26 0.31 4.97 0.20 5.64c 0.48b 0.68 0.39 0.94 0.62 0.20 35.1b 0.95 26.1 8.95c 2.92a 0.57 0.67a 67.0b 1.94

0.26 1.23 0.89 0.89 1.39 0.56 1.28 0.07 0.07 1.45 1.05 0.07 0.09 0.45 0.56 0.56 0.09 0.15 0.08 0.15 0.17 0.09 1.54 0.10 1.65 0.98 0.67 0.04 0.09 1.15 0.21

Pooled SE

N = 20 per group; a..c: P b 0,05. Peroxidability index = (% monoenoic × 0.025) + (% dienoic × 1) + (% trienoic × 2) + (% tetraenoic × 4) + (% pentaenoic × 6) + (% hexaenoic × 8). Atherogenicity index = (C12:0 + 4xC14:0 + C16:0) / [(ΣMUFA + Σn-6) + Σ n-3)]; Thrombogenicity index = (C14:0 + C16:0 + C18:0) / [(0.5 × ΣMUFA + 0.5 × n-6 + 3 × n3) + (n-3) / n-6)]; Hypochol./hyperchol. fatty acid ratio = [(C18:1n-9 + C18:2n-6 + C20:4n-6 + C18:3n-3 + C20:5n-3 + C22:5 n-3 + C22:6n-3) / (C14:0 + C16:0.

The phytochemical contents of the Longissimus lumborum muscle are shown in Table 7. Few studies have examined phytoestrogen

C: Control; A: Alfalfa; F:Flax Fig. 1. Factor loading and score variables. C: Control; A: Alfalfa; F: Flax.

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A. Dal Bosco et al. / Meat Science 106 (2015) 31–37

Acknowledgements

Table 7 Phytochemical contents in Longissimus dorsi muscle.

Secoisolariciresinol diglucoside Isolariciresinol Hydroxymatairesinol Secoisolariciresinol Matairesinol Daidzein Cumestrol Total phytoestrogens

μg 100 g−1 f.m. " " " " " " "

Control

Alfalfa

Flax

Pooled SE

78.8a

115.1b

126.9b

11.0

9.13 8.15 9.56 1.50 4.50 5.17 5.10 0.25 21.9 23.7 22.8 2.5 3.11 3.34 2.96 0.38 15.1c 12.4b 1.3 1.88a 47.8c 25.1a 2.19 39.2b 215.0b 204.8b 11.2 158.5a

N = 20 per group; a..c: P b 0.05.

concentrations in tissues or body fluids other than plasma and urine. Despite the potential importance of lignans in reducing the risk of disease and cancer, little is known of the metabolic fate of these compounds or direct metabolites and their distribution in the body tissues resulting from the ingestion of the precursor compounds (Saarinen and Thompson, 2010; Whitten and Patisau, 2001). Dietary supplementation with sprouts produced higher total phytoestrogen content (approximately 30%) than was found in the control group. This change was mainly due to secoisolariciresinol diglucoside. Despite the fact that the secoisolariciresinol diglucoside content was definitively higher in flaxseed than in alfalfa sprouts, the difference in the tissue levels of animals fed sprouts was not significant. A similar trend was observed for other lignans such as hydroxymatairesinol and secoisolariciresinol with differences even less pronounced. The isolariciresinol content was lower in the alfalfa sprouts than in the controls, and this difference was also found in the respective muscle tissues. In contrast, the supplementation with flaxseed sprouts seemed to induce a slight increase in isolariciresinol levels whereas no difference in the matairesinol content was observed. The major classes of phytochemical compounds are isoflavones, lignans and coumestans (Bingham, Atkinson, Liggins, Bluck & Coward, 1998). In plants, these compounds occur as glycosides, which are deconjugated by intestinal glucosidases to aglycones (Webb and McCullough, 2005) and further metabolized by the intestinal microflora into hormone-like compounds with weak estrogenic activity. Lignans can be converted into the mammalian lignans enterodiol and enterolactone, whereas the isoflavone daidzein can be converted into O-desmethylangolensin (O-DMA) and equol. The phytoestrogen content of rabbit meat (150–220 μg/100 g) is lower than that of soybased foods (approximately 6 mg/100 g) but similar to that of commonly consumed meat and vegetables (Kuhnle et al. 2008). A number of studies have reported a correlation between lignan intake and risk of certain illnesses such as cancer and CHD. The benefits of lignan and lignan metabolites are thought to be associated with the estrogen-like activity of these compounds; however, the exact mechanisms are not entirely known. Recently, Almario and Karakas (2013) found that diets high in lignan decreased total cholesterol, LDL-C, and oxidized (Ox)-LDL levels by 25%, thus contributing to a decrease in one of the main risk factors for cardiovascular disease. 4. Conclusion This study represents the first evidence on the possibility to transfer bioactive compounds from sprouts to rabbit meat and, considering the lack of studies on this specific topic, these results provide a foundation for the pursuit of investigations in order to improve nutritional quality of rabbit meat. Further studies should be conducted to understand phytochemical metabolism in rabbit and the effect of these dietary compounds on human health.

We gratefully acknowledge Dr. Oriana Porfiri of CGS Seeds (Acquasparta, Terni, Italy) for providing seeds, Mr. Silvano Locchi for his assistance in sprout production in the Seed Laboratory and Giovanni Migni and Osvaldo Mandoloni for animal handling.

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