Fatty acid profile, color and lipid oxidation of meat from young bulls fed ground soybean or rumen protected fat with or without monensin

Meat Science 96 (2014) 597–605

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Fatty acid profile, color and lipid oxidation of meat from young bulls fed ground soybean or rumen protected fat with or without monensin☆,☆☆ M.M. Ladeira a,⁎, L.C. Santarosa a, M.L. Chizzotti a, E.M. Ramos b, O.R. Machado Neto a, D.M. Oliveira a, J.R.R. Carvalho a, L.S. Lopes c, J.S. Ribeiro d a

Department of Animal Science, Universidade Federal de Lavras, Lavras, Minas Gerais 37.200-000, Brazil Department of Food Science, Universidade Federal de Lavras, Lavras, Minas Gerais 37.200-000, Brazil Centro de Educação Superior do Oeste, Universidade do Estado de Santa Catarina, Chapecó, Santa Catarina 89.800-000, Brazil d Universidade Federal de Alagoas, Arapiraca, Alagoas 57.309-005, Brazil b c

a r t i c l e

i n f o

Article history: Received 29 October 2012 Received in revised form 29 April 2013 Accepted 30 April 2013 Available online 19 August 2013 Keywords: Calcium salts Conjugated linoleic acid Ionophore Lipids Oilseed TBARS

a b s t r a c t The objective of the present study was to evaluate the meat quality and fatty acid (FA) profile of the muscle and subcutaneous fat of young bulls fed ground soybean grain (SB) or rumen protected fat (RPF) with (230 mg head− 1 day−1) or without monensin. Forty animals with an initial weight of 359 kg were allotted in a 2 × 2 factorial arrangement of treatments. The use of monensin increased the arachidonic and α-linolenic acids in the longissimus dorsi (LD) muscle and subcutaneous fat, respectively (P b 0.05). The meat from the animals receiving RPF had greater C18:1 content (P b 0.01). The CLA and C18:2 contents were greater in the LD muscle of the animals fed SB (P b 0.01). However, α-C18:3 was greater in the LD muscle of animals fed RPF (P b 0.01). In the subcutaneous fat, SB reduced C12:0 and C14:0 contents (P b 0.01) and increased C18:0 (P b 0.05). The inclusion of RPF increased the C18:1 and CLA contents (P b 0.01) in the subcutaneous fat. Soybean elevated PUFA contents and increased susceptibility of muscle and subcutaneous fat to lipid oxidation. © 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

1. Introduction Changing the fatty acid profile of beef to obtain a lower proportion of saturated fatty acids (SFA) is an important way to produce a healthier meat for the consumer. According to the literature, this can be achieved through the use of lipid sources in the diet (Daley, Abbott, Doyle, Nader, & Larson, 2010; Wood et al., 2008). However, the best lipid sources to achieve this manipulation have not been well described. Despite of soybean meal is considered the major protein source for animal nutrition, in some parts of the world, the whole soybean grain is used to increase the diet lipid contents, and may be an option to improve the fatty acid profile of ruminant products (Oliveira et al., 2011). Another important lipid source is the rumen protected fat or calcium salts from soybean oil, which exhibit low fatty acid release in the

☆ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ☆☆ Project financed by Conselho Nacional de Desenvolvimento Científico e Tecnológico CNPq (Brasília, Brazil) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais Fapemig (Belo Horizonte, Minas Gerais, Brazil). ⁎ Corresponding author. Tel.: +55 35 3829 1244; fax: +55 35 3829 1231. E-mail address: [email protected]fla.br (M.M. Ladeira).

rumen (Gulati, Scott, & Ashe, 1997; Jenkins & Bridges, 2007), and may result in increased monounsaturated fatty acids (MUFA) deposition in meat. When the beef cattle diets have high concentration of unsaturated fatty acid (UFA), the use of ionophores may alter the meat lipid composition by affecting ruminal biohydrogenation. In vitro studies have shown that the rates of triglyceride hydrolysis and fatty acid biohydrogenation were reduced in the presence of monensin (Fellner, Sauer, & Kramer, 1997). Additionally, UFA (Eifert et al., 2006) and cis9, trans-11 C18:2 (Silva-Kazama et al., 2010) were greater in the milk of cows supplemented with monensin. Increase concentration of UFA improves human health due to the participation of these fatty acids in vital metabolic processes, such as membrane structure and immunological processes (Cook, Whigham, & Yang, 2001; Kremmyda, Tvrzicka, Stankova, & Zak, 2011). However, greater UFA concentrations in meat can lead to problems related to shelf life and sensory characteristics, such as color and flavor. According to Wood et al. (2008), the muscle fatty acid composition affects its oxidative stability, and polyunsaturated fatty acids (PUFA) are more prone to oxidation than MUFA and SFA. Therefore, the objective of this study was to evaluate the fatty acid profile and qualitative characteristics of meat from young bulls fed ground soybean grain or rumen protected fat from soybean oil, with or without monensin.

0309-1740/$ – see front matter © 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2013.04.062

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2. Materials and methods The animal care and handling were approved by the Federal University of Lavras Animal Care and Use Committee before the research was initiated. The experiment was conducted at the Department of Animal Science of the Federal University of Lavras from June to September 2009. 2.1. Animals and diet Forty young bulls of Red Norte breeding (10 per treatment), with an initial average age of 20 mo and an initial average weight of 359 ± 47 kg were allotted in a completely randomized design using a 2 × 2 factorial arrangement. The animals were housed in group pens, according to the diets, with 30 m2 per animal, and each animal was the experimental unit. The experimental period lasted 84 days and was preceded by an adaption period of 14 days, during which the animals received the same diet. At the beginning of the adaption period, the animals were treated for internal and external parasites (Ivomec, Paulína, Brazil). The animals were weighed at the beginning and ending of the experimental period, after a 16-h fasting period. Experimental diets were formulated to be isonitrogenous according to the NRC (1996) and provided ad libitum twice daily, at 0700 and 1400 h. Corn silage was used as roughage, and two types of concentrates, one containing ground soybean and the other rumen protected fat from soybean oil (MEGALAC-E®, calcium salts of long-chain fatty acids: Arm & Hammer, Church & Dwight Company, QGN, Brazil), were used as lipid sources (Table 1). Half of the animals that received each concentrate (soybean or rumen protected fat) were supplemented with 230 mg head−1 day−1 of the ionophore monensin (Rumenpac®, M.Cassab, São Paulo, Brazil) during the experimental period. Therefore, we assessed the following treatments: SB, diet containing ground soybean without monensin; SBM, diet containing ground soybean and monensin; RPF, diet containing rumen protected fat without monensin; and RPFM, diet containing rumen protected fat and monensin. Samples of concentrate ingredients and corn silage were collected once a week. These samples were dried in a forced-air oven at 65 °C for 72 h and then were ground to a mesh size of 1 mm. Analysis of the dry matter (DM), crude protein (CP), and ether extract (EE) from the diets were conducted according to the AOAC (1990). The concentration of neutral detergent fiber (NDF) was analyzed according to the method Table 1 Composition of ingredients and experimental diets: ground soybean (SB), ground soybean + monensin (SBM), rumen protected fat from soybean oil (RPF), and rumen protected fat from soybean oil + monensin (RPFM). Diets SB

SBM

RPF

RPFM

Ingredients, % of DM Corn silage Ground corn grain Soybean meal Ground soybean grain Premixed minerala MEGALAC-E® (RPF) Monensin, mg day−1

40.0 38.2 – 20.0 1.8 – –

40.0 38.2 – 20.0 1.8 – 230

40.0 40.2 13.8 – 1.8 4.2 –

40.0 40.2 13.8 – 1.8 4.2 230

Nutrients, % of DM Dry matterb Crude protein NDF NFC Ether extract ME, MJ/kg

66.0 12.9 27.0 46.0 6.5 11.5

66.0 12.9 27.0 46.0 6.5 11.5

64.9 12.5 28.0 48.5 6.7 12.3

64.9 12.5 28.0 48.5 6.7 12.3

a Guaranteed content per kilogram of product is as follows: Ca, 235 g; P, 45 g; S, 23 g; Na, 80.18 g; Zn, 2.38 mg; Cu, 625 mg; Fe, 1.18 mg; Mn, 312 mg; Co, 32 mg; I, 41.6 mg; Se, 11.25 mg; vitamin A, 70,000 IU; vitamin D3, 5000 IU; vitamin E, 15 IU; and niacin, 3.33 mg. b As-is basis.

of Goering and Van Soest (1970), and the NDF in the concentrates was analyzed according to the procedure described by Van Soest, Robertson, & Lewis, (1991). The non-fibrous carbohydrates (NFC) and metabolizable energy were calculated according to the NRC (2001). The objective of the final body weight was around 500 kg and the animals were slaughtered at an average weight of 497 ± 18.7 kg (P N 0.05) by captive bolt and exsanguination, followed by hide removal and evisceration, without electrical stimulus. The carcasses were identified, washed, and divided into halves, which were individually weighed and then refrigerated at 2 °C for 24 h. 2.2. Tissue collection and meat analysis At 24 h post-mortem, six samples of the longissimus dorsi (LD) muscle from each animal, approximately 200 g, and 2.54 cm thick, were collected from the left side of the carcass from the muscle section at the 13th rib toward the head. These samples were stored at −20 °C until subsequent analysis. For determination of the chemical composition, one LD sample (sample-1; S1) was lyophilized to extract the muscle water. Crude protein was quantified by N analysis using the Kjeldahl method, EE was extracted by the Soxhlet method, moisture content was determined in an oven at 105 °C until a constant weight was reached, and ash was determined in a muffle furnace at 550 °C (AOAC, 1990). 2.3. Fatty acid extraction and gas chromatography analysis Lipids from the LD muscle and subcutaneous fat were extracted from other sample (S2; between 12 and 13th rib section) of the longissimus dorsi (LD) following the method described by Folch, Less, & Stanley, (1957) and methylated according to Hara and Radim (1978). The transmethylated samples were analyzed via gas chromatography (GC2010, Shimadzu, Kyoto, Japan) with a flame ionization detector and the following capillary column dimensions: 100 m × 0.25 mm × 0.20 μm (SP2560, Supelco, Bellefonte, PA). The chromatographic conditions were as follows: the initial column was incubated at 140 °C for 5 min, and the temperature increased by 4 °C/min until reaching 240 °C. Following this, a temperature of 240 °C was maintained for 30 min. The injector and detector were both maintained at 260 °C. Main fatty acids were identified by comparison of the retention times of methyl esters in the samples with standards of fatty acids from butter. Fatty acids were quantified by normalizing the areas of methyl esters. Fatty acid results were expressed as percentage of the area (%) obtained using ChromQuest 4.1 software (Thermo Electron, Milan, Italy). The Δ9 desaturase and elongase enzymatic activities were determined as described by Malau-Aduli, Siebert, Bottema, and Pitchford (1997), using mathematical indices. The atherogenicity index was calculated according to Ulbricht and Southgate (1991), as an indicator of the risk of cardiovascular disease. The calculations were performed as follows: Δ9 desaturase 16 index = 100 [(C16:1cis9) / (C16:1cis9 + C16:0)]; Δ9 desaturase 18 index = 100 [(C18:1cis9) / (C18:1cis9 + C18:0)]; elongase index = 100 [(C18:0 + C18:1cis9) / (C16:0 + C16:1cis9 + C18:0 + C18:1cis9)]; and atherogenicity index = [C12:0 + 4(C14:0) + C16:0] / ∑UFA. 2.4. Meat color Four samples of the LD muscle (S3–S6; between 11 and 12th rib section) were vacuum stored in polyethylene bags (estimated permeability to O2 of 5.5 × 1012 cm3 cm cm−2 s−1, at 30 °C) for determination of meat color at four aging times (0, 7, 14, and 21 days) at 2 °C. Each package was evacuated (−700 mm Hg) and sealed using a Selovac CV8 (São Paulo, Brazil) gas/vacuum packaging machine. Determination of the L*,

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a*, and b* color components during blooming was done after removing the filets from the packaging and exposing them to air for 30 min for oxygenation of myoglobin (Tapp III, Yancey, & Apple, 2011). Color was measured on the surface of the filets using the CIE L*a*b* system with illuminant D65 and 10° as the standard observing point. A Minolta CR-400 device (Konica Minolta, Osaka, Japan) was used for the color measurement and was calibrated with a white standard. The following color indices were used: L* is an index associated with luminosity (0 = black and 100 = white); a* is an index that varies from green (−) to red (+); and b* is an index that varies from blue (−) to yellow (+) (Houben, Van Dijk, Eikelenboom, & Hoving-Bolink, 2000). Six measurements were taken per slice, and the averages were used in the statistical analysis. Determinations of the chroma (C*) and hue angle (h*) were conducted according to the procedure described by MacDougall (1994), using the L*, a*, and b* obtained in the colorimetric determinations, with the following formulas: C* = [(a*)2 + (b*)2]0.5; and h* = arctan (b* / a*). 2.5. TBARS To analyze lipid stability, the content of thiobarbituric acid-reactive substances (TBARS) was determined using the acid precipitation technique described by Tarladgis, Watts, and Younathan (1960), with minor modifications. We used 50 g of LD (S3–S6) after vacuum aging at 2 °C for 0, 7, 14, and 21 days postmortem (after meat color reading). Of this amount, 10 g was produced, containing 8 g of meat and 2 g of fat. These samples were crushed in a multiprocessor, and 0.2 mL of BHT antioxidant (0.03%) and 50 mL of distilled water were added. The samples were then ground again and homogenized for 1 min. After homogenization, the samples were transferred to a 250-mL volumetric flask containing pieces of porcelain, which 50 mL of a 4 M HCl solution was added. Subsequently, the samples were distilled in a blanket heater at 100 °C, until 50 mL of the distillate was collected. From the distillate, 5 mL was transferred to a test tube, and 5 mL of 0.02 M thiobarbituric acid (TBA) was added. The test tubes remained in a boiling water bath for 35 min and cooled under tap water. Absorbance was measured at 530 nm with a spectrophotometer (Hitachi High Technologies America, Inc., Model U-2900, Pleasanton, USA). TBARS values were expressed as mg of malonaldehyde (MDA) per kg of meat plus fat using a standard curve prepared from 1,1,3,3 tetraethoxypropane (TEP). 2.6. Statistical analysis The LD muscle chemical composition, fatty acid profile, and enzyme activity indices were analyzed using the GLM procedure of the SAS 9.1 statistical software (SAS Inst. Inc., Cary, NC), considering the effects of oilseed, monensin addition and the interaction. Color and lipid oxidation were analyzed using the MIXED procedure of SAS 9.1, as suggested by Littel, Henry, and Ammerman (1998). For these characteristics the model included diet and aging period, along with the interaction, as fixed effects and animals as random effect. Animals within treatment were considered as random variable and aging period as a repeated measure in time. The Kenward–Roger option was used to adjust the degrees of freedom. The covariance structures tested were unstructured (UN), variance components (VC), autoregressive 1 (AR [1]), heterogeneous autoregressive (ARH [1]), Toeplitz (TOEP), heterogeneous compound symmetry (CSH), and heterogeneous Toeplitz (TOEPH). The covariance structure that resulted in the smallest Akaike information criterion of value was selected. 3. Results and discussion 3.1. Performance and chemical composition of meat The average daily gain of the animals was not affected by the diets (P N 0.11), as well as no effect of including monensin was detected on meat composition (P N 0.41) (Table 2). The amounts of fat in the muscle

599

Table 2 Daily gain and chemical composition of the LD muscle from young bulls fed ground soybean (SB), ground soybean + monensin (SBM), rumen protected fat from soybean oil (RPF), and rumen protected fat from soybean oil + monensin (RPFM). Attributes

Intake, kg Daily gain, kg Moisture, % Ash, % Protein, % Ether extract, %

Diets

SEM

P-value

SB

SBM

RPF

RPFM

L

M

L×M

11.56 1.71 74.94 1.19 20.41 2.09

11.51 1.67 75.19 118 20.46 1.74

11.47 1.83 73.97 1.14 21.03 2.27

11.07 1.83 73.95 1.13 20.67 2.37

– 0.11 b0.01 b0.01 0.02 0.10

– 0.81 0.72 0.59 0.41 0.60

– 0.81 0.68 0.99 0.27 0.35

0.08 0.32 0.01 0.18 0.25

L: effect of the lipid source; M: effect of monensin; L × M: interaction between lipid and monensin.

typically result from a balance between dietary energy and metabolic requirements of the animal (Oliveira et al., 2011). The use of monensin increases propionate production and decreases methane production, which makes the energy absorption through the rumen greater. Furthermore, because propionate is gluconeogenic, the greater production of this volatile fatty acid (VFA) could increase deposition of intramuscular fat, which uses a high proportion of glucose for the fatty acid synthesis, compared to subcutaneous adipose tissue (Gilbert, Lunt, Miller, & Smith, 2003). However, it is most probable that the increase of energy availability and propionate production, when ionophores are used, was not enough to increase intramuscular fat synthesis in this study. The meat from animals fed ground soybean diet showed greater moisture and ash contents than the meat from animals fed rumen protected fat (P b 0.01). However, the use of rumen protected fat increased the protein content (P = 0.02) and shows a tendency (P = 0.10) toward increased ether extract content in the meat. These results can be explained by the reduction in the LD muscle moisture. According to Olivo and Olivo (2006), the moisture and fat contents exhibit a negative correlation, i.e., when the fat content is greater, the moisture is less and vice versa. Despite exhibiting differences, the protein contents in the meat were within the expected range (Rotta et al., 2009). The literature suggests that the total protein content is less variable in bovine meat, with values of approximately 20% observed in the longissimus dorsi muscle without the fat cover, and this is independent of food, breed, the genetic group, and the physiological condition (Abrahão, Prado, Perotto, & Moletta, 2005; Marques et al., 2006; Menezes et al., 2006; Moreira, Souza, Matsushita, Prado, & Nascimento, 2003; Silva, Prado, Matsushita, & Souza, 2001).

3.2. Fatty acid profile The composition of the main fatty acids from the ground soybean grain and rumen protected fat used in the diets is presented in Table 3. It can be observed that ground soybean contains greater levels of linoleic acid (C18:2), α-linolenic acid (C18:3), and total PUFA compared to rumen protected fat. Otherwise, the rumen protected fat contains greater levels of palmitic (C16:0), stearic (C18:0), and oleic (C18:1) acids. Overall, monensin did not alter (P N 0.29) the fatty acid profile of the LD muscle from the animals fed ground soybean or rumen protected fat, as only the arachidonic acid content increased when the additive was used (Table 4). Therefore, although monensin alters the ruminal microorganisms, the dose used in this experiment may not have been sufficient to significantly alter ruminal biohydrogenation. The potential of ionophores to decrease biohydrogenation has been demonstrated in vitro by Van Nevel and Demeyer (1995). Monensin, for example, inhibited the growth of Butyrivibrio fibrisolvens, which resulted in an increased in proportion of cis-9, trans-11 C18:2 when incubated with C18:2 in vitro (Fellner, Sauer, & Kramer, 1995). Nonetheless, Martineau et al. (2008) reported that the supplementation with

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were found by Oliveira et al. (2012), who reported that rumen protected fat (MEGALAC-E®) produced meat with less CLA content compared to soybean oil. The increase in stearic acid concentration when the ground soybean diet was used could also be explained by larger exposure of UFA during ruminal biohydrogenation. In the diet of ruminants without added lipids, the major fatty acid component is linoleic acid; and stearic acid represents just around 2%. However, stearic acid is a large fraction of fatty acids arriving to the small intestine; while linoleic is a small portion, just over 10% (Duckett & Andrade, 2000). The ground soybean diet promoted greater linoleic acid concentrations in the LD muscle (P = 0.01). Despite the greater biohydrogenation of UFA in the rumen of animals fed oils or ground oilseeds, the greater linoleic acid content in soybean, compared to rumen protected fat (Table 3) may explain this result, as ruminal escape probably also occurred. According to Bauman, Perfield, Veth, and Lock (2003), for most diets, the extent of linoleic acid biohydrogenation would be between 70 and 95%. However, biohydrogenation could be lower in high concentrate diets; effects attributed to reduced ruminal pH and reduced lipolysis (Van Nevel & Demeyer, 1996). Biohydrogenation is also affected by an excess of non-protected lipids, i.e., using oils in the diet (Chilliard et al., 2007; Jenkins, Wallace, Moate, & Mosley, 2008; Lourenço, Ramos Morales, & Wallace, 2010). Another factor that may affect biohydrogenation is the processing of oilseeds (Oliveira et al., 2011; Peng, Brown, Wua, & Liu, 2010; Xu et al., 2006). In the work of Oliveira et al. (2012), linoleic acid content was greater when rumen protected fat were used, compared to soybean oil (not protected from rumen biohydrogenation). Therefore, the method of lipid supplementation is crucial for the manipulation of the fatty acid profile. In contrast to linoleic acid, the α-linolenic acid content was greater (P b 0.01) in the LD muscle of animals fed rumen protected fat, despite greater concentrations of this FA in soybean. This result could be explained by the lower biohydrogenation of the UFA from calcium salts and the greater extent of biohydrogenation that occurs with α-linolenic acid (85 to 100%), compared to linoleic acid (Bauman et al., 2003). Similar to what occurred in the LD muscle, monensin did not alter (P N 0.10) the proportion of most fatty acids analyzed in the subcutaneous fat (Table 5). Only the α-linolenic acid content was on average greater when the animals fed ionophores. There were some interactions (P b 0.04) in the fatty acid profile between lipid sources and monensin in the subcutaneous fat. When soybean grain was used, myristoleic, palmitoleic and heptadecenoic acids increased with monensin. On the other hand, the ionophore reduced the concentrations of these fatty acids when MEGALAC-E was the lipid

Table 3 Proportion (%) of the main fatty acids from lipid sources used in the diets. Fatty acids Myristic Palmitic Stearic Oleic Linoleic α-Linolenic ΣSFA ΣUFA ΣMUFA ΣPUFA

C14:0 C16:0 C18:0 C18:1 C18:2 c9–c12 C18:3 n3

Maize silage

Ground soybean

Rumen protected fat

1.42 21.80 12.86 24.31 31.12 4.29 37.46 61.35 25.94 35.41

0.21 12.00 4.20 21.99 49.64 5.13 19.38 79.81 23.31 56.51

0.94 19.48 6.62 27.11 31.03 2.02 27.04 60.56 27.11 33.45

monensin or lasalocid had no effect on in situ ruminal biohydrogenation of UFA at various times from 2 to 48 h of incubation. In agreement with our results, the monensin was not an effective method in increasing the cis-9, trans-11 C18:2 content in the intramuscular fat of the longissimus dorsi of Hanwoo steers (Song et al., 2010). Different from dairy cattle (Eifert et al., 2006; and Silva-Kazama et al., 2010), further studies are necessary to evaluate the effect of monensin on fatty acid profile of beef. Lipid sources did not affect the myristic or palmitic acid content in the LD muscle, although rumen protected fat contains greater concentrations of these fatty acids, particularly palmitic acid. These results have important implications for human health, because according to Woollett, Spady, & Dietschy, (1992), myristic and palmitic acids interfere with the normal function of LDL receptors in the liver, reducing LDL removal and increasing its concentration in plasma. Animals that received rumen protected fat exhibited greater oleic acid contents in the LD muscle, which can be explained by the partial protection of this lipid source from ruminal biohydrogenation. In the study of Fiorentini et al. (2012), the oleic acid concentrations in the muscle of the animals fed a diet containing 60% corn silage and 40% concentrate based on protected fat (MEGALAC-E®) were similar (38.7%) to those observed in this study. According to Gilmore et al. (2011), oleic acid helps to reduce plasma LDL and triglyceride concentrations, which is important for human health. Moreover, these authors concluded that consumption of beef with high MUFA content is likely or would increase plasma HDL concentrations. The CLA (cis-9, trans-11 C18:2) content was greater (15%) in the LD muscle of animals fed ground soybean (P = 0.03), which can be explained by a greater biohydrogenation of linoleic acid as a consequence of grinding the oilseed. Incomplete biohydrogenation of linoleic acid results in CLA (Harfoot & Hazlewood, 1997), which subsequently is absorbed in the small intestine and incorporated into fat. Similar results

Table 4 Proportion (%) of the main fatty acids present in the LD muscle of young bulls fed ground soybean (SB), ground soybean + monensin (SBM), rumen protected fat from soybean oil (RPF), and rumen protected fat from soybean oil + monensin (RPFM). Fatty acid

Lauric Myristic Myristoleic Pentadecanoic Palmitic Palmitoleic Margaric Heptadecenoic Stearic Oleic CLA Linoleic α-Linolenic Arachidonic

Diets

C12:0 C14:0 C14:1 c9 C15:0 C16:0 C16:1 c9 C17:0 C17:1 C18:0 C18:1 c9 C18:2 c9–t11 C18:2 c9–c12 C18:3 n3 C20:4 n6

SEM

SB

SBM

RPF

RPFM

0.29 2.02 0.32 1.73 20.92 2.33 0.70 1.62 16.40 33.97 0.76 10.77 0.31 0.03

0.26 2.36 0.31 1.52 23.63 3.24 0.74 1.36 14.87 34.98 0.68 9.54 0.32 0.04

0.39 2.55 0.56 1.74 23.73 3.54 0.54 0.94 12.42 37.68 0.60 7.32 0.49 0.05

0.34 2.27 0.48 1.71 22.68 3.27 0.54 1.02 13.06 38.48 0.61 7.70 0.47 0.08

L: effect of the lipid source; M: effect of monensin use; L × M: interaction between lipid and monensin.

0.06 0.18 0.05 0.19 1.02 0.31 0.05 0.10 0.63 1.33 0.05 1.02 0.02 0.01

P-value L

M

L×M

0.12 0.23 b0.01 0.57 0.35 0.04 b0.01 b0.01 b0.01 b0.01 0.03 0.01 b0.01 b0.01

0.52 0.89 0.41 0.52 0.40 0.29 0.71 0.34 0.47 0.48 0.42 0.66 0.83 0.02

0.83 0.09 0.58 0.62 0.06 0.06 0.67 0.10 0.08 0.93 0.40 0.42 0.56 0.34

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Table 5 Proportion (%) of the main fatty acids in the subcutaneous fat of young bulls fed ground soybean (SB), ground soybean + monensin (SBM), rumen protected fat from soybean oil (RPF), and rumen protected fat from soybean oil + monensin (RPFM). Fatty acid

Lauric Myristic Myristoleic Pentadecanoic Palmitic Palmitoleic Margaric Heptadecenoic Stearic Oleic CLA Linoleic α-Linolenic Arachidonic

Diets

C12:0 C14:0 C14:1 c9 C15:0 C16:0 C16:1 c9 C17:0 C17:1 C18:0 C18:1 c9 C18:2 c9–t11 C18:2 c9–c12 C18:3 n3 C20:4 n6

SEM

SB

SBM

RPF

RPFM

0.07 3.15 1.04 0.23 22.73 3.77 0.89 0.69 22.88 37.35 0.41 3.39 0.64 0.01

0.07 3.09 1.16 0.17 23.63 4.25 0.89 0.83 19.38 39.22 0.33 3.27 0.92 0.01

0.08 3.86 1.16 0.21 25.21 4.19 0.74 0.66 17.76 41.05 0.91 1.22 0.27 0.02

0.09 3.54 0.88 0.19 24.46 3.78 0.77 0.53 18.79 41.50 0.77 1.46 0.40 0.01

P-value

0.01 0.21 0.08 0.02 0.63 0.21 0.03 0.05 1.25 1.48 0.06 0.21 0.07 0.01

L

M

L×M

b0.01 b0.01 0.31 0.99 0.01 0.90 b0.01 b0.01 0.02 0.04 b0.01 b0.01 b0.01 0.01

0.67 0.38 0.36 0.18 0.90 0.85 0.64 0.91 0.31 0.42 0.10 0.80 0.02 0.30

0.52 0.54 0.01 0.36 0.18 0.04 0.61 0.01 0.06 0.62 0.62 0.39 0.27 0.98

L: effect of the lipid source; M: effect of monensin use; L × M: interaction between lipid and monensin.

source. Therefore, the monensin can be more effective to impair the last step of the biohydrogenation when the lipids are not protected. The ground soybean diet decreased the concentrations of lauric, myristic, and palmitic acids. In contrast, the stearic acid content increased when this oilseed was used. Therefore, these results demonstrate that diets containing ground soybean reduce the hypercholesterolemic SFA concentration of subcutaneous fat, compared to diets containing rumen protected fat. Stearic acid, according to Sinclair (1993), would have no effect because it can be converted into oleic acid in the organism without influencing the blood cholesterol levels. In addition, stearic acid is quite an important factor in terms of meat quality, because the cuts with greater concentrations of this fatty acid have been observed to achieve the highest scores in tasting panels (Mir, Paterson, & Mir, 2000). Similar to the LD muscle, the addition of rumen protected fat increased the oleic acid and CLA contents in subcutaneous fat. The concentration of CLA was 2.3 times greater when rumen protected fat was used (0.84 versus 0.37%), which can be explained by the greater index of the Δ9-desaturase 18 enzyme in this tissue, further the content of oleic acid in the rumen protected fat. The greater index of the Δ9-desaturase presented in Table 7 is consistent with this finding. The concentrations of the essential fatty acids, linoleic acids, and αlinolenic acids were greater in subcutaneous fat (P b 0.01) from animals fed the ground soybean diet. Even with the grinding process, the

soybean hull prevents some of the fatty acids from undergoing lipolysis and biohydrogenation, and some UFA absorption occurs in the small intestine. According to Duckett and Andrade (2000), fat protection can be the result of the formation of complexes between fatty acids and calcium salts, protection by protected protein, which makes the fat chemically unavailable to ruminal biohydrogenation, or oilseeds, which are physically protected from biohydrogenation by their coats. The rumen protected fat diet promoted greater concentrations of MUFA in the LD muscle (Table 6). However, the ground soybean diet manifested greater PUFA concentrations in the LD muscle and subcutaneous fat of the animals. Oliveira et al. (2011) reported that greater PUFA concentrations in the LD muscle and subcutaneous fat of animals fed soybean can be attributed to greater linoleic acid intake. The addition of ground soybean to the diet led to reduced omega-3 concentrations in the LD muscle, resulting in an omega-6:omega-3 ratio about two times greater than that in the LD muscle of animals fed rumen protected fat. Nonetheless, this ratio was not affected in subcutaneous fat, and the values in this tissue were lower than in the LD muscle. The diets influenced the desaturase 16 and 18 enzyme activity indices in the LD muscle, and only the desaturase 18 in the subcutaneous fat, with the greater index occurring when the animals fed rumen protected fat diet (Table 7). The greater palmitic and stearic acid contents in the

Table 6 Proportions (% of total lipid) of fatty acids in the LD muscle and subcutaneous fat of young bulls fed ground soybean (SB), ground soybean + monensin (SBM), rumen protected fat from soybean oil (RPF), and rumen protected fat from soybean oil + monensin (RPFM). Fatty acid

Diets

SEM

P-value

SB

SBM

RPF

RPFM

LD muscle ∑Saturated ∑Unsaturated ∑Monounsaturated ∑Polyunsaturated ∑UFA/∑SFA ∑Omega-3 ∑Omega-6 ∑Omega-6/∑Omega-3

43.22 54.65 40.95 13.70 1.27 0.38 10.80 28.42

44.41 54.49 42.40 12.09 1.22 0.36 9.58 26.61

42.80 56.12 45.44 10.68 1.31 0.56 7.38 13.18

43.62 55.04 46.02 9.02 1.28 0.54 7.78 14.41

1.30 0.71 1.36 1.06 0.04 0.02 1.02 3.84

0.17 0.05 b0.01 0.01 0.07 b0.01 001 b0.01

0.83 0.36 0.41 0.65 0.70 0.83 0.68 0.51

0.40 0.67 0.67 0.41 0.44 0.56 0.41 0.48

Subcutaneous fat ∑Saturated ∑Unsaturated ∑Monounsaturated ∑Polyunsaturated ∑UFA/∑SFA ∑Omega-3 ∑Omega-6 ∑Omega-6/∑Omega-3

50.79 48.35 43.87 4.48 0.96 0.64 3.40 5.32

48.08 51.03 46.49 4.54 1.06 0.92 3.28 3.57

48.73 50.51 48.07 2.44 1.04 0.27 1.24 4.61

48.70 50.34 47.72 2.62 1.03 0.40 1.47 3.68

1.57 1.58 1.61 0.21 0.06 0.07 0.21 0.83

0.63 0.61 0.08 b0.01 0.72 b0.01 b0.01 0.67

0.37 0.40 0.47 0.49 0.40 b0.01 0.81 0.21

0.37 0.36 0.34 0.72 0.37 0.44 0.39 0.25

L: effect of the lipid source; M: effect of monensin use; L × M: interaction between lipid and monensin.

L

M

L×M

602

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Table 7 Indices of enzymes involved in fatty acid metabolism and the atherogenicity index of LD muscle and subcutaneous fat of young bulls fed ground soybean (SB), ground soybean + monensin (SBM), rumen protected fat from soybean oil (RPF), and rumen protected fat from soybean oil + monensin (RPFM). Index

Diets

SEM

SB

SBM

RPF

RPFM

LD muscle Δ9 desaturase 16 Δ9 desaturase 18 Elongase Atherogenicity

10.01 67.36 68.38 0.584

11.55 70.14 65.25 0.660

12.96 75.08 64.67 0.670

12.65 74.68 66.46 0.616

Subcutaneous fat Δ9 desaturase 16 Δ9 desaturase 18 Elongase Atherogenicity

14.22 61.89 69.43 0.748

15.31 66.80 67.73 0.721

14.31 69.83 66.66 0.823

13.38 68.85 68.08 0.784

P-value L

M

L×M

0.60 1.03 1.17 0.02

b0.01 b0.01 0.27 0.23

0.29 0.23 0.55 060

0.11 0.11 0.03 0.04

0.73 2.12 0.82 0.02

0.19 0.01 0.13 b0.01

0.90 0.34 0.86 0.90

0.15 0.15 0.05 0.17

L: effect of the lipid source; M: effect of monensin use; L × M: interaction between lipid and monensin. Δ9 desaturase 16: 100 [(C16:1cis9) / (C16:1cis9 + C16:0)]. Δ9 desaturase 18: 100 [(C18:1cis9) / (C18:1cis9 + C18:0)]. Elongase: 100 [(C18:0 + C18:1cis9) / (C16:0 + C16:1cis9 + C18:0 + C18:1cis9)]. Atherogenicity: [C12:0 + 4(C14:0) + C16:0] / ∑UFA.

MEGALAC-E® indicate that this ingredient may have favored the gene expression of this enzyme in the LD muscle. However, the same phenomenon did not occur for the Δ9 desaturase 16 index in the subcutaneous fat. These results demonstrate the possible influence of nutrition on the expression of these enzymes, but further research is necessary to support this hypothesis. For atherogenicity index, an interaction between the lipid source and monensin was found in the LD muscle. Monensin reduced this index in the meat of animals fed diets containing rumen protected fat. In contrast, the opposite occurred in the LD muscle when ground soybean was used. Therefore, as discussed before, the ionophore was more effective to impair the biohydrogenation when the rumen protected fat was used, because the UFA of the ground soybean can reduce the biohydrogenation too (Lourenço et al., 2010; Maia et al., 2010). The subcutaneous fat of animals fed rumen protected fat also exhibited greater atherogenicity. 3.3. Oxidation of lipids The oxidation of lipids is one of the most important changes that occur during food storage because it can affect the color, aroma, flavor, texture, and even the nutritive value of the food (Fernández, PérezÁlvarez, & Fernández-López, 1997). The TBA method, and its different variations, is the most widely used test for measuring the extent of lipid oxidation in the muscle foods. The TBARS test quantifies the malonaldehyde levels, one of the main products of hydroperoxide decomposition formed during the oxidation processing of PUFA (Raharjo & Sofos, 1993), and allows the estimation of rancidity development in meat. There was no effect of monensin, interactions between monensin and aging, or triple interaction (P N 0.05) on the TBARS values at the different aging times (data not shown). However, in the present study, greater oxidation was observed in animals receiving ground soybean as a lipid source (P b 0.01) after 21 days of aging (Fig. 1). This greater oxidation possibly occurred due to the greater PUFA concentrations found in the LD muscle from these animals. According to Morrissey, Sheeny, Galvin, Kerry, & Buckley, (1998), although unsaturated lipids are desirable for human consumption, with increasing degrees of lipid unsaturation, their susceptibility to oxidation increases, which makes meat preservation more difficult. Wood et al. (2004) have reported that increased muscle linoleic and α-linolenic acid concentrations resulted in significant reductions in lipid stability levels after 10 days of storage, a shorter duration than that observed in this study. According to Djenane, Sánchez-Escalante, Beltrán, and Roncalés (2001), Trout (2003), and McKenna et al. (2005), the muscular rate of lipid oxidation may also act as an indicator of the degree of meat

pigment susceptibility to oxidation, bearing in mind the close relationship between these two oxidation processes. 3.4. Meat color The meat color data are discussed with regard to the effects of the interaction between the lipid source and aging time because the addition of monensin did not affect these characteristics (P N 0.10). For the L*, a* and b* values, similar behavior could be observed through aging (Fig. 2), and therefore, the changes in meat color are probably best represented by variations in lightness (L*) and chroma (C*). Chroma (C*) represents the color intensity and is a good indicator of blooming (i.e., myoglobin becomes oxygenated as oxygen is absorbed by the meat to form oxymyoglobin, a characteristically red pigment that causes the meat to turn from purple to the appropriate shade of red) in meat freshly exposed to the air. The color indices (L*, a* and C*) of LD muscle from the animals that consumed ground soybean exhibited a consistent decrease on the first seven days of aging. Contrary, in the LD muscle from the animals fed rumen protected fat, the color values increased during blooming until seven days of aging and then exhibited a significant drop. The decline of the meat color indices over the aging can be attributed to the oxidation

Fig. 1. Lipid oxidation (mg of malonaldehyde/kg of meat plus fat) after 0, 7, 14, and 21 days of aging in the meat from young bulls fed ground soybean (SB) or rumen protected fat (RPF). P values for the lipid source (L), monensin (M), and day (D): lipid P b 0.01; monensin P = 0.81; L × M P = 0.18; day P = 0.41; D × L P = 0.02; D × M P = 0.53; D × L × M P = 0.40.

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603

Fig. 2. Lightness index (L*), redness index (a*), yellowness index (b*), chroma index (C*), and hue index (h*) at 0, 7, 14, and 21 days of aging in the meat from young bulls fed ground soybean (SB) or rumen protected fat (PF).

of myoglobin (deoxymyoglobin or oxymyoglobin) to metmyoglobin, probably due to decreased metmyoglobin reducing activity (MRA), resulting in metmyoglobin accumulation in meat (Faustman & Cassens, 1990; McKenna et al., 2005). MRA is thought to prolong the color stability of muscles by reducing metmyoglobin to myoglobin. According to Gill and McGinnis (1995), MRA is clearly important for the stability of the color of meat that is temporarily exposed to low concentrations of oxygen in waste

gases from vacuum packaging. When fresh meat is vacuum packed, metmyoglobin formed in the muscle tissue before or during the absorption of residual oxygen by the tissue after packaging will be converted to deoxymyoglobin by MRA during storage. The subsequent oxygenation of myoglobin maintains the bright cherry-red color desired by consumers. However, with storage, NADH eventually becomes depleted and the rate of metmyoglobin formation begins to exceed the rate of its reduction to myoglobin.

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The observed increase in L*, a*, b* and, consequently, C* values on the seventh day in meat from the animals fed rumen protected fat can be attributed to increased formation of oxymyoglobin. However, this increase was not observed throughout storage in meat from animals fed ground soybean. This was probably due to the higher oxidation of the myoglobin pigment caused by the increased susceptibility to lipid oxidation of the LD muscle from these animals (Fig. 1), as they exhibited higher polyunsaturated fatty acid concentrations (Table 6). Radicals generated by lipid oxidation can promote the accumulation of metmyoglobin (Faustman, Sun, Mancini, & Suman, 2010; Mancini & Hunt, 2005; Trout, 2003), which can be reduced back to the oxygenbinding form while MRA is active. However, in the degradative, oxidative environment of muscle with free radicals formed in lipid oxidation, the NADH depletes faster, reducing MRA activity and leading to metmyoglobin accumulation. This fact is consistent with the statement that the effect of nutrition on meat color, especially the redness index (a*), is associated with the instability of heme pigments (Mancini & Hunt, 2005) in the secondary products (alpha- and beta-aldehydes) of lipid oxidation, causing the decreased stability of oxymyoglobin redox (Faustman, Liebler, McClure, & Sun, 1999; Lynch & Faustman, 2000). According to Zakrys, Hogan, O'Sullivan, Allen, and Kerry (2008), changes in the a* and the oxymyoglobin values appear to be driven by lipid oxidation and are strongly correlated with the TBARS values. Faustman and Cassens (1990) also reported a strong relationship between lipid oxidation and myoglobin oxidation; however, it is unclear whether the oxidation of myoglobin catalyzes lipid oxidation or vice-versa. In this study, although the TBARS index was higher in meat from animals fed the ground soybean diet after 21 days of aging, there were no differences (P N 0.05) in the color indices of this meat, compared to those from animals fed rumen protected fat at the same aging time. According to Faustman et al. (2010), the extent of lipid oxidation in meat is proportional to the concentration of oxygen present, and would be expected to be minimal in environments with low partial oxygen pressures (pO2). Thus, atmospheres containing very low concentrations of oxygen (such as vacuum packaging) provide conditions in which the oxidative interactions between lipids and myoglobin are not tightly linked. In contrast, because the TBARS index evaluates the secondary products (malonaldehyde) of lipid oxidation (Fernández et al., 1997; Raharjo & Sofos, 1993), higher values after 21 days of aging in meat from the ground soybean diet indicate a more advanced oxidative rancidity in these meats. These observations allow us to assume that the primary products (free radicals and hydroperoxides) generated during lipid oxidation act directly by oxidizing the pigment and/or indirectly by antagonizing the reducing systems (Faustman et al., 2010; McKenna et al., 2005) in meat from animals fed ground soybean, which explains the behavior of the color indices in this meat. 4. Conclusions Monensin, in the level used, had virtually no effect on the fatty acid profile or qualitative aspects of the longissimus dorsi muscle and subcutaneous fat. The inclusion of ground soybean grain in the diet increased the polyunsaturated fatty acid content in the longissimus dorsi muscle, compared to the longissimus dorsi muscle of animals fed rumen protected fat, which rendered it more susceptible to lipid oxidation. Changes in the fatty acid profile by the use ground soybean interfered with longissimus dorsi muscle color during aging, which caused it to appear darker and less red, than the muscle of animals fed rumen protected fat. References Abrahão, J. J. S., Prado, I. N., Perotto, D., & Moletta, J. L. (2005). Características de carcaças e da carne de tourinhos submetidos a dietas com diferentes níveis de substituição do milho por resíduo úmido da extração da fécula de mandioca. Revista Brasileira de Zootecnia, 34, 1640–1650.

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