- Email: [email protected]
Conjugated Linoleic Acid and Fish Oil in Laying Hen Diets: Effects on Egg Fatty Acids, Thiobarbituric Acid Reactive Substances, and Tocopherols During Storage
PROCESSING, PRODUCTS, AND FOOD SAFETY Conjugated Linoleic Acid and Fish Oil in Laying Hen Diets: Effects on Egg Fatty Acids, Thiobarbituric Acid Reactive Substances, and Tocopherols During Storage G. Cherian,*1 M. G. Traber,† M. P. Goeger,* and S. W. Leonard† *Department of Animal Sciences, and †Linus Pauling Institute, Oregon State University, Corvallis 97331
Key words: egg, tocopherol, conjugated linoleic acid, thiobarbituric acid reactive substance, n-3 fatty acid 2007 Poultry Science 86:953–958
provide positive health effects (Fritsche et al., 1999; Ip et al., 1999). Because Americans are opting for low-fat dairy and beef products and choosing more poultry products than beef, it is likely that the dietary contribution of CLA will further be reduced in a typical US diet. Similarly, consumption of n-3 fatty acids in the United States is 1.6 g/d (USDA, 2006). Although no official dietary recommendations have been made in the United States, nutritional scientists suggest including 2.9 g/d of n-3 (KrisEtherton et al., 2000). Therefore an additional 1.3 g/d of n-3 is needed in the current US diet. Increasing the concentration of CLA and n-3 fatty acids in poultry foods is a possible way for humans to increase their intake of these compounds and obtain potential health benefits of CLA and n-3 fatty acids. Dietary manipulation by feeding synthetic CLA oil has been the documented method in enriching eggs with CLA (Cherian, 2005). Feeding flax and marine oils to hens has been used to enrich eggs with n-3 fatty acids (Cherian, 2002; Rymer and Givens, 2005). Altering the n-3 and CLA content in eggs also increases the degree of unsaturation leading to changes in egg fatty acid quality and oxidative stability.
INTRODUCTION Recently, there has been a great interest in the role of foods for minimizing the risks of cardiovascular disease or ameliorating the progression of atherosclerosis. Such food or food ingredients that offer health-enhancing properties are called functional foods. Conjugated linoleic acids (CLA), n-3 fatty acids, and antioxidant vitamins (vitamin E) have received considerable attention as functional nutrients for their anticarcinogenic, antiatherogenic, hypocholesterolemic, immunomodulatory, and body fat reduction properties (Traber, 1999; Belury, 2002; Kris-Etherton et al., 2003; Terpstra, 2004). Dietary CLA is contributed by ruminant foods, and n-3 fatty acids are contributed through marine foods and oils from terrestrial sources such as flax. The consumption of CLA is less than 600 mg/d compared with the 3 g/d needed to
©2007 Poultry Science Association Inc. Received September 30, 2006. Accepted January 23, 2007. 1 Corresponding author: [email protected]
953
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
fatty acids were lower (>19%) in YG-CLA and YG-CLAFO compared with YG. Egg n-3 was highest in YG-FO eggs and lowest in YG eggs (P < 0.0001). Storage over 60 d led to a 20 and 67% depletion of CLA in the YG-CLA and YG-CLA-FO eggs (P < 0.0001). A 29% reduction was observed in the total n-3 fatty acid content of YG-CLAFO eggs at d 60 of storage when compared with d 0 of storage (P < 0.0001). Diet and storage increased TBARS (P < 0.0001), which was highest in YG-CLA eggs at 60 d of storage. The YG-CLA and YG-CLA-FO diets reduced α and γ-tocopherol contents at all days of storage compared with YG eggs (P < 0.05). Regardless of diet, egg storage for 40 d or longer depleted egg tocopherol contents (P < 0.05). These data demonstrate that healthy eggs with increased n-3 fatty acids and CLA can be generated by minor diet modifications, but added tocopherol supplementation may be needed to reduce lipid peroxidation when n-3 or CLA is included in the hen diet.
ABSTRACT The effects of incorporating conjugated linoleic acid (CLA) and fish oil in laying hen diets on egg CLA, n-3 fatty acid, tocopherol, and TBA reactive substances (TBARS) during 60 d of storage were investigated. Hens were fed corn-soybean meal-based diets containing 3% yellow grease (YG), 2.75% yellow grease + 0.25% CLA (YG-CLA), 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (YG-CLA-FO), or 2.75% yellow grease + 0.25% fish oil (YG-FO). Eggs were collected and stored at 4°C up to 60 d. On storage d 0, 20, 40, and 60, eggs (n = 8) from each treatment were selected randomly, and tocopherol and TBARS contents were measured. Egg total lipid and fatty acids were determined on d 0 and 60 of storage. Feeding YG-CLA-FO led to a 5.4 and 7.7% reduction in egg total lipids on d 0 and 60 (P < 0.05) when compared with YG eggs. The YG-CLA and YG-CLA-FO diets led to a 12% increase in egg saturated fatty acids compared with YG eggs. The content of monounsaturated
954
CHERIAN ET AL. Table 1. Tocopherol content and fatty acid composition of the laying hen diet Dietary treatments1 Item
YG-CLA
YG-CLA-FO
YG-FO
19.1 ± 0.09 5.7 ± 0.33 24.8 ± 0.37
17.3 ± 0.31 4.3 ± 0.07 21.6 ± 0.30
18.2 ± 0.02 4.3 ± 0.16 22.5 ± 0.17
17.7 ± 0.33 4.1 ± 0.12 21.7 ± 0.44
16.2 4.9 34.5 37.7 0.0 0.0 3.1 0.0 0.0 23.7 35.5 3.1 0.0
± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.35 1.69 1.65 0.00 0.00 0.29 0.00 0.00 0.26 2.19 0.29 0.00
15.5 4.5 32.0 35.7 1.0 0.7 3.1 0.0 0.0 24.5 32.9 3.1 1.7
± ± ± ± ± ± ± ± ± ± ± ± ±
0.29 0.21 0.71 1.21 0.05 0.00 0.20 0.00 0.00 0.71 1.10 0.20 0.05
16.3 4.9 32.7 34.5 0.8 0.7 2.9 0.5 0.4 23.8 34.8 3.8 1.5
± ± ± ± ± ± ± ± ± ± ± ± ±
0.05 0.74 2.68 2.33 0.14 0.17 0.37 0.02 0.01 0.08 2.94 0.38 0.31
15.9 4.5 31.4 36.4 0.0 0.0 2.6 0.6 0.4 26.9 33.1 3.6 0.0
± ± ± ± ± ± ± ± ± ± ± ± ±
0.61 0.06 0.27 1.56 0.00 0.01 0.27 0.01 0.01 1.83 0.26 0.28 0.00
1 YG = 3.0% yellow grease;YG-CLA= 2.75% yellow grease + 0.25 conjugated linoleic acid (CLA); YG-CLAFO = 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (FO); YG-FO = 2.75% yellow grease +0.25% FO. Means ± SE of 3 replicate feed samples.
Consumer acceptance of eggs depends upon storage stability and nutritional quality. The effect of feeding n-3 and CLA to hens on egg CLA, n-3 fatty acid, tocopherols, and lipid peroxidation products during 60 d of storage was investigated. The length of storage times was selected to simulate likely duration of consumer storage of shell eggs.
maintained on a 16L:8D photoperiod and standard conditions of temperature and ventilation as per Oregon State University Poultry Farm standard operating procedures. Water and feed were provided ad libitum. Feed intake was measured on a weekly basis. The experiment was conducted for 320 d.
Egg Collection MATERIALS AND METHODS These experiments were approved by the Oregon State University Animal Care and Use Committee to ensure adherence to animal care guidelines.
Diets Isocaloric (2,900 kcal/kg of feed) and isonitrogenous (16% CP) corn-soybean meal-based experimental rations were formulated with 3% yellow grease (YG), 2.75% yellow grease + 0.25% CLA (YG-CLA), 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (YG-CLA-FO), or 2.75% yellow grease + 0.25% fish oil (YG-FO). The composition of the diet is shown in Table 1. Menhaden fish oil was used as a source of n-3 fatty acid (Omega Protein Inc., Reedville, VA). The CLA oil containing 75% free fatty acids was made up of equal amounts of cis9, trans11, and trans10, cis12 CLA isomers (Pharmanutrients, Lake Bluff, IL). The diets were mixed weekly and were stored in a cold room (4°C) in airtight containers.
Birds A total of 112 twenty-week-old Single Comb White Leghorn laying hens were weighed and distributed randomly to the experimental diets (4 replications of 7 birds each). The birds were kept individually in cages and were
A total of 128 eggs were collected (32/diet, 8 eggs/ replicate) during the peak production period (wk 30 to 31) and were kept in fiber flats at 4°C. Length of storage times was selected to simulate likely duration of consumer storage of shell eggs. On d 0, 20, 40, and 60 of storage, 2 eggs were selected randomly from each replicate, totaling 8 eggs/treatment for tocopherol and lipid peroxidation assays as measured by TBA reactive substances (TBARS). Total lipids and fatty acids were determined for eggs stored for 0 and 60 d.
Lipid Analyses Total lipids were extracted by the method of Folch et al. (1957). One gram of yolk sample was weighed into a screw-capped test tube with 20 mL of chloroform:methanol (2:1, vol/vol) and homogenized with a polytron homogenizer (PT10/35, Brinkman Instruments, Westbury, NY) for 15 to 20 s at high speed. After an overnight incubation at 4°C, the homogenate was filtered through Whatman no. 1 filter paper into a 100-mL graduated cylinder, and 4 mL of 0.88% NaCl solution was added and mixed. After phase separation, the volume of lipid layer was recorded, and the top layer was completely siphoned off. Total lipids were determined gravimetrically. Analysis of fatty acid composition was performed with a Agilent 6890 gas chromatograph (Agilent Technologies
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
Tocopherols (mg/kg) α-Tocopherol γ-Tocopherol Σ Tocopherols Fatty acids (% of total fatty acids) 16:0 18:0 18:1 18:2 n-6 cis9, trans11 CLA trans10, cis12 CLA 18:3n-3 20:5n-3 22:6n-3 Σ Saturates Σ Monounsaturates Σ Total n-3 Σ CLA
YG
955
CONJUGATED LINOLEIC ACID AND FISH OIL Table 2. Effect of dietary conjugated linoleic and fish oil on the total lipids and fatty acid content of eggs during storage Dietary treatments1 d 0 of storage Items2
YG
YG-CLA
YG-CLA-FO
YG-FO
YG
YG-CLA
YG-CLA-FO
YG-FO
Diet
Storage
Diet × storage
31.2a 0.4 27.8b 3.4a 9.3b 40.4b 15.4b 0.2b 0.0 2.2a 0.8b 37.6b 43.8b 17.6b 1.3b 0.0b
30.3ab 0.5 29.9a 1.8b 12.8a 33.4c 17.5a 0.4ab 0.5a 2.2a 0.7b 43.2a 35.3c 19.8a 1.4b 0.6a
29.5b 0.5 29.2a 2.0b 12.3a 33.3c 16.5ab 0.4a 0.6a 1.8b 1.5a 42.1a 35.4c 18.2ab 2.4a 0.6a
30.0b 0.6 27.8b 3.8a 8.6b 42.1a 13.5c 0.4ab 0.1b 1.6b 1.4a 37.1b 45.9a 15.2c 2.2a 0.1b
27.3a 0.1 24.1c 3.2b 9.5b 44.9a 15.0c 0.6bc 0.0b 1.8 0.7c 33.7b 48.7a 16.8c 0.8b 0.0b
26.5a 0.2 25.6ab 2.1d 13.2a 36.6b 18.5a 1.0a 0.4a 1.8 0.6c 39.0a 39.7b 20.3a 1.4a 0.4a
25.2b 0.1 26.6a 2.4c 12.7a 37.5b 16.8b 0.8ab 0.2ab 1.6 1.2b 39.5a 40.7b 18.3b 1.7a 0.2ab
27.1a 0.1 25.0bc 3.5a 9.5b 44.6a 13.7d 0.4c 0.0b 1.6 1.5a 34.6b 48.6a 15.2d 1.5a 0.0a
0.0541 0.7248 0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0037 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
0.1059 <0.0001 <0.0001 0.6044 0.0950 <0.0001 0.4249 0.0002 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.8790 <0.0001 <0.0068
0.0110 0.2385 0.2704 0.1194 0.8166 0.2160 0.5099 0.0396 0.0510 <0.0001 <0.0001 0.4705 0.0848 0.5508 0.0914 0.1583
Means significantly different within each day of storage (P < 0.05). YG = 3.0% yellow grease; YG-CLA = 2.75% yellow grease + 0.25 conjugated linoleic acid (CLA); YG-CLA-FO = 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (FO); YG-FO = 2.75% yellow grease + 0.25% FO. 2 SFA = saturated fatty acids; MUFA = monounsaturated fatty acids. a–d 1
Inc., Palo Alto, CA) equipped with an autosampler, flame ionization detector and fused silica capillary column, 100 m × 0.25 mm × 0.2 m film thickness (Sp-2560, Supelco, Bellefonte, PA). Sample (1 L) was injected with He as a carrier gas onto the column programmed for ramped oven temperatures (initial temperature was 110°C, held for 1 min, then ramped at 15°C/min to 190°C and held for 55 min, then ramped at 5°C/min to 230°C and held for 5 min). Inlet and detector temperatures were both 220°C. Peak areas and percentages were calculated using Agilent ChemStation software. Fatty acid methyl esters were identified by comparison with retention times of authentic standards (Matreya, Pleasant Gap, PA). Fatty acid values and total lipids were expressed as weight percentages.
Tocopherol Assay For analysis of egg yolk α- and γ-tocopherols, a modification of the method by Podda et al. (1996) was used. Briefly, a known weight (∼50 mg) of individual yolk sample (n = 8) was saponified with alcoholic KOH, extracted with hexane, dried under N, resuspended in 1:1 ethanol:methanol, then injected into an HPLC system (Shimadzu, Columbia, MD). The HPLC system consisted of a Shimadzu LC-10ADVP controller and a SIL-10ADVP auto injector with a 50-L sample loop. Tocopherols were detected with a LC-4B amperometric electrochemical detector (Bioanalytical Systems Inc., West Lafayette, IN) with a glassy C working electrode and silver chloride reference electrode. The column used was a Waters Symmetry Shield RP18 column, 100 × 4.6 mm, 3.5-m particle size with a Waters Symmetry Shield RP18 precolumn, 20 × 3.9 mm, 3.5 m (Waters Inc., Watford, Hertfordshire, UK). An isocratic mobile phase delivery system [99:1 (vol/vol) methanol:water containing 0.1% (wt/vol) lithium perchlorate] was used, with a total run time of 6 min.
The electrochemical detector was in the oxidizing mode, potential 500 mV, full recorder scale at 500 nA. Peak areas were integrated using a Shimadzu Scientific 4.2 Class VP software package, and α- and γ-tocopherols were individually quantitated using authentic standards.
TBARS Assay Egg yolk samples (2 g) were weighed into 50-mL test tubes, and 18 mL of 3.86% perchloric acid was added. The samples were homogenized with a polytron homogenizer for 15 s. Butylated hydroxytoluene (50 L, in 4.5% ethanol) was added to each sample during homogenization to control lipid oxidation. The homogenate was filtered through Whatman no. 1 filter paper. Filtrate (2 mL) was mixed with 2 mL of 20 mM TBA in distilled water and incubated in the dark at room temperature for 15 to 17 h. Absorbance was determined at 531 nm. The TBARS were expressed as milligrams of malondialdehyde/gram of yolk (Cherian et al., 1996a).
Statistical Analysis A 2-way ANOVA was used to analyze effects of diet and storage on egg tocopherols, TBARS, fatty acids, and total lipids using eggs within diet by storage as the error term (SAS Institute, 2001). Diet and storage were the fixed effects. Significant differences among treatment means were analyzed by the Student-Newman-Keuls multiple range test at P < 0.05 (Steel and Torrie, 1980). Means of interaction were analyzed by comparing the storage time separately for each diet by the Student-Newman-Keuls multiple range test. Computations were done using the GLM procedure of SAS Institute (2001). Mean values are reported.
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
Σ Lipids 14:0 16:0 16:1 18:0 18:1 18:2 18:3 Cis9, trans11 CLA 20:4 22:6 Σ SFA Σ MUFA Σ n-6 Σ n-3 Σ CLA
P-value
d 60 of storage
956
CHERIAN ET AL. Table 3. Effect of dietary conjugated linoleic acid and fish oil feeding on the α-, γ-, and total tocopherol content of egg yolk during storage Dietary treatments1 Tocopherol (nmol/g)
YG-CLA
YG-CLA-FO
YG-FO
78.0 ± 5.89a 2.4 ± 0.29a 80.7 ± 6.10a
56.6 ± .36b 1.2 ± 0.14b 57.9 ± 5.44b
62.7 ± 2.96b 1.2 ± 0.15b 63.9 ± 2.94b
67.0 ± 2.53ab 1.4 ± 0.15b 68.4 ± 2.60ab
88.1 ± 5.95a 2.1 ± 0.07a 90.2 ± 5.99a
65.3 ± 1.36bc 1.1 ± 0.12b 66.4 ± 1.32bc
62.0 ± 5.13c 1.2 ± 0.16b 63.2 ± 5.21c
74.9 ± 2.79b 1.1 ± 0.07b 76.0 ± 2.80b
67.7 ± 2.85a 2.8 ± 0.09a 70.5 ± 2.86a
49.5 ± 4.00b 1.7 ± 0.13b 51.2 ± 4.10b
49.0 ± 3.34b 1.5 ± 0.08b 50.5 ± 3.35b
53.6 ± 1.69b 1.4 ± 0.11b 54.9 ± 1.63b
58.7 ± 3.63a 2.4 ± 0.13a 61.1 ± 3.73a
45.9 ± 4.02b 1.4 ± 0.08b 47.3 ± 4.04b
46.6 ± 4.55b 1.5 ± 0.11b 48.1 ± 4.61b
52.5 ± 3.91ab 1.4 ± 0.13b 53.9 ± 3.99ab
ANOVA Diet α-Tocopherol γ-Tocopherol Σ Tocopherols Storage α-Tocopherol γ-Tocopherol Σ Tocopherols Diet × storage α-Tocopherol γ-Tocopherol Σ Tocopherols
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.7845 0.6296 0.7888
Means significantly different for each dietary treatment (P < 0.05). YG = 3.0% yellow grease; YG-CLA = 2.75% yellow grease + 0.25 conjugated linoleic acid (CLA); YG-CLAFO = 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (FO); YG-FO = 2.75% yellow grease + 0.25% FO. a–c 1
RESULTS AND DISCUSSION The fatty acid composition and tocopherol contents of the diet are shown in Table 1. Conjugated linoleic acid was present only in the CLA-supplemented diets (YGCLA and YG-CLA-FO) and consisted of cis9, trans11, and trans10, cis12 isomers at 1.0, 0.8, 0.7, and 0.7, respectively. Inclusion of fish oil resulted in the incorporation of longchain n-3 fatty acids, such as eicosapentaenoic acid (20:5n3) and docosahexaenoic acid (22:6 n-3) in YG-CLA-FO and YG-FO diets. α-Linolenic acid (18:3 n-3) was the only source of n-3 fatty acids in the YG and YG-CLA diets. αTocopherol was the predominant form of tocopherol in the diet (17.3 to 19.1 mg/kg), followed by γ-tocopherol (4.1 to 5.7 mg/kg). The total tocopherol content of the diet varied from 21.6 to 24.8 mg/kg. The total lipid content of the diet was 3%.
Diet Effects on Yolk Fatty Acids The egg total lipid content and fatty acid composition at storage d 0 and 60 is shown in Table 2. Egg total lipids were affected by dietary oils (P < 0.05). Feeding YG-CLAFO led to a 5.4% reduction in total lipids when compared with YG eggs. The yolk fatty acid profile clearly reflected the dietary fatty acid composition. Conjugated linoleic
acid was present only in eggs from YG-CLA and YGCLA-FO (P < 0.05). The only isomer detected in egg yolk was cis9, trans11. A significant effect of diet was observed on egg saturated fatty acids (SFA; 16:0, 18:0; Table 2). Addition of 0.25% CLA resulted in a significant increase in SFA with a concomitant reduction in monounsaturated fatty acids (MUFA). The increase in SFA in the YG-CLA eggs was over 12% higher than YG eggs. The content of MUFA was over 19% lower in YG-CLA eggs than YG eggs. These results also corroborate our previous reported results and also of others (Du et al., 1999; Cherian et al., 2002). Inclusion of fish oil along with CLA did not overcome the MUFA-suppressing effect of CLA in YG-CLA-FO eggs. The ⌬9-desaturase enzyme is responsible for the conversion of stearic acid (18:0) to oleic acid (18:1). Dietary CLA may have an inhibitory action on desaturases, thereby leading to the reduction of MUFA. A recent study also reported a decrease in mRNA expression of stearoyl coenzyme A in CLA-fed rats affecting the synthesis of MUFA and accumulation of SFA (Choi et al., 2000). Higher levels of SFA in yolk lipids due to CLA supplementation may be of concern, because increased consumption of SFA, especially 14:0 and 16:0, is associated with increasing plasma total and low-density lipoprotein cholesterol concentrations in blood plasma and changes related to car-
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
d0 α-Tocopherol γ-Tocopherol Σ Tocopherols d 20 α-Tocopherol γ-Tocopherol Σ Tocopherols d 40 α-Tocopherol γ-Tocopherol Σ Tocopherols d 60 α-Tocopherol γ-Tocopherol Σ Tocopherols
YG
CONJUGATED LINOLEIC ACID AND FISH OIL
Storage Effects on Yolk Fatty Acids Irrespective of the lipid source, storage reduced fatty acids in eggs (Table 2). Total lipids were lowest in the YG-CLA-FO eggs stored for 60 d compared with other eggs (P < 0.05). A significant decrease due to storage was observed for all fatty acids except 16:1, 18:0, and 18:2 n6. Storage over 60 d led to a 20 and 67% depletion of CLA in the YG-CLA and YG-CLA-FO eggs (P < 0.0001). The presence of long-chain n-3 fatty acids in the YG-CLAFO eggs enhanced the depletion of CLA when compared with YG-CLA eggs. A 29% reduction was observed in the total n-3 fatty acid content of YG-CLA-FO eggs at d 60 of storage when compared with d 0 of storage (P < 0.0001). The decrease in total lipids, CLA, and n-3 fatty acids may suggest a degradation of egg lipids and polyunsaturated fatty acids (PUFA) during storage. In addition, oxidative stability of eggs based on TBARS revealed a significant effect of diet and storage (Figure 1). At d 0, the eggs from the YG-CLA-FO and YG-FO regimen had higher TBARS values (P < 0.05) than those from YG or YG-CLA, suggesting that the onset of lipid oxidation may be enhanced in these eggs due to the high content of longchain (>20-C) PUFA. However, during storage, accumulation of TBARS was higher in YG-CLA than all the other treatments. We have previously reported that CLA incorporation is preferentially in the egg triglycerides (Cherian, 2005) compared with long-chain n-3 PUFA in the phospholipids (Cherian and Sim, 1992). In the egg yolk, phospholipids and protein are interwoven in the exterior sur-
Figure 1. Effect of dietary conjugated linoleic and fish oil feeding on the TBA reactive substances in egg yolk during storage. The values reported are in milligrams of malondialdehyde per gram of yolk. YG = 3.0% yellow grease; YG-CLA = 2.75% yellow grease + 0.25 conjugated linoleic acid (CLA); YG-CLA-FO = 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (FO); YG-FO = 2.75% yellow grease + 0.25% FO. a,bLetters above a day indicate a significant difference (P < 0.05).
face of low-density lipoprotein. This compact surface layer can partly exclude O2 from the lipid core of the particle (Burley and Vadehra, 1989), thus impeding oxidation. Thus, the structural configuration of yolk phospholipids may have aided in preventing oxidation of longchain PUFA in YG-CLA-FO or YG-FO eggs. The results of TBARS further attests to the tocopherol content of eggs (Table 3). Diet and storage reduced the tocopherol content of eggs (P < 0.0001). Eggs from YGCLA and YG-CLA-FO had reduced α and γ- tocopherol contents at all days of storage compared with YG eggs (P < 0.05). In a previous study, we reported that the inclusion of tocopherols resulted in a significant reduction (P < 0.05) in TBARS in the egg yolk from hens fed menhaden fish oil during storage (Cherian et al., 1996b). Therefore, it appears that the longer-chain n-3 PUFA in fish oil-fed eggs were protected from undergoing deterioration by tocopherol supplementation. Regardless of dietary oils, egg storage for 40 d or longer depleted tocopherol contents promoting lipid oxidation and the accumulation of TBARS. Thus, incorporating tocopherols into chicken eggs may increase the oxidative stability and also provide a source of tocopherols for the human diet. Several authors have reported CLA enrichment through diet manipulation (Cherian, 2005). However, no study has reported the effect of CLA enrichment on egg tocopherol content and lipid oxidation products during storage. These results from the current study demonstrate that healthy eggs with increased n-3 fatty acids and CLA can be generated by minor diet modifications, and added tocopherol supplementation may be needed when n-3 or CLA is included in the hen diet to maintain product quality and nutritional value.
ACKNOWLEDGMENTS The Ott professorship awarded to G. Cherian is acknowledged. The CLA used in this study was supplied
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
diovascular disease (Bonanome and Grundy, 1988). However, if the antiatherogenic activity of CLA found in rabbits (Lee et al., 1994), hamsters (Nicolosi et al., 1997), and mice (Munday et al., 1999) could be extrapolated to humans, the adverse consequences of increased SFA levels could be counteracted by CLA. A significant increase in linoleic acid was observed in YG-CLA eggs (P < 0.0001). However, CLA along with fish oil led to a reduction in arachidonic acid content in YG-CLA-FO eggs. ⌬6-Desaturase enzyme is involved in the formation of arachidonic acid (20:4 n-6) from linoleic acid (18:2 n-6). The increase in linoleic acid observed in the current study (YG-CLA) may suggest an inhibitory effect of CLA on ⌬6-desaturase leading to its accumulation. Higher dietary CLA (>0.5%) has been reported to reduce egg arachidonic acid (Cherian et al., 2002). Higher dietary CLA has also been reported to cause rubbery and hard egg yolks upon cooking (Cherian, 2005). Low levels of CLA were used in the current study to minimize such negative yolk textural properties. Fish oil supplementation increased n-3 fatty acids and caused a reduction in n-6 fatty acids such as linoleic and arachidonic acid. The increase in n-3 fatty acids with a concomitant decrease in arachidonic acid by fish oil was consistent with our previous results (Cherian and Sim, 1991). The presence of CLA did not affect the content of n-3 fatty acids in YGCLA-FO eggs.
957
958
CHERIAN ET AL.
by Pharmanutrients. The generous donation of menhaden oil from Omega Protein Inc. is appreciated.
REFERENCES
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
Belury, M. A. 2002. Dietary conjugated linoleic acid in health: Physiological effects and mechanisms of action. Annu. Rev. Nutr. 22:505–531. Bonanome, A., and S. M. Grundy. 1988. Effect of dietary stearic acid on plasma cholesterol and lipoprotein. N. Engl. J. Med. 318:1244–1248. Burley, R. W., and D. V. Vadehra. 1989. Egg yolk: Structure and properties. Pages 171–233 in The Avian Egg. R. W. Burley and D. V. Vadehra, ed. John Wiley and Sons, New York, NY. Cherian, G. 2002. Lipid modification strategies and nutritionally functional poultry foods. Pages 77–92 in Food Science and Product Technology. T. Nakano and L. Ozimek, ed. Res. Signpost, Trivandrum, India. Cherian, G. 2005. Conjugated linoleic acid (CLA) for health: Chicken eggs as alternative sources of CLA. Pages 49–64 in The Amazing Egg: Nature’s Perfect Functional Food for Health Promotion. J. S. Sim and H. H. Sunwoo, ed. Univ. Alberta, Edmonton, Canada. Cherian, G., T. B. Holsonbake, M. P. Goeger, and R. Bildfell. 2002. Dietary conjugated linoleic acid alters yolk and tissue fatty acid composition and hepatic histopathology of laying hens. Lipids 37:751–757. Cherian, G., and J. S. Sim. 1991. Effect of feeding full fat flax and canola seeds to laying hens on the fatty acid composition of eggs, embryos, and newly hatched chicks. Poult. Sci. 70:917–922. Cherian, G., and J. S. Sim. 1992. Preferential accumulation of n3 fatty acids in the brain tissue of chicks from n-3 fatty acid enriched eggs. Poult. Sci. 71:1658–1668. Cherian, G., F. H. Wolfe, and J. S. Sim. 1996a. Dietary oils with added tocopherols: Effects on egg or tissue tocopherols, fatty acids and oxidative stability. Poult. Sci. 75:423–432. Cherian, G., F. H. Wolfe, and J. S. Sim. 1996b. Dietary oils with tocopherols: Effects on internal qualities of eggs during storage. J. Food Sci. 61:15–18. Choi, Y., Y. Kim, Y. Han, Y. Park, M. W. Pariza, and J. M. Ntambi. 2000. The trans-10, cis-2 isomer of conjugated linoleic acid downregulates stearoyl-CoA desaturase 1 gene expression in 3T3-L1 adipocytes. J. Nutr. 130:1920–1924. Du, M., D. U. Ahn, and J. L. Sell. 1999. Effect of dietary conjugated linoleic acid on the composition of egg yolk lipids. Poult. Sci. 7:1639–1645.
Folch, J., M. Lees, and G. H. Sloane-Stanely. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497–507. Fritsche, J., R. Rickert, H. Steinhart, M. P. Yurawecz, M. M. Mossaba, N. Sehat, J. A. Roach, J. K. Kramer, and Y. Ku. 1999. Conjugated linoleic acid (CLA) isomers: Formation, analysis, amounts in foods and dietary intake. Fett-Lipid 101:272–276. Ip, C., S. Banni, E. Angioni, G. Carta, J. McGinley, H. J. Thompson, D. Barbano, and D. E. Bauman. 1999. Conjugated linoleic acid-enriched butterfat alters mammary gland morphogenesis and reduces cancer risk in rats. J. Nutr. 129:2135–2142. Kris-Etherton, P. M., W. S. Harris, and L. J. Appel. 2003. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Arterioscler. Thromb. Vasc. Biol. 23:20–30. Kris-Etherton, P. M., D. S. Taylor, S. Yu-Poth, P. Huth, K. Moriarty, V. Fishell, and R. L. Hargrove. 2000. The polyunsaturated fatty acids in the food chain in the United States. Am. J. Clin. Nutr. 71:179S–188S. Lee, K. N., D. Kritchevevsky, and M. W. Pariza. 1994. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 108:19–25. Munday, J. S., K. G. Thompson, and K. A. James. 1999. Dietary conjugated linoleic acids promote fatty streak formation in the C57BL/6 mouse atherosclerosis model. Br. J. Nutr. 81:251–255. Nicolosi, R. J., E. J. Rogers, D. Kritchevsky, J. A. Scimeca, and P. J. Huth. 1997. Dietary conjugated linoleic acid reduces plasma lipoproteins and early atherosclerosis in hypercholesterolemic hamsters. Artery 22:266–277. Podda, M., C. Weber, M. G. Traber, and L. Packer. 1996. Simultaneous determination of tissue tocopherols, tocotrienols, ubiquinols and ubiquinones. J. Lipid Res. 37:893–901. Rymer, C., and D. I. Givens. 2005. n-3 Fatty acid enrichment of edible tissue of poultry: A review. Lipids 40:121–130. SAS Institute. 2001. SAS User’s Guide: Statistics, Release 8.2. SAS Inst. Inc, Cary, NC. Steel, R. G. D., and J. H. Torrie. 1980. Principles and procedures of statistics: A biometrical approach. 2nd ed. McGraw-Hill Book Co., Toronto, Canada. Terpstra, A. H. 2004. Effect of conjugated linoleic acid on body composition and plasma lipids in humans: An overview of the literature. J. Clin. Nutr. 79:352–361. Traber, M. G. 1999. Vitamin E. Pages 347–362 in Nutrition in Health and Disease. 9th ed. M. Shils, J. A. Olson, M. Shike, and A. C. Ross, ed. Williams & Wilkins, Baltimore, MD. USDA. 2006. Food consumption and nutrient intake. http:// www.ers.usda.gov Accessed Jul. 2006.
Key words: egg, tocopherol, conjugated linoleic acid, thiobarbituric acid reactive substance, n-3 fatty acid 2007 Poultry Science 86:953–958
provide positive health effects (Fritsche et al., 1999; Ip et al., 1999). Because Americans are opting for low-fat dairy and beef products and choosing more poultry products than beef, it is likely that the dietary contribution of CLA will further be reduced in a typical US diet. Similarly, consumption of n-3 fatty acids in the United States is 1.6 g/d (USDA, 2006). Although no official dietary recommendations have been made in the United States, nutritional scientists suggest including 2.9 g/d of n-3 (KrisEtherton et al., 2000). Therefore an additional 1.3 g/d of n-3 is needed in the current US diet. Increasing the concentration of CLA and n-3 fatty acids in poultry foods is a possible way for humans to increase their intake of these compounds and obtain potential health benefits of CLA and n-3 fatty acids. Dietary manipulation by feeding synthetic CLA oil has been the documented method in enriching eggs with CLA (Cherian, 2005). Feeding flax and marine oils to hens has been used to enrich eggs with n-3 fatty acids (Cherian, 2002; Rymer and Givens, 2005). Altering the n-3 and CLA content in eggs also increases the degree of unsaturation leading to changes in egg fatty acid quality and oxidative stability.
INTRODUCTION Recently, there has been a great interest in the role of foods for minimizing the risks of cardiovascular disease or ameliorating the progression of atherosclerosis. Such food or food ingredients that offer health-enhancing properties are called functional foods. Conjugated linoleic acids (CLA), n-3 fatty acids, and antioxidant vitamins (vitamin E) have received considerable attention as functional nutrients for their anticarcinogenic, antiatherogenic, hypocholesterolemic, immunomodulatory, and body fat reduction properties (Traber, 1999; Belury, 2002; Kris-Etherton et al., 2003; Terpstra, 2004). Dietary CLA is contributed by ruminant foods, and n-3 fatty acids are contributed through marine foods and oils from terrestrial sources such as flax. The consumption of CLA is less than 600 mg/d compared with the 3 g/d needed to
©2007 Poultry Science Association Inc. Received September 30, 2006. Accepted January 23, 2007. 1 Corresponding author: [email protected]
953
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
fatty acids were lower (>19%) in YG-CLA and YG-CLAFO compared with YG. Egg n-3 was highest in YG-FO eggs and lowest in YG eggs (P < 0.0001). Storage over 60 d led to a 20 and 67% depletion of CLA in the YG-CLA and YG-CLA-FO eggs (P < 0.0001). A 29% reduction was observed in the total n-3 fatty acid content of YG-CLAFO eggs at d 60 of storage when compared with d 0 of storage (P < 0.0001). Diet and storage increased TBARS (P < 0.0001), which was highest in YG-CLA eggs at 60 d of storage. The YG-CLA and YG-CLA-FO diets reduced α and γ-tocopherol contents at all days of storage compared with YG eggs (P < 0.05). Regardless of diet, egg storage for 40 d or longer depleted egg tocopherol contents (P < 0.05). These data demonstrate that healthy eggs with increased n-3 fatty acids and CLA can be generated by minor diet modifications, but added tocopherol supplementation may be needed to reduce lipid peroxidation when n-3 or CLA is included in the hen diet.
ABSTRACT The effects of incorporating conjugated linoleic acid (CLA) and fish oil in laying hen diets on egg CLA, n-3 fatty acid, tocopherol, and TBA reactive substances (TBARS) during 60 d of storage were investigated. Hens were fed corn-soybean meal-based diets containing 3% yellow grease (YG), 2.75% yellow grease + 0.25% CLA (YG-CLA), 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (YG-CLA-FO), or 2.75% yellow grease + 0.25% fish oil (YG-FO). Eggs were collected and stored at 4°C up to 60 d. On storage d 0, 20, 40, and 60, eggs (n = 8) from each treatment were selected randomly, and tocopherol and TBARS contents were measured. Egg total lipid and fatty acids were determined on d 0 and 60 of storage. Feeding YG-CLA-FO led to a 5.4 and 7.7% reduction in egg total lipids on d 0 and 60 (P < 0.05) when compared with YG eggs. The YG-CLA and YG-CLA-FO diets led to a 12% increase in egg saturated fatty acids compared with YG eggs. The content of monounsaturated
954
CHERIAN ET AL. Table 1. Tocopherol content and fatty acid composition of the laying hen diet Dietary treatments1 Item
YG-CLA
YG-CLA-FO
YG-FO
19.1 ± 0.09 5.7 ± 0.33 24.8 ± 0.37
17.3 ± 0.31 4.3 ± 0.07 21.6 ± 0.30
18.2 ± 0.02 4.3 ± 0.16 22.5 ± 0.17
17.7 ± 0.33 4.1 ± 0.12 21.7 ± 0.44
16.2 4.9 34.5 37.7 0.0 0.0 3.1 0.0 0.0 23.7 35.5 3.1 0.0
± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.35 1.69 1.65 0.00 0.00 0.29 0.00 0.00 0.26 2.19 0.29 0.00
15.5 4.5 32.0 35.7 1.0 0.7 3.1 0.0 0.0 24.5 32.9 3.1 1.7
± ± ± ± ± ± ± ± ± ± ± ± ±
0.29 0.21 0.71 1.21 0.05 0.00 0.20 0.00 0.00 0.71 1.10 0.20 0.05
16.3 4.9 32.7 34.5 0.8 0.7 2.9 0.5 0.4 23.8 34.8 3.8 1.5
± ± ± ± ± ± ± ± ± ± ± ± ±
0.05 0.74 2.68 2.33 0.14 0.17 0.37 0.02 0.01 0.08 2.94 0.38 0.31
15.9 4.5 31.4 36.4 0.0 0.0 2.6 0.6 0.4 26.9 33.1 3.6 0.0
± ± ± ± ± ± ± ± ± ± ± ± ±
0.61 0.06 0.27 1.56 0.00 0.01 0.27 0.01 0.01 1.83 0.26 0.28 0.00
1 YG = 3.0% yellow grease;YG-CLA= 2.75% yellow grease + 0.25 conjugated linoleic acid (CLA); YG-CLAFO = 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (FO); YG-FO = 2.75% yellow grease +0.25% FO. Means ± SE of 3 replicate feed samples.
Consumer acceptance of eggs depends upon storage stability and nutritional quality. The effect of feeding n-3 and CLA to hens on egg CLA, n-3 fatty acid, tocopherols, and lipid peroxidation products during 60 d of storage was investigated. The length of storage times was selected to simulate likely duration of consumer storage of shell eggs.
maintained on a 16L:8D photoperiod and standard conditions of temperature and ventilation as per Oregon State University Poultry Farm standard operating procedures. Water and feed were provided ad libitum. Feed intake was measured on a weekly basis. The experiment was conducted for 320 d.
Egg Collection MATERIALS AND METHODS These experiments were approved by the Oregon State University Animal Care and Use Committee to ensure adherence to animal care guidelines.
Diets Isocaloric (2,900 kcal/kg of feed) and isonitrogenous (16% CP) corn-soybean meal-based experimental rations were formulated with 3% yellow grease (YG), 2.75% yellow grease + 0.25% CLA (YG-CLA), 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (YG-CLA-FO), or 2.75% yellow grease + 0.25% fish oil (YG-FO). The composition of the diet is shown in Table 1. Menhaden fish oil was used as a source of n-3 fatty acid (Omega Protein Inc., Reedville, VA). The CLA oil containing 75% free fatty acids was made up of equal amounts of cis9, trans11, and trans10, cis12 CLA isomers (Pharmanutrients, Lake Bluff, IL). The diets were mixed weekly and were stored in a cold room (4°C) in airtight containers.
Birds A total of 112 twenty-week-old Single Comb White Leghorn laying hens were weighed and distributed randomly to the experimental diets (4 replications of 7 birds each). The birds were kept individually in cages and were
A total of 128 eggs were collected (32/diet, 8 eggs/ replicate) during the peak production period (wk 30 to 31) and were kept in fiber flats at 4°C. Length of storage times was selected to simulate likely duration of consumer storage of shell eggs. On d 0, 20, 40, and 60 of storage, 2 eggs were selected randomly from each replicate, totaling 8 eggs/treatment for tocopherol and lipid peroxidation assays as measured by TBA reactive substances (TBARS). Total lipids and fatty acids were determined for eggs stored for 0 and 60 d.
Lipid Analyses Total lipids were extracted by the method of Folch et al. (1957). One gram of yolk sample was weighed into a screw-capped test tube with 20 mL of chloroform:methanol (2:1, vol/vol) and homogenized with a polytron homogenizer (PT10/35, Brinkman Instruments, Westbury, NY) for 15 to 20 s at high speed. After an overnight incubation at 4°C, the homogenate was filtered through Whatman no. 1 filter paper into a 100-mL graduated cylinder, and 4 mL of 0.88% NaCl solution was added and mixed. After phase separation, the volume of lipid layer was recorded, and the top layer was completely siphoned off. Total lipids were determined gravimetrically. Analysis of fatty acid composition was performed with a Agilent 6890 gas chromatograph (Agilent Technologies
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
Tocopherols (mg/kg) α-Tocopherol γ-Tocopherol Σ Tocopherols Fatty acids (% of total fatty acids) 16:0 18:0 18:1 18:2 n-6 cis9, trans11 CLA trans10, cis12 CLA 18:3n-3 20:5n-3 22:6n-3 Σ Saturates Σ Monounsaturates Σ Total n-3 Σ CLA
YG
955
CONJUGATED LINOLEIC ACID AND FISH OIL Table 2. Effect of dietary conjugated linoleic and fish oil on the total lipids and fatty acid content of eggs during storage Dietary treatments1 d 0 of storage Items2
YG
YG-CLA
YG-CLA-FO
YG-FO
YG
YG-CLA
YG-CLA-FO
YG-FO
Diet
Storage
Diet × storage
31.2a 0.4 27.8b 3.4a 9.3b 40.4b 15.4b 0.2b 0.0 2.2a 0.8b 37.6b 43.8b 17.6b 1.3b 0.0b
30.3ab 0.5 29.9a 1.8b 12.8a 33.4c 17.5a 0.4ab 0.5a 2.2a 0.7b 43.2a 35.3c 19.8a 1.4b 0.6a
29.5b 0.5 29.2a 2.0b 12.3a 33.3c 16.5ab 0.4a 0.6a 1.8b 1.5a 42.1a 35.4c 18.2ab 2.4a 0.6a
30.0b 0.6 27.8b 3.8a 8.6b 42.1a 13.5c 0.4ab 0.1b 1.6b 1.4a 37.1b 45.9a 15.2c 2.2a 0.1b
27.3a 0.1 24.1c 3.2b 9.5b 44.9a 15.0c 0.6bc 0.0b 1.8 0.7c 33.7b 48.7a 16.8c 0.8b 0.0b
26.5a 0.2 25.6ab 2.1d 13.2a 36.6b 18.5a 1.0a 0.4a 1.8 0.6c 39.0a 39.7b 20.3a 1.4a 0.4a
25.2b 0.1 26.6a 2.4c 12.7a 37.5b 16.8b 0.8ab 0.2ab 1.6 1.2b 39.5a 40.7b 18.3b 1.7a 0.2ab
27.1a 0.1 25.0bc 3.5a 9.5b 44.6a 13.7d 0.4c 0.0b 1.6 1.5a 34.6b 48.6a 15.2d 1.5a 0.0a
0.0541 0.7248 0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0037 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
0.1059 <0.0001 <0.0001 0.6044 0.0950 <0.0001 0.4249 0.0002 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.8790 <0.0001 <0.0068
0.0110 0.2385 0.2704 0.1194 0.8166 0.2160 0.5099 0.0396 0.0510 <0.0001 <0.0001 0.4705 0.0848 0.5508 0.0914 0.1583
Means significantly different within each day of storage (P < 0.05). YG = 3.0% yellow grease; YG-CLA = 2.75% yellow grease + 0.25 conjugated linoleic acid (CLA); YG-CLA-FO = 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (FO); YG-FO = 2.75% yellow grease + 0.25% FO. 2 SFA = saturated fatty acids; MUFA = monounsaturated fatty acids. a–d 1
Inc., Palo Alto, CA) equipped with an autosampler, flame ionization detector and fused silica capillary column, 100 m × 0.25 mm × 0.2 m film thickness (Sp-2560, Supelco, Bellefonte, PA). Sample (1 L) was injected with He as a carrier gas onto the column programmed for ramped oven temperatures (initial temperature was 110°C, held for 1 min, then ramped at 15°C/min to 190°C and held for 55 min, then ramped at 5°C/min to 230°C and held for 5 min). Inlet and detector temperatures were both 220°C. Peak areas and percentages were calculated using Agilent ChemStation software. Fatty acid methyl esters were identified by comparison with retention times of authentic standards (Matreya, Pleasant Gap, PA). Fatty acid values and total lipids were expressed as weight percentages.
Tocopherol Assay For analysis of egg yolk α- and γ-tocopherols, a modification of the method by Podda et al. (1996) was used. Briefly, a known weight (∼50 mg) of individual yolk sample (n = 8) was saponified with alcoholic KOH, extracted with hexane, dried under N, resuspended in 1:1 ethanol:methanol, then injected into an HPLC system (Shimadzu, Columbia, MD). The HPLC system consisted of a Shimadzu LC-10ADVP controller and a SIL-10ADVP auto injector with a 50-L sample loop. Tocopherols were detected with a LC-4B amperometric electrochemical detector (Bioanalytical Systems Inc., West Lafayette, IN) with a glassy C working electrode and silver chloride reference electrode. The column used was a Waters Symmetry Shield RP18 column, 100 × 4.6 mm, 3.5-m particle size with a Waters Symmetry Shield RP18 precolumn, 20 × 3.9 mm, 3.5 m (Waters Inc., Watford, Hertfordshire, UK). An isocratic mobile phase delivery system [99:1 (vol/vol) methanol:water containing 0.1% (wt/vol) lithium perchlorate] was used, with a total run time of 6 min.
The electrochemical detector was in the oxidizing mode, potential 500 mV, full recorder scale at 500 nA. Peak areas were integrated using a Shimadzu Scientific 4.2 Class VP software package, and α- and γ-tocopherols were individually quantitated using authentic standards.
TBARS Assay Egg yolk samples (2 g) were weighed into 50-mL test tubes, and 18 mL of 3.86% perchloric acid was added. The samples were homogenized with a polytron homogenizer for 15 s. Butylated hydroxytoluene (50 L, in 4.5% ethanol) was added to each sample during homogenization to control lipid oxidation. The homogenate was filtered through Whatman no. 1 filter paper. Filtrate (2 mL) was mixed with 2 mL of 20 mM TBA in distilled water and incubated in the dark at room temperature for 15 to 17 h. Absorbance was determined at 531 nm. The TBARS were expressed as milligrams of malondialdehyde/gram of yolk (Cherian et al., 1996a).
Statistical Analysis A 2-way ANOVA was used to analyze effects of diet and storage on egg tocopherols, TBARS, fatty acids, and total lipids using eggs within diet by storage as the error term (SAS Institute, 2001). Diet and storage were the fixed effects. Significant differences among treatment means were analyzed by the Student-Newman-Keuls multiple range test at P < 0.05 (Steel and Torrie, 1980). Means of interaction were analyzed by comparing the storage time separately for each diet by the Student-Newman-Keuls multiple range test. Computations were done using the GLM procedure of SAS Institute (2001). Mean values are reported.
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
Σ Lipids 14:0 16:0 16:1 18:0 18:1 18:2 18:3 Cis9, trans11 CLA 20:4 22:6 Σ SFA Σ MUFA Σ n-6 Σ n-3 Σ CLA
P-value
d 60 of storage
956
CHERIAN ET AL. Table 3. Effect of dietary conjugated linoleic acid and fish oil feeding on the α-, γ-, and total tocopherol content of egg yolk during storage Dietary treatments1 Tocopherol (nmol/g)
YG-CLA
YG-CLA-FO
YG-FO
78.0 ± 5.89a 2.4 ± 0.29a 80.7 ± 6.10a
56.6 ± .36b 1.2 ± 0.14b 57.9 ± 5.44b
62.7 ± 2.96b 1.2 ± 0.15b 63.9 ± 2.94b
67.0 ± 2.53ab 1.4 ± 0.15b 68.4 ± 2.60ab
88.1 ± 5.95a 2.1 ± 0.07a 90.2 ± 5.99a
65.3 ± 1.36bc 1.1 ± 0.12b 66.4 ± 1.32bc
62.0 ± 5.13c 1.2 ± 0.16b 63.2 ± 5.21c
74.9 ± 2.79b 1.1 ± 0.07b 76.0 ± 2.80b
67.7 ± 2.85a 2.8 ± 0.09a 70.5 ± 2.86a
49.5 ± 4.00b 1.7 ± 0.13b 51.2 ± 4.10b
49.0 ± 3.34b 1.5 ± 0.08b 50.5 ± 3.35b
53.6 ± 1.69b 1.4 ± 0.11b 54.9 ± 1.63b
58.7 ± 3.63a 2.4 ± 0.13a 61.1 ± 3.73a
45.9 ± 4.02b 1.4 ± 0.08b 47.3 ± 4.04b
46.6 ± 4.55b 1.5 ± 0.11b 48.1 ± 4.61b
52.5 ± 3.91ab 1.4 ± 0.13b 53.9 ± 3.99ab
ANOVA Diet α-Tocopherol γ-Tocopherol Σ Tocopherols Storage α-Tocopherol γ-Tocopherol Σ Tocopherols Diet × storage α-Tocopherol γ-Tocopherol Σ Tocopherols
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.7845 0.6296 0.7888
Means significantly different for each dietary treatment (P < 0.05). YG = 3.0% yellow grease; YG-CLA = 2.75% yellow grease + 0.25 conjugated linoleic acid (CLA); YG-CLAFO = 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (FO); YG-FO = 2.75% yellow grease + 0.25% FO. a–c 1
RESULTS AND DISCUSSION The fatty acid composition and tocopherol contents of the diet are shown in Table 1. Conjugated linoleic acid was present only in the CLA-supplemented diets (YGCLA and YG-CLA-FO) and consisted of cis9, trans11, and trans10, cis12 isomers at 1.0, 0.8, 0.7, and 0.7, respectively. Inclusion of fish oil resulted in the incorporation of longchain n-3 fatty acids, such as eicosapentaenoic acid (20:5n3) and docosahexaenoic acid (22:6 n-3) in YG-CLA-FO and YG-FO diets. α-Linolenic acid (18:3 n-3) was the only source of n-3 fatty acids in the YG and YG-CLA diets. αTocopherol was the predominant form of tocopherol in the diet (17.3 to 19.1 mg/kg), followed by γ-tocopherol (4.1 to 5.7 mg/kg). The total tocopherol content of the diet varied from 21.6 to 24.8 mg/kg. The total lipid content of the diet was 3%.
Diet Effects on Yolk Fatty Acids The egg total lipid content and fatty acid composition at storage d 0 and 60 is shown in Table 2. Egg total lipids were affected by dietary oils (P < 0.05). Feeding YG-CLAFO led to a 5.4% reduction in total lipids when compared with YG eggs. The yolk fatty acid profile clearly reflected the dietary fatty acid composition. Conjugated linoleic
acid was present only in eggs from YG-CLA and YGCLA-FO (P < 0.05). The only isomer detected in egg yolk was cis9, trans11. A significant effect of diet was observed on egg saturated fatty acids (SFA; 16:0, 18:0; Table 2). Addition of 0.25% CLA resulted in a significant increase in SFA with a concomitant reduction in monounsaturated fatty acids (MUFA). The increase in SFA in the YG-CLA eggs was over 12% higher than YG eggs. The content of MUFA was over 19% lower in YG-CLA eggs than YG eggs. These results also corroborate our previous reported results and also of others (Du et al., 1999; Cherian et al., 2002). Inclusion of fish oil along with CLA did not overcome the MUFA-suppressing effect of CLA in YG-CLA-FO eggs. The ⌬9-desaturase enzyme is responsible for the conversion of stearic acid (18:0) to oleic acid (18:1). Dietary CLA may have an inhibitory action on desaturases, thereby leading to the reduction of MUFA. A recent study also reported a decrease in mRNA expression of stearoyl coenzyme A in CLA-fed rats affecting the synthesis of MUFA and accumulation of SFA (Choi et al., 2000). Higher levels of SFA in yolk lipids due to CLA supplementation may be of concern, because increased consumption of SFA, especially 14:0 and 16:0, is associated with increasing plasma total and low-density lipoprotein cholesterol concentrations in blood plasma and changes related to car-
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
d0 α-Tocopherol γ-Tocopherol Σ Tocopherols d 20 α-Tocopherol γ-Tocopherol Σ Tocopherols d 40 α-Tocopherol γ-Tocopherol Σ Tocopherols d 60 α-Tocopherol γ-Tocopherol Σ Tocopherols
YG
CONJUGATED LINOLEIC ACID AND FISH OIL
Storage Effects on Yolk Fatty Acids Irrespective of the lipid source, storage reduced fatty acids in eggs (Table 2). Total lipids were lowest in the YG-CLA-FO eggs stored for 60 d compared with other eggs (P < 0.05). A significant decrease due to storage was observed for all fatty acids except 16:1, 18:0, and 18:2 n6. Storage over 60 d led to a 20 and 67% depletion of CLA in the YG-CLA and YG-CLA-FO eggs (P < 0.0001). The presence of long-chain n-3 fatty acids in the YG-CLAFO eggs enhanced the depletion of CLA when compared with YG-CLA eggs. A 29% reduction was observed in the total n-3 fatty acid content of YG-CLA-FO eggs at d 60 of storage when compared with d 0 of storage (P < 0.0001). The decrease in total lipids, CLA, and n-3 fatty acids may suggest a degradation of egg lipids and polyunsaturated fatty acids (PUFA) during storage. In addition, oxidative stability of eggs based on TBARS revealed a significant effect of diet and storage (Figure 1). At d 0, the eggs from the YG-CLA-FO and YG-FO regimen had higher TBARS values (P < 0.05) than those from YG or YG-CLA, suggesting that the onset of lipid oxidation may be enhanced in these eggs due to the high content of longchain (>20-C) PUFA. However, during storage, accumulation of TBARS was higher in YG-CLA than all the other treatments. We have previously reported that CLA incorporation is preferentially in the egg triglycerides (Cherian, 2005) compared with long-chain n-3 PUFA in the phospholipids (Cherian and Sim, 1992). In the egg yolk, phospholipids and protein are interwoven in the exterior sur-
Figure 1. Effect of dietary conjugated linoleic and fish oil feeding on the TBA reactive substances in egg yolk during storage. The values reported are in milligrams of malondialdehyde per gram of yolk. YG = 3.0% yellow grease; YG-CLA = 2.75% yellow grease + 0.25 conjugated linoleic acid (CLA); YG-CLA-FO = 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (FO); YG-FO = 2.75% yellow grease + 0.25% FO. a,bLetters above a day indicate a significant difference (P < 0.05).
face of low-density lipoprotein. This compact surface layer can partly exclude O2 from the lipid core of the particle (Burley and Vadehra, 1989), thus impeding oxidation. Thus, the structural configuration of yolk phospholipids may have aided in preventing oxidation of longchain PUFA in YG-CLA-FO or YG-FO eggs. The results of TBARS further attests to the tocopherol content of eggs (Table 3). Diet and storage reduced the tocopherol content of eggs (P < 0.0001). Eggs from YGCLA and YG-CLA-FO had reduced α and γ- tocopherol contents at all days of storage compared with YG eggs (P < 0.05). In a previous study, we reported that the inclusion of tocopherols resulted in a significant reduction (P < 0.05) in TBARS in the egg yolk from hens fed menhaden fish oil during storage (Cherian et al., 1996b). Therefore, it appears that the longer-chain n-3 PUFA in fish oil-fed eggs were protected from undergoing deterioration by tocopherol supplementation. Regardless of dietary oils, egg storage for 40 d or longer depleted tocopherol contents promoting lipid oxidation and the accumulation of TBARS. Thus, incorporating tocopherols into chicken eggs may increase the oxidative stability and also provide a source of tocopherols for the human diet. Several authors have reported CLA enrichment through diet manipulation (Cherian, 2005). However, no study has reported the effect of CLA enrichment on egg tocopherol content and lipid oxidation products during storage. These results from the current study demonstrate that healthy eggs with increased n-3 fatty acids and CLA can be generated by minor diet modifications, and added tocopherol supplementation may be needed when n-3 or CLA is included in the hen diet to maintain product quality and nutritional value.
ACKNOWLEDGMENTS The Ott professorship awarded to G. Cherian is acknowledged. The CLA used in this study was supplied
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
diovascular disease (Bonanome and Grundy, 1988). However, if the antiatherogenic activity of CLA found in rabbits (Lee et al., 1994), hamsters (Nicolosi et al., 1997), and mice (Munday et al., 1999) could be extrapolated to humans, the adverse consequences of increased SFA levels could be counteracted by CLA. A significant increase in linoleic acid was observed in YG-CLA eggs (P < 0.0001). However, CLA along with fish oil led to a reduction in arachidonic acid content in YG-CLA-FO eggs. ⌬6-Desaturase enzyme is involved in the formation of arachidonic acid (20:4 n-6) from linoleic acid (18:2 n-6). The increase in linoleic acid observed in the current study (YG-CLA) may suggest an inhibitory effect of CLA on ⌬6-desaturase leading to its accumulation. Higher dietary CLA (>0.5%) has been reported to reduce egg arachidonic acid (Cherian et al., 2002). Higher dietary CLA has also been reported to cause rubbery and hard egg yolks upon cooking (Cherian, 2005). Low levels of CLA were used in the current study to minimize such negative yolk textural properties. Fish oil supplementation increased n-3 fatty acids and caused a reduction in n-6 fatty acids such as linoleic and arachidonic acid. The increase in n-3 fatty acids with a concomitant decrease in arachidonic acid by fish oil was consistent with our previous results (Cherian and Sim, 1991). The presence of CLA did not affect the content of n-3 fatty acids in YGCLA-FO eggs.
957
958
CHERIAN ET AL.
by Pharmanutrients. The generous donation of menhaden oil from Omega Protein Inc. is appreciated.
REFERENCES
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
Belury, M. A. 2002. Dietary conjugated linoleic acid in health: Physiological effects and mechanisms of action. Annu. Rev. Nutr. 22:505–531. Bonanome, A., and S. M. Grundy. 1988. Effect of dietary stearic acid on plasma cholesterol and lipoprotein. N. Engl. J. Med. 318:1244–1248. Burley, R. W., and D. V. Vadehra. 1989. Egg yolk: Structure and properties. Pages 171–233 in The Avian Egg. R. W. Burley and D. V. Vadehra, ed. John Wiley and Sons, New York, NY. Cherian, G. 2002. Lipid modification strategies and nutritionally functional poultry foods. Pages 77–92 in Food Science and Product Technology. T. Nakano and L. Ozimek, ed. Res. Signpost, Trivandrum, India. Cherian, G. 2005. Conjugated linoleic acid (CLA) for health: Chicken eggs as alternative sources of CLA. Pages 49–64 in The Amazing Egg: Nature’s Perfect Functional Food for Health Promotion. J. S. Sim and H. H. Sunwoo, ed. Univ. Alberta, Edmonton, Canada. Cherian, G., T. B. Holsonbake, M. P. Goeger, and R. Bildfell. 2002. Dietary conjugated linoleic acid alters yolk and tissue fatty acid composition and hepatic histopathology of laying hens. Lipids 37:751–757. Cherian, G., and J. S. Sim. 1991. Effect of feeding full fat flax and canola seeds to laying hens on the fatty acid composition of eggs, embryos, and newly hatched chicks. Poult. Sci. 70:917–922. Cherian, G., and J. S. Sim. 1992. Preferential accumulation of n3 fatty acids in the brain tissue of chicks from n-3 fatty acid enriched eggs. Poult. Sci. 71:1658–1668. Cherian, G., F. H. Wolfe, and J. S. Sim. 1996a. Dietary oils with added tocopherols: Effects on egg or tissue tocopherols, fatty acids and oxidative stability. Poult. Sci. 75:423–432. Cherian, G., F. H. Wolfe, and J. S. Sim. 1996b. Dietary oils with tocopherols: Effects on internal qualities of eggs during storage. J. Food Sci. 61:15–18. Choi, Y., Y. Kim, Y. Han, Y. Park, M. W. Pariza, and J. M. Ntambi. 2000. The trans-10, cis-2 isomer of conjugated linoleic acid downregulates stearoyl-CoA desaturase 1 gene expression in 3T3-L1 adipocytes. J. Nutr. 130:1920–1924. Du, M., D. U. Ahn, and J. L. Sell. 1999. Effect of dietary conjugated linoleic acid on the composition of egg yolk lipids. Poult. Sci. 7:1639–1645.
Folch, J., M. Lees, and G. H. Sloane-Stanely. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497–507. Fritsche, J., R. Rickert, H. Steinhart, M. P. Yurawecz, M. M. Mossaba, N. Sehat, J. A. Roach, J. K. Kramer, and Y. Ku. 1999. Conjugated linoleic acid (CLA) isomers: Formation, analysis, amounts in foods and dietary intake. Fett-Lipid 101:272–276. Ip, C., S. Banni, E. Angioni, G. Carta, J. McGinley, H. J. Thompson, D. Barbano, and D. E. Bauman. 1999. Conjugated linoleic acid-enriched butterfat alters mammary gland morphogenesis and reduces cancer risk in rats. J. Nutr. 129:2135–2142. Kris-Etherton, P. M., W. S. Harris, and L. J. Appel. 2003. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Arterioscler. Thromb. Vasc. Biol. 23:20–30. Kris-Etherton, P. M., D. S. Taylor, S. Yu-Poth, P. Huth, K. Moriarty, V. Fishell, and R. L. Hargrove. 2000. The polyunsaturated fatty acids in the food chain in the United States. Am. J. Clin. Nutr. 71:179S–188S. Lee, K. N., D. Kritchevevsky, and M. W. Pariza. 1994. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 108:19–25. Munday, J. S., K. G. Thompson, and K. A. James. 1999. Dietary conjugated linoleic acids promote fatty streak formation in the C57BL/6 mouse atherosclerosis model. Br. J. Nutr. 81:251–255. Nicolosi, R. J., E. J. Rogers, D. Kritchevsky, J. A. Scimeca, and P. J. Huth. 1997. Dietary conjugated linoleic acid reduces plasma lipoproteins and early atherosclerosis in hypercholesterolemic hamsters. Artery 22:266–277. Podda, M., C. Weber, M. G. Traber, and L. Packer. 1996. Simultaneous determination of tissue tocopherols, tocotrienols, ubiquinols and ubiquinones. J. Lipid Res. 37:893–901. Rymer, C., and D. I. Givens. 2005. n-3 Fatty acid enrichment of edible tissue of poultry: A review. Lipids 40:121–130. SAS Institute. 2001. SAS User’s Guide: Statistics, Release 8.2. SAS Inst. Inc, Cary, NC. Steel, R. G. D., and J. H. Torrie. 1980. Principles and procedures of statistics: A biometrical approach. 2nd ed. McGraw-Hill Book Co., Toronto, Canada. Terpstra, A. H. 2004. Effect of conjugated linoleic acid on body composition and plasma lipids in humans: An overview of the literature. J. Clin. Nutr. 79:352–361. Traber, M. G. 1999. Vitamin E. Pages 347–362 in Nutrition in Health and Disease. 9th ed. M. Shils, J. A. Olson, M. Shike, and A. C. Ross, ed. Williams & Wilkins, Baltimore, MD. USDA. 2006. Food consumption and nutrient intake. http:// www.ers.usda.gov Accessed Jul. 2006.