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Enrichment of conjugated linoleic acid (CLA) in hen eggs and broiler chickens meat by lactic acid bacteria S. M. Herzallah
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Department of Nutrition and Food Science, Faculty of Agriculture , Mu'tah University Accepted author version posted online: 25 Aug 2013.
To cite this article: British Poultry Science (2013): Enrichment of conjugated linoleic acid (CLA) in hen eggs and broiler chickens meat by lactic acid bacteria, British Poultry Science, DOI: 10.1080/00071668.2013.836734 To link to this article: http://dx.doi.org/10.1080/00071668.2013.836734
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CBPS-2012-282 Ed. Kjaer, July 2013; MacLeod, August 2013
Enrichment of conjugated linoleic acid (CLA) in hen eggs and broiler chickens meat by
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S.M. HERZALLAH
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Department of Nutrition and Food Science, Faculty of Agriculture, Mu tah University, Karak-
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Jordan
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Running title: CLA ENRICHMENT BY BACTERIA
Correspondence to: S.M. Herzallah, Department of Nutrition and Food Science, Faculty of
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Agriculture, Mu tah University, Karak-Jordan, Postal code: 61710. Email: [email protected] , [email protected] Tel: +9632372380, +962785103354.
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lactic acid bacteria
Fax: +962 3 2323154.
Accepted for publication 28th June 2013
1
Abstract.
1. The aim of this work was to compare conjugated linoleic acid (CLA)
concentrations in chickens supplemented with 4 American Tissue Culture Collection (ATCC) bacterial strains, Lactobacillus plantarum, Lactobacillus lactis, Lactobacillus casei and Lactobacillus fermentum and 4 isolates of Lactobacillus reuteri from camel, cattle, sheep and goat rumen extracts.
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2. Microorganisms were grown anaerobically in MRS broth and 106 CFU/ml of bacteria were
administered orally to mixed-sex 1-d-old broiler chickens weekly for 4 weeks and to 23-
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3. The 5 strains were evaluated for their effects on synthesis of CLA in hen eggs and broiler meat cuts.
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4. Administration of pure Lactobacillus and isolated L. reuteri strains from camel, cattle, goat and sheep led to significantly increased CLA concentrations of 0.2–1.2 mg/g of fat in eggs
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and 0.3–1.88 mg/g of fat in broiler chicken flesh homogenates of leg, thigh and breast.
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5. These data demonstrate that lactic acid bacteria of animal origin (L. reuteri) significantly
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enhanced CLA synthesis in both eggs and broiler meat cuts. INTRODUCTION
Conjugated linoleic acid (CLA) is a positional and geometric linoleic fatty acid (LA) of cis
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and trans isomers (cis–9- and cis-12-octadecanoic). Linoleic acid is a natural food component found in plant oils (Sebedio et al.,1998; Bauman et al., 2003), whereas, dietary CLA is found in meat and dairy products from ruminants (Chin et al., 1992; Bovet et al., 2007). Poultry
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week-old layer hens weekly for 6 weeks.
meat and associated products represent about 30% of total global meat consumption (FAO, 2006). Recent studies examining nutritional compounds of public interest have revealed a role for CLA in reducing cancer cell growth and body fat accumulation (Wang and Jones, 2004). Conjugated linoleic acid (CLA) is used to improve immune function, reduce plasma
cholesterol and fatness and prevent arteriosclerosis (Schultz et al., 1992; Schonberg and Krokan, 1995; Park et al., 1997; West et al., 1998). Several bacteria express enzymes that 2
convert linoleic acid to CLA, including Propionibacterium, Eubacterium, Butyrivibrio and Lactobacillus strains (Kepler et al., 1966; Eyssen and Verhulst, 1984; Jiang et al., 1998). Moreover, isomerisation of linoleic acid to CLA acts as a defence mechanism against bacteria by inhibiting bacterial growth (Verhulst et al., 1986; Jiang et al., 1998). However, direct inhibition of bacterial growth by LA and CLA has not been reported to date. Bacterial species
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that produce lactic acid are often used in food fermentation, and those present in the
mammalian gastrointestinal tract provide several health benefits (Holzapfel et al., 1998).
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effects and immune stimulation (Mallet et al., 1984; Hill, 1995, 1998; Morotomi et al., 1997; Vonk et al., 1997; Ringø et al., 1998). In the present study, a variety of lactic acid bacteria
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(LAB) strains was administered, and subsequent CLA enrichment in eggs and broiler chicken meat was determined.
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MATERIAL AND METHOD
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Lactobacillus plantarum (ATCC 8014), Lactobacillus casei (ATCC 393), Lactobacillus lactis
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(ATCC 7830) and Lactobacillus fermentum (ATCC 9338) were purchased from American Tissue Culture Collection (ATCC). L. reuteri strains were isolated from goat, sheep, cattle and camel rumen. Then the isolated bacteria were cultured in MRS broth (Difco, Detroit,
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USA).
Management of birds Experimental design I
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These intestinal microflora liberate free fatty acids from unabsorbed fat, leading to anticancer
A total of 180 one-d-old Hubbard broiler chickens were purchased from Hammoudeh Poultry Company, Amman, Jordan. Chickens were randomly divided into 9 groups of 10 chickens and were housed in rearing cages at the agriculture experimental laboratory. Chickens were
reared under standard hygienic conditions with 24 h lighting and free access to water. Chickens were given commercially balanced rations of starter diet for 4 weeks, and a finisher diet was provided in week 5 (Table 1). Group I were given a balanced diet and were not 3
treated with bacteria (control). The remaining groups (II–IX) received weekly supplements of 1 ml of microbial cultures containing 106 CFU/ml of L. plantarum, L. lactis, L. casei, L. fermentum or L. reuteri from camel, L. reuiteri from cattle, L. reuteri from sheep or L. reuteri from goat. At the end of every week, two chickens were slaughtered in the commercial poultry slaughter plant. Chicken breasts and legs were deboned, ground with skin and
Experimental design II
Table 1 near here
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subcutaneous fat and stored at −20 ± 1°C until analysis.
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each of 9 bacterial treatment groups. The chickens were reared under standard hygienic
conditions in cages and were given a balanced commercial layer hen’s ration (Table 2) for 3
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weeks of acclimatisation. Layer hens of group I were kept as controls and were not administered any microbial strains. Hens in groups II–IX were orally administered 1 ml of
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bacterial cultures containing 106 CFU/ml of L. plantarum, L. lactis, L. casei, L. fermentum or
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L. reuteri from camel, L. reuteri from cattle, L. reuteri from sheep or L. reuteri from goat.
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Eggs were collected, broken, homogenised and subsampled every week for 5 weeks, stored at 4 ± 1°C and were assayed for CLA concentration in triplicate within 3 d.
Table 2 near here
Fat extraction, CLA derivatisation and gas chromatography analysis
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Lipids were extracted from homogenised deboned raw poultry meat (breast and leg muscles with skin) or eggs using chloroform and methanol as described by Bligh and Dyer (1959) and Herzallah et al. (2005), and were stored at −20 ± 1°C until analysis. CLA methyl esters were
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A total of 27 23-week-old commercial Hisex white layer hens were assigned in triplicate to
prepared by dissolving 60 mg of extracted fat in 100 µl of hexane, and were then vortexed with 100 µl of 2 N methanolic KOH (ISO-IDF, 2002) for 30 s. Formation of CLA methyl ester was measured by injecting 1µl aliquots into capillary columns (TR-CN100, 60 m, 0.2 µm, 0.25 mm i.d., Teknokroma, Barcelona, Spain) of a Shimadzu gas chromatograph (Shimadzu 2010, Kyoto, Japan) equipped with a flame ionisation detector (GC–FID). Injector and detector temperatures were set at 250 and 300°C, respectively, carrier gas was N 2 (0.3 4
ml/min) with a split ratio of 1:50. The oven temperature was 150°C for the first 5 min, was increased at a rate of 5°C/min to 240°C and was held for a final 5 min. CLA isomers were determined and identified using a CLA standard purchased from Sigma-Aldrich (St Louis, MO, USA). Analyses of CLA cis and trans isomers of 9,11- and 10,12-octadecadienoic acid methyl ester were conducted using response areas of 3 replicate injections, and the mean ±
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standard deviation (SD) was calculated.
Preparation of the CLA methyl ester standard calibration curve
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hexane (gas chromatography grade) and concentrations of 0.0, 1.0, 2.0, 4.0 and 10 µg/ml were injected into the gas chromatography system (Shimadzu 2010, Tokyo, Japan).
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CLA recovery
CLA recovery was determined by mixing 30 µl of 50-µg/ml CLA standard with 10 ml of
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hexane and then incorporating 5 g samples of finely ground, homogenised, fat free oven dried
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meat and/or eggs. CLA spiked samples were homogenised and stored under nitrogen in 20-ml
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brown vials and were kept for 3 d at 4.0 ± 1.0°C. Subsequently, CLA content was re-extracted from 1 g samples in triplicate using 5 ml of hexane, and the 3 hexane portions were combined and dried over anhydrous sodium sulphate (99%; Sigma-Aldrich, MO, US). CLA was
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determined in 5 replicates; 5 µl of CLA methyl ester was injected in triplicate into a GC–FID and the average CLA recovery values were 96.4% and 97.3%, respectively.
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The 250-mg CLA-methyl ester standard (Sigma, O5632, USA) was dissolved in 10 ml of
Statistical analysis Mean changes in CLA content of eggs and broiler flesh were compared to that in control animals using PC-SAS version 9.0 (SAS Institute, 2002). The least significant difference (LSD) test was performed with mean values of CLA measurements using a Completely Randomised Design (CRD) and differences were considered significant when P < 0.05. RESULTS
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CLA contents of poultry meat from chickens administered LAB are presented in Table 3. All LAB strains enhanced formation of CLA and increased concentrations in poultry meat, particularly L. reuteri. Table 4 shows that weekly administration of 106 CFU/ml of LAB strains to laying hens increased CLA contents of eggs. All strains led to increased concentrations of CLA in eggs, and L. reuteri, L. fermentum and L. plantarum were the most effective. DISCUSSION
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Tables 3 and 4 near here
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and 1.85 mg/g fat) after 4 and 5 weeks compared to 0.03 and 0.08 mg/g fat found in control chickens in week 1 and 3, respectively. CLA concentrations were significantly higher in
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poultry meat of broiler chickens that were administered L. reuteri strains from rumen digests of camel, cattle, goat and sheep than in control and LAB supplemented chickens. CLA was
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found at 0.83 mg/g fat in poultry meat from chickens administered L. plantarum, and at 1.8
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mg/g fat in meat samples from chickens reared with L. reuteri from sheep rumen digests. This
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CLA enrichment in poultry meat samples from LAB-reared chickens is in agreement with data from Crespo and Esteve-Garcia (2001) showing that feeding broilers on dietary fat (6 to 10%) produced high concentrations of n-3 PUFA, which met > 70% of the recommended
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daily allowance (RDI) for humans. Moreover, CLA concentrations were increased by 46% in breast meat and 36% in thigh meat. Grashorn (2007) also showed enrichment of poultry meat with CLA and omega-3 fatty acids, meeting 3–11% and 70–130% of human RDIs,
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CLA concentrations in broiler meat were higher in L. reuteri supplemented chickens (1.65
respectively, in broiler chickens receiving rapeseed oil and -tocopherol supplements. These
observations agree with data showing that supplementation of the LAB L. zeae ATCC 393, L. reuteri ATCC 23272 and L. acidophilus (NCFM strain) with fatty acids increased the concentration of cis-9, trans-11-CLA by 20% in bacterial cell membranes (Jenkins and Courtney, 2003). Hence CLA content in poultry meat (breast, thigh) could be increased by about 40% with dietary CLA supplementation (Du and Ahn, 2002; Aletor et al., 2003; Sirri et 6
al., 2003; Grashorn, 2005; Suksombat et al., 2007; Shin et al., 2011). In addition, Blankson et al. (2000) reported that CLA (3.4 g/d) aided loss of 28.3–30.3 kg of body fat in overweight and obese humans after 12 weeks of exercise training. Both cis-9 and trans-11 CLA isomers were increased in egg lipids, and varied with the type and source of LAB administered to Hisex white laying hens. The concentrations of CLA
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in eggs collected during the first week did not differ significantly (P > 0.05) between bacterial supplement groups, but were significantly higher than in control animals (P < 0.05). The
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supplementation, with higher concentrations in hens given isolated bacterial strains from camel, cattle, sheep and goats. Indeed, the highest CLA concentrations were detected after 5
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weeks supplementation with L. reuteri from rumen digests, with CLA concentrations of 1.25, 1.12, 1.11 and 1.03 mg/g fat in eggs from hens administered L. reuteri from camel, sheep,
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goat and cattle, respectively. In comparison, CLA concentrations were 0.2 and 0.46 mg/g fat
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in control and L. plantarum supplemented eggs, respectively. High CLA production by L.
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reuteri was also reported by Jenkins and Courtney (2003), who showed that supplementation of L. reuteri ATCC 55739 growth medium with exogenous CLA and linoleic fatty acids strongly induced detoxification of linoleic acid by isomerisation to CLA in bacterial cell
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membranes. Narahari (2003) reported enrichment of eggs with fatty acids and omega-3, with 30 fold increases in linoleic (LA) and docosahexanoic (DHA) acids in egg yolk of laying hens supplemented with special oil blends. CLA can also be enriched in eggs by supplementing
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enrichment of CLA in eggs from Hisex laying hens increased with the duration of microbial
layer hens with CLA (Sirri et al., 2003) or oil blends (linseed, rapeseed; Du et al., 2001). Yin et al. (2008) showed that CLA supplementation of hens increased CLA deposition in egg yolk lipids, even with soybean-based diets containing < 5% CLA. Additionally, Szymczyk and Pisulewski (2003) found increased CLA and fatty acids in eggs yolk from laying hens given < 20 g/kg CLA. In conclusion, the present results suggest that CLA production in both meat and eggs can be enhanced by LAB, particularly by isolated L. reuteri. 7
ACKNOWLEDGEMENTS The authors wish to thank the Dean of the University of Jordan Research for financial support of this work. The authors also thank the University of Mu'tah for the use of their gas chromatography and HPLC equipment. REFERENCES
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Table 1. Basic composition of the experimental broiler diets Finisher
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Ingredients g/kg Maize 585 670.5 Soybean meal (48% protein) 356.5 260.0 Vegetable oil (Palm oil) 16.9 30.0 Limestone 18.4 16.8 Dicalcium phosphate (DCP, Ca, 10.0 10.2 28% and P, 19%) Sodium chloride 4.1 4.2 DL-Methionine (99%) 2.0 2.0 L-Lysine (98%) 1.1 1.3 Coccidiostat (Hindal, 66 g/kg) 1.0 0.00 Vitamin Premix1 1.0 1.0 Mineral Premix2 1.0 1.0 Choline chloride (60%) 1.0 1.0 Antioxidant (Fintox, HA) 1.0 1.0 Antifungal (Fintox) 1.0 1.0 Calculated nutrient Composition: Metabolisable energy (MJ/kg of 12.47 13.21 dry matter) Crude protein (g/kg) 223.0 182.0 3 NPP (g/kg) 4.5 4.0 Ca (g/kg) 10.3 9.5 Na (g/kg) 1.8 1.8 1 Vitamin premix (Hindal Extra, Jordan) contained the following nutrients per kg of feed: vitamin A, 12000 IU (retinyl acetate); Cholecalciferol (D3), 3000 IU; vitamin E, 20 IU (DLα-tocopheryl acetate); vitamin K (menadione sodium bisulphate, 3.3 mg; thiamin, 2.5 mg; riboflavin, 5 mg; pantathenic acid, 13 mg; niacin, 30 mg; vitamin C, 100 mg; vitamin B12, 0.02 mg; biotin, 0.05 mg; pyridoxine, 4 mg; folic acid, 1 mg, ethoxyquin, 13 mg. 2 Mineral mix contained (Hindal Extra): Iron, 30 mg; copper, 15 mg; manganese, 15 mg; zinc, 50 mg; iodine, 1.5 mg; selenium, 0.2 mg; cobalt, 0.2 mg 3 NPP, non phytate phosphorous
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Starter
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Ingredients
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Maize 605 Soybean meal 215 Vegetable Oil 30.0 Limestone 86.6 1 DCP 04.9 Sodium chloride 2 DL-Methionine 1.1 L-Lysine 9.4 Coccidiostat 0.00 Vitamin Premix2 2.5 Mineral Premix3 1 Choline chloride 1 Antioxidant 1 Antifungal 1 Analysis: Metabolisable energy (MJ/kg of dry matter) 12.17 CP4 180.0 NPP4 4.3 Ca 38 Na 1.8 1 DCP, dicalcium phosphate 2 Each kg of vitamin premix contained 2.4 × 106 IU vitamin A; 3.2 × 105 vitamin D3; 5.6 × 103 mg vitamin E, 640 mg vitamin K3; 500 mg vitamin B1; 1120 mg vitamin B2, 3200 mg niacin;1600 mg Ca-D-pantothenate; 800 mg vitamin C; 2.4 mg vitamin B12; 160 mg folic acid; 7.2 mg D-biotin; 8000 mg vitamin C and 20000 mg choline chloride. 3 Each kg of mineral premix contained 8 × 104 mg manganese; 6 × 104 mg iron; 6 × 104 mg zinc; 200 mg cobalt; 100 mg iodine and 150 mg selenium. 4 CP, crude protein; NPP, non phytate phosphorous
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Table 2. Feed composition of commercially balanced rations for laying hens
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Total CLA1 mg/g fat) Age (week) 3
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Treatment
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1 2 4 5 0.41cd 0.013 0.49 d 0.020 0.50 d 0.02 0.48 cd 0.09 0.50 c 0.06 Control 0.45 c 0.016 0.36 e 0.080 0.40 e 0.03 0.60 b 0.11 0.83 b 0.03 L. plantarum 0.33 d 0.021 0.41de 0.052 0.48 de 0.03 0.51 c 0.12 0.50 c 0.11 L. lactis cd c cd d 0.41 0.011 0.60 0.108 0.60 0.04 0.45 0.03 0.78 bc 0.12 L. casei 0.56 ab 0.012 0.71 b 0.110 0.70 b 0.066 0.61 b 0.06 0.85 b 0.086 L.fermentum 0.51b 0.032 0.82 a 0.093 0.89 a 0.112 1.62 a 0.081 1.66 ab 0.113 L.reuteri from camel a b ab a 0.60 0.091 0.73 0.081 0.76 0.081 1.60 0.083 1.63 ab 0.112 L. reuteri from cattle ab 0.63 c 0.033 0.68 c 0.088 1.58 a 0.10 1.61 ab 0.21 L. reuteri from sheep 0.53 0.120 a a a a 0.62 0.110 0.83 0.088 0.85 0.061 1.65 0.113 1.81 a 0.113 L. reuteri from goat 1 Data are expressed as the mean ± standard deviation (SD) of triplicate analysis. a,b,c,d,e Within a column, values not sharing a common superscript letter are significantly different (least significant difference tests, P < 0.05).
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Table 3. Effect of lactic acid bacterial strains on conjugated linoleic acid (CLA) content in broiler chicken meat
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Table 4. Effect of lactic acid bacterial strains on conjugated linoleic acid (CLA) concentrations in chicken eggs Total CLA 1 mg/g fat)
Treatment 1 0.03 c
L. lactis L. casei L.fermentum
0.011
0.08 c
0.011
0.12 e
0.010
0.20d
0.012
0.23a b 0.010 0.12 bc 0.011
0.21 ab
0.020
0.31 bc
0.012
0.33 d
0.012
0.46 cd
0.011
0.28 a
0.036
0.43 b
0.022
0.42 c
0.011
0.44 cd
0.016
ab
0.029
0.31
bc
0.032
c
0.013
0.42
cd
0.019
0.20 b
0.071
0.46 b
0.018
0.40 c
0.011
0.53 c
0.011
0.31 a
0.052
0.85 a
0.022
0.95 ab
0.022
1.25 a
0.033
b
0.022
0.21
0.24 ab 0.025 0.21 ab 0.011 0.29 a 0.060
0.41
ce
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d
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0.30 a 0.093 0.89 a 0.041 0.82 b 0.021 1.03 bc 0.081 L. reuteri from cattle 0.92 a 0.012 0.83 b 0.011 1.12 ab 0.062 L. reuteri from sheep 0.18 b 0.090 0.28 a 0.065 b b a a 0.19 0.012 0.21 0.028 0.81 0.091 1.08 0.071 1.11 b 0.090 L. reuteri from goat 1 Data are presented as the mean ± standard deviation (SD) of triplicate analysis. a,b,c,d,e Within a column, values not sharing a common superscript letter are significantly different (least significant difference tests, P < 0.05).
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5
0.05 c
0.18
L.reuteri from camel
2 0.010
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Control L. plantarum
Collection time (week) 3 4
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British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Enrichment of conjugated linoleic acid (CLA) in hen eggs and broiler chickens meat by lactic acid bacteria S. M. Herzallah
a
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Department of Nutrition and Food Science, Faculty of Agriculture , Mu'tah University Accepted author version posted online: 25 Aug 2013.
To cite this article: British Poultry Science (2013): Enrichment of conjugated linoleic acid (CLA) in hen eggs and broiler chickens meat by lactic acid bacteria, British Poultry Science, DOI: 10.1080/00071668.2013.836734 To link to this article: http://dx.doi.org/10.1080/00071668.2013.836734
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CBPS-2012-282 Ed. Kjaer, July 2013; MacLeod, August 2013
Enrichment of conjugated linoleic acid (CLA) in hen eggs and broiler chickens meat by
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S.M. HERZALLAH
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Department of Nutrition and Food Science, Faculty of Agriculture, Mu tah University, Karak-
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Jordan
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Running title: CLA ENRICHMENT BY BACTERIA
Correspondence to: S.M. Herzallah, Department of Nutrition and Food Science, Faculty of
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Agriculture, Mu tah University, Karak-Jordan, Postal code: 61710. Email: [email protected] , [email protected] Tel: +9632372380, +962785103354.
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lactic acid bacteria
Fax: +962 3 2323154.
Accepted for publication 28th June 2013
1
Abstract.
1. The aim of this work was to compare conjugated linoleic acid (CLA)
concentrations in chickens supplemented with 4 American Tissue Culture Collection (ATCC) bacterial strains, Lactobacillus plantarum, Lactobacillus lactis, Lactobacillus casei and Lactobacillus fermentum and 4 isolates of Lactobacillus reuteri from camel, cattle, sheep and goat rumen extracts.
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2. Microorganisms were grown anaerobically in MRS broth and 106 CFU/ml of bacteria were
administered orally to mixed-sex 1-d-old broiler chickens weekly for 4 weeks and to 23-
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3. The 5 strains were evaluated for their effects on synthesis of CLA in hen eggs and broiler meat cuts.
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4. Administration of pure Lactobacillus and isolated L. reuteri strains from camel, cattle, goat and sheep led to significantly increased CLA concentrations of 0.2–1.2 mg/g of fat in eggs
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and 0.3–1.88 mg/g of fat in broiler chicken flesh homogenates of leg, thigh and breast.
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5. These data demonstrate that lactic acid bacteria of animal origin (L. reuteri) significantly
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enhanced CLA synthesis in both eggs and broiler meat cuts. INTRODUCTION
Conjugated linoleic acid (CLA) is a positional and geometric linoleic fatty acid (LA) of cis
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and trans isomers (cis–9- and cis-12-octadecanoic). Linoleic acid is a natural food component found in plant oils (Sebedio et al.,1998; Bauman et al., 2003), whereas, dietary CLA is found in meat and dairy products from ruminants (Chin et al., 1992; Bovet et al., 2007). Poultry
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week-old layer hens weekly for 6 weeks.
meat and associated products represent about 30% of total global meat consumption (FAO, 2006). Recent studies examining nutritional compounds of public interest have revealed a role for CLA in reducing cancer cell growth and body fat accumulation (Wang and Jones, 2004). Conjugated linoleic acid (CLA) is used to improve immune function, reduce plasma
cholesterol and fatness and prevent arteriosclerosis (Schultz et al., 1992; Schonberg and Krokan, 1995; Park et al., 1997; West et al., 1998). Several bacteria express enzymes that 2
convert linoleic acid to CLA, including Propionibacterium, Eubacterium, Butyrivibrio and Lactobacillus strains (Kepler et al., 1966; Eyssen and Verhulst, 1984; Jiang et al., 1998). Moreover, isomerisation of linoleic acid to CLA acts as a defence mechanism against bacteria by inhibiting bacterial growth (Verhulst et al., 1986; Jiang et al., 1998). However, direct inhibition of bacterial growth by LA and CLA has not been reported to date. Bacterial species
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that produce lactic acid are often used in food fermentation, and those present in the
mammalian gastrointestinal tract provide several health benefits (Holzapfel et al., 1998).
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effects and immune stimulation (Mallet et al., 1984; Hill, 1995, 1998; Morotomi et al., 1997; Vonk et al., 1997; Ringø et al., 1998). In the present study, a variety of lactic acid bacteria
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(LAB) strains was administered, and subsequent CLA enrichment in eggs and broiler chicken meat was determined.
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MATERIAL AND METHOD
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Lactobacillus plantarum (ATCC 8014), Lactobacillus casei (ATCC 393), Lactobacillus lactis
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(ATCC 7830) and Lactobacillus fermentum (ATCC 9338) were purchased from American Tissue Culture Collection (ATCC). L. reuteri strains were isolated from goat, sheep, cattle and camel rumen. Then the isolated bacteria were cultured in MRS broth (Difco, Detroit,
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USA).
Management of birds Experimental design I
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These intestinal microflora liberate free fatty acids from unabsorbed fat, leading to anticancer
A total of 180 one-d-old Hubbard broiler chickens were purchased from Hammoudeh Poultry Company, Amman, Jordan. Chickens were randomly divided into 9 groups of 10 chickens and were housed in rearing cages at the agriculture experimental laboratory. Chickens were
reared under standard hygienic conditions with 24 h lighting and free access to water. Chickens were given commercially balanced rations of starter diet for 4 weeks, and a finisher diet was provided in week 5 (Table 1). Group I were given a balanced diet and were not 3
treated with bacteria (control). The remaining groups (II–IX) received weekly supplements of 1 ml of microbial cultures containing 106 CFU/ml of L. plantarum, L. lactis, L. casei, L. fermentum or L. reuteri from camel, L. reuiteri from cattle, L. reuteri from sheep or L. reuteri from goat. At the end of every week, two chickens were slaughtered in the commercial poultry slaughter plant. Chicken breasts and legs were deboned, ground with skin and
Experimental design II
Table 1 near here
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subcutaneous fat and stored at −20 ± 1°C until analysis.
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each of 9 bacterial treatment groups. The chickens were reared under standard hygienic
conditions in cages and were given a balanced commercial layer hen’s ration (Table 2) for 3
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weeks of acclimatisation. Layer hens of group I were kept as controls and were not administered any microbial strains. Hens in groups II–IX were orally administered 1 ml of
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bacterial cultures containing 106 CFU/ml of L. plantarum, L. lactis, L. casei, L. fermentum or
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L. reuteri from camel, L. reuteri from cattle, L. reuteri from sheep or L. reuteri from goat.
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Eggs were collected, broken, homogenised and subsampled every week for 5 weeks, stored at 4 ± 1°C and were assayed for CLA concentration in triplicate within 3 d.
Table 2 near here
Fat extraction, CLA derivatisation and gas chromatography analysis
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Lipids were extracted from homogenised deboned raw poultry meat (breast and leg muscles with skin) or eggs using chloroform and methanol as described by Bligh and Dyer (1959) and Herzallah et al. (2005), and were stored at −20 ± 1°C until analysis. CLA methyl esters were
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A total of 27 23-week-old commercial Hisex white layer hens were assigned in triplicate to
prepared by dissolving 60 mg of extracted fat in 100 µl of hexane, and were then vortexed with 100 µl of 2 N methanolic KOH (ISO-IDF, 2002) for 30 s. Formation of CLA methyl ester was measured by injecting 1µl aliquots into capillary columns (TR-CN100, 60 m, 0.2 µm, 0.25 mm i.d., Teknokroma, Barcelona, Spain) of a Shimadzu gas chromatograph (Shimadzu 2010, Kyoto, Japan) equipped with a flame ionisation detector (GC–FID). Injector and detector temperatures were set at 250 and 300°C, respectively, carrier gas was N 2 (0.3 4
ml/min) with a split ratio of 1:50. The oven temperature was 150°C for the first 5 min, was increased at a rate of 5°C/min to 240°C and was held for a final 5 min. CLA isomers were determined and identified using a CLA standard purchased from Sigma-Aldrich (St Louis, MO, USA). Analyses of CLA cis and trans isomers of 9,11- and 10,12-octadecadienoic acid methyl ester were conducted using response areas of 3 replicate injections, and the mean ±
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standard deviation (SD) was calculated.
Preparation of the CLA methyl ester standard calibration curve
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hexane (gas chromatography grade) and concentrations of 0.0, 1.0, 2.0, 4.0 and 10 µg/ml were injected into the gas chromatography system (Shimadzu 2010, Tokyo, Japan).
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CLA recovery
CLA recovery was determined by mixing 30 µl of 50-µg/ml CLA standard with 10 ml of
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hexane and then incorporating 5 g samples of finely ground, homogenised, fat free oven dried
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meat and/or eggs. CLA spiked samples were homogenised and stored under nitrogen in 20-ml
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brown vials and were kept for 3 d at 4.0 ± 1.0°C. Subsequently, CLA content was re-extracted from 1 g samples in triplicate using 5 ml of hexane, and the 3 hexane portions were combined and dried over anhydrous sodium sulphate (99%; Sigma-Aldrich, MO, US). CLA was
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determined in 5 replicates; 5 µl of CLA methyl ester was injected in triplicate into a GC–FID and the average CLA recovery values were 96.4% and 97.3%, respectively.
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The 250-mg CLA-methyl ester standard (Sigma, O5632, USA) was dissolved in 10 ml of
Statistical analysis Mean changes in CLA content of eggs and broiler flesh were compared to that in control animals using PC-SAS version 9.0 (SAS Institute, 2002). The least significant difference (LSD) test was performed with mean values of CLA measurements using a Completely Randomised Design (CRD) and differences were considered significant when P < 0.05. RESULTS
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CLA contents of poultry meat from chickens administered LAB are presented in Table 3. All LAB strains enhanced formation of CLA and increased concentrations in poultry meat, particularly L. reuteri. Table 4 shows that weekly administration of 106 CFU/ml of LAB strains to laying hens increased CLA contents of eggs. All strains led to increased concentrations of CLA in eggs, and L. reuteri, L. fermentum and L. plantarum were the most effective. DISCUSSION
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Tables 3 and 4 near here
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and 1.85 mg/g fat) after 4 and 5 weeks compared to 0.03 and 0.08 mg/g fat found in control chickens in week 1 and 3, respectively. CLA concentrations were significantly higher in
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poultry meat of broiler chickens that were administered L. reuteri strains from rumen digests of camel, cattle, goat and sheep than in control and LAB supplemented chickens. CLA was
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found at 0.83 mg/g fat in poultry meat from chickens administered L. plantarum, and at 1.8
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mg/g fat in meat samples from chickens reared with L. reuteri from sheep rumen digests. This
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CLA enrichment in poultry meat samples from LAB-reared chickens is in agreement with data from Crespo and Esteve-Garcia (2001) showing that feeding broilers on dietary fat (6 to 10%) produced high concentrations of n-3 PUFA, which met > 70% of the recommended
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daily allowance (RDI) for humans. Moreover, CLA concentrations were increased by 46% in breast meat and 36% in thigh meat. Grashorn (2007) also showed enrichment of poultry meat with CLA and omega-3 fatty acids, meeting 3–11% and 70–130% of human RDIs,
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CLA concentrations in broiler meat were higher in L. reuteri supplemented chickens (1.65
respectively, in broiler chickens receiving rapeseed oil and -tocopherol supplements. These
observations agree with data showing that supplementation of the LAB L. zeae ATCC 393, L. reuteri ATCC 23272 and L. acidophilus (NCFM strain) with fatty acids increased the concentration of cis-9, trans-11-CLA by 20% in bacterial cell membranes (Jenkins and Courtney, 2003). Hence CLA content in poultry meat (breast, thigh) could be increased by about 40% with dietary CLA supplementation (Du and Ahn, 2002; Aletor et al., 2003; Sirri et 6
al., 2003; Grashorn, 2005; Suksombat et al., 2007; Shin et al., 2011). In addition, Blankson et al. (2000) reported that CLA (3.4 g/d) aided loss of 28.3–30.3 kg of body fat in overweight and obese humans after 12 weeks of exercise training. Both cis-9 and trans-11 CLA isomers were increased in egg lipids, and varied with the type and source of LAB administered to Hisex white laying hens. The concentrations of CLA
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in eggs collected during the first week did not differ significantly (P > 0.05) between bacterial supplement groups, but were significantly higher than in control animals (P < 0.05). The
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supplementation, with higher concentrations in hens given isolated bacterial strains from camel, cattle, sheep and goats. Indeed, the highest CLA concentrations were detected after 5
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weeks supplementation with L. reuteri from rumen digests, with CLA concentrations of 1.25, 1.12, 1.11 and 1.03 mg/g fat in eggs from hens administered L. reuteri from camel, sheep,
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goat and cattle, respectively. In comparison, CLA concentrations were 0.2 and 0.46 mg/g fat
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in control and L. plantarum supplemented eggs, respectively. High CLA production by L.
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reuteri was also reported by Jenkins and Courtney (2003), who showed that supplementation of L. reuteri ATCC 55739 growth medium with exogenous CLA and linoleic fatty acids strongly induced detoxification of linoleic acid by isomerisation to CLA in bacterial cell
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membranes. Narahari (2003) reported enrichment of eggs with fatty acids and omega-3, with 30 fold increases in linoleic (LA) and docosahexanoic (DHA) acids in egg yolk of laying hens supplemented with special oil blends. CLA can also be enriched in eggs by supplementing
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enrichment of CLA in eggs from Hisex laying hens increased with the duration of microbial
layer hens with CLA (Sirri et al., 2003) or oil blends (linseed, rapeseed; Du et al., 2001). Yin et al. (2008) showed that CLA supplementation of hens increased CLA deposition in egg yolk lipids, even with soybean-based diets containing < 5% CLA. Additionally, Szymczyk and Pisulewski (2003) found increased CLA and fatty acids in eggs yolk from laying hens given < 20 g/kg CLA. In conclusion, the present results suggest that CLA production in both meat and eggs can be enhanced by LAB, particularly by isolated L. reuteri. 7
ACKNOWLEDGEMENTS The authors wish to thank the Dean of the University of Jordan Research for financial support of this work. The authors also thank the University of Mu'tah for the use of their gas chromatography and HPLC equipment. REFERENCES
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Table 1. Basic composition of the experimental broiler diets Finisher
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Ingredients g/kg Maize 585 670.5 Soybean meal (48% protein) 356.5 260.0 Vegetable oil (Palm oil) 16.9 30.0 Limestone 18.4 16.8 Dicalcium phosphate (DCP, Ca, 10.0 10.2 28% and P, 19%) Sodium chloride 4.1 4.2 DL-Methionine (99%) 2.0 2.0 L-Lysine (98%) 1.1 1.3 Coccidiostat (Hindal, 66 g/kg) 1.0 0.00 Vitamin Premix1 1.0 1.0 Mineral Premix2 1.0 1.0 Choline chloride (60%) 1.0 1.0 Antioxidant (Fintox, HA) 1.0 1.0 Antifungal (Fintox) 1.0 1.0 Calculated nutrient Composition: Metabolisable energy (MJ/kg of 12.47 13.21 dry matter) Crude protein (g/kg) 223.0 182.0 3 NPP (g/kg) 4.5 4.0 Ca (g/kg) 10.3 9.5 Na (g/kg) 1.8 1.8 1 Vitamin premix (Hindal Extra, Jordan) contained the following nutrients per kg of feed: vitamin A, 12000 IU (retinyl acetate); Cholecalciferol (D3), 3000 IU; vitamin E, 20 IU (DLα-tocopheryl acetate); vitamin K (menadione sodium bisulphate, 3.3 mg; thiamin, 2.5 mg; riboflavin, 5 mg; pantathenic acid, 13 mg; niacin, 30 mg; vitamin C, 100 mg; vitamin B12, 0.02 mg; biotin, 0.05 mg; pyridoxine, 4 mg; folic acid, 1 mg, ethoxyquin, 13 mg. 2 Mineral mix contained (Hindal Extra): Iron, 30 mg; copper, 15 mg; manganese, 15 mg; zinc, 50 mg; iodine, 1.5 mg; selenium, 0.2 mg; cobalt, 0.2 mg 3 NPP, non phytate phosphorous
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Starter
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Ingredients
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Maize 605 Soybean meal 215 Vegetable Oil 30.0 Limestone 86.6 1 DCP 04.9 Sodium chloride 2 DL-Methionine 1.1 L-Lysine 9.4 Coccidiostat 0.00 Vitamin Premix2 2.5 Mineral Premix3 1 Choline chloride 1 Antioxidant 1 Antifungal 1 Analysis: Metabolisable energy (MJ/kg of dry matter) 12.17 CP4 180.0 NPP4 4.3 Ca 38 Na 1.8 1 DCP, dicalcium phosphate 2 Each kg of vitamin premix contained 2.4 × 106 IU vitamin A; 3.2 × 105 vitamin D3; 5.6 × 103 mg vitamin E, 640 mg vitamin K3; 500 mg vitamin B1; 1120 mg vitamin B2, 3200 mg niacin;1600 mg Ca-D-pantothenate; 800 mg vitamin C; 2.4 mg vitamin B12; 160 mg folic acid; 7.2 mg D-biotin; 8000 mg vitamin C and 20000 mg choline chloride. 3 Each kg of mineral premix contained 8 × 104 mg manganese; 6 × 104 mg iron; 6 × 104 mg zinc; 200 mg cobalt; 100 mg iodine and 150 mg selenium. 4 CP, crude protein; NPP, non phytate phosphorous
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Table 2. Feed composition of commercially balanced rations for laying hens
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cr ip t
Total CLA1 mg/g fat) Age (week) 3
us
Treatment
ce
pt e
d
M
an
1 2 4 5 0.41cd 0.013 0.49 d 0.020 0.50 d 0.02 0.48 cd 0.09 0.50 c 0.06 Control 0.45 c 0.016 0.36 e 0.080 0.40 e 0.03 0.60 b 0.11 0.83 b 0.03 L. plantarum 0.33 d 0.021 0.41de 0.052 0.48 de 0.03 0.51 c 0.12 0.50 c 0.11 L. lactis cd c cd d 0.41 0.011 0.60 0.108 0.60 0.04 0.45 0.03 0.78 bc 0.12 L. casei 0.56 ab 0.012 0.71 b 0.110 0.70 b 0.066 0.61 b 0.06 0.85 b 0.086 L.fermentum 0.51b 0.032 0.82 a 0.093 0.89 a 0.112 1.62 a 0.081 1.66 ab 0.113 L.reuteri from camel a b ab a 0.60 0.091 0.73 0.081 0.76 0.081 1.60 0.083 1.63 ab 0.112 L. reuteri from cattle ab 0.63 c 0.033 0.68 c 0.088 1.58 a 0.10 1.61 ab 0.21 L. reuteri from sheep 0.53 0.120 a a a a 0.62 0.110 0.83 0.088 0.85 0.061 1.65 0.113 1.81 a 0.113 L. reuteri from goat 1 Data are expressed as the mean ± standard deviation (SD) of triplicate analysis. a,b,c,d,e Within a column, values not sharing a common superscript letter are significantly different (least significant difference tests, P < 0.05).
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Table 3. Effect of lactic acid bacterial strains on conjugated linoleic acid (CLA) content in broiler chicken meat
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Table 4. Effect of lactic acid bacterial strains on conjugated linoleic acid (CLA) concentrations in chicken eggs Total CLA 1 mg/g fat)
Treatment 1 0.03 c
L. lactis L. casei L.fermentum
0.011
0.08 c
0.011
0.12 e
0.010
0.20d
0.012
0.23a b 0.010 0.12 bc 0.011
0.21 ab
0.020
0.31 bc
0.012
0.33 d
0.012
0.46 cd
0.011
0.28 a
0.036
0.43 b
0.022
0.42 c
0.011
0.44 cd
0.016
ab
0.029
0.31
bc
0.032
c
0.013
0.42
cd
0.019
0.20 b
0.071
0.46 b
0.018
0.40 c
0.011
0.53 c
0.011
0.31 a
0.052
0.85 a
0.022
0.95 ab
0.022
1.25 a
0.033
b
0.022
0.21
0.24 ab 0.025 0.21 ab 0.011 0.29 a 0.060
0.41
ce
pt e
d
M
an
us
0.30 a 0.093 0.89 a 0.041 0.82 b 0.021 1.03 bc 0.081 L. reuteri from cattle 0.92 a 0.012 0.83 b 0.011 1.12 ab 0.062 L. reuteri from sheep 0.18 b 0.090 0.28 a 0.065 b b a a 0.19 0.012 0.21 0.028 0.81 0.091 1.08 0.071 1.11 b 0.090 L. reuteri from goat 1 Data are presented as the mean ± standard deviation (SD) of triplicate analysis. a,b,c,d,e Within a column, values not sharing a common superscript letter are significantly different (least significant difference tests, P < 0.05).
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5
0.05 c
0.18
L.reuteri from camel
2 0.010
cr ip t
Control L. plantarum
Collection time (week) 3 4
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