Animal Feed Science and Technology 123–124 (2005) 643–653 Effect of pH and oxygen on conjugated linoleic acid (CLA) production by mixed rumen bacteria from cows fed high concentrate and high forage diets Nag-Jin Choi a, Jee Young Imm b, Sejong Oh c, Byoung-Chul Kim d, Han-Joon Hwang e, Young Jun Kim e,∗ a National Livestock Research Institute, Suwon, Kyonggi 441-706, Republic of Korea b Department of Food and Nutrition, Kookmin University, Seoul 136-702, Republic of Korea c Department of Animal Science, Chonnam University, Gwangju 500-600, Republic of Korea d Division of Food Science, Korea University, 5-1 Anam, Sungbuk-gu, Seoul 136-701, Republic of Korea e Department of Food and Biotechnology, Korea University, Chochiwon, Yeon-ki kun, Chungnam 339-700, Republic of Korea Received 19 March 2004; received in revised form 21 March 2005; accepted 15 April 2005 Abstract Conjugated linoleic acid (CLA) production from linoleic acid (LA) was characterized, in a rela- tionship to biohydrogenation (BH), with mixed rumen bacteria obtained from three cows fed a high concentrate diet (HCD; 630 g/kg) or a high forage diet (HFD; 770 g/kg) for 5 weeks. Mixed rumen bacteria were incubated with LA in a rumen fluid medium under various conditions. Rumen bacteria from cows fed HCD were more active (P < 0.05) in BH than those from cows fed the HFD at most tested pHs. Rumen bacteria from HFD fed cows produced mostly cis-9, trans-11 CLA at pHs higher than 6.2, but those from HCD fed cows produced more (P < 0.05) trans-10, cis-12 CLA than cis-9, trans-11 CLA at lower pHs. Production of cis-9, trans-11 CLA, positively correlated, and trans-10, cis-12 CLA inversely correlated, to pH with rumen bacteria from cows fed both diets (r2 = 0.88). Rumen bacteria from HCD fed cows accumulated about four times more cis-9, trans-11 CLA in aerobic conditions, versus anaerobic conditions, after 1 h of incubation. Overall, trans-10, cis-12 Abbreviations: BH, biohydrogenation; CLA, conjugated linoleic acid; FA, fatty acid; HCD, high concentrate diet; HFD, high forage diet; LA, linoleic acid; PUFAs, polyunsaturated fatty acids ∗ Corresponding author. Tel.: +82 41 860 1435; fax: +82 41 865 0220. E-mail address: [email protected] (Y.J. Kim). 0377-8401/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2005.04.054 644 N.-J. Choi et al. / Animal Feed Science and Technology 123–124 (2005) 643–653 CLA producing rumen bacteria may be more acid-tolerant and aero-tolerant than cis-9, trans-11 CLA producing rumen bacteria. © 2005 Elsevier B.V. All rights reserved. Keywords: Conjugated linoleic acid; Rumen bacteria; Diet; pH; Aerobic condition 1. Introduction Conjugated linoleic acid (CLA) refers to a group of conjugated fatty acid (FA) isomers of octadecadienoate (C18:2) with double bonds with different positional and geometric configurations (Sehat et al., 1998). Since Ha et al. (1987) first identified the antimutagenic properties of CLA, it has been the object of intensive research for its health promoting roles and the uniqueness of its dietary sources (Chin et al., 1992). Numerous potential health promoting effects have been reported and reviewed (Belury, 2002; Lee et al., 2004). Based on these reports, a great deal of effort has been expended to increase the CLA content in animal products (Banni et al., 2001; Choi et al., 2002; Weill et al., 2002). Biohydrogenation (BH) of polyunsaturated FA (PUFA) is a unique biochemical pro- cess carried out by membrane associated enzymes of some rumen bacteria, through which some CLA isomers emerge as transient intermediates (Kepler and Tove, 1967; Hughes et al., 1982). Earlier studies have shown that cow rations have strong impacts on the rumen environment, which could also affect the yield and isomer profiles of CLA, as well as other FAs of ruminant origin (Latham et al., 1972; Corl et al., 2001). Unsaturated fat supplements are often added to diets of lactating cows to alter the CLA profile in milk fat (Chouinard et al., 2001). Concentrations, and the duration of feeding fat substrates and energy sources, could affect microflora, and FA metabolism in the rumen. These dietary factors could be major determinants at ruminal CLA production (Bessa et al., 2000). In previous studies, we found that the ruminal CLA profile could be affected by the diet of cows, and that some environmental factors affect CLA accumulation by some rumen bacteria. In this study, we determined effects of ruminal pH, and the presence of oxygen, on production of cis-9, trans-11 and trans-10, cis-12 CLA isomers by mixed rumen bacteria from cows on two distinct dietary regimens, being a high concentrate diet (HCD) and a high forage diet (HFD). 2. Materials and methods 2.1. Chemicals Organic solvents and reagents were purchased from Fisher Co. (Fair Lawn, NJ, USA). cis-9, trans-11 and trans-10, cis-12 CLA isomers (>95% purity, Lipozen, Pyong Taek, Korea) were used to identify and quantify each CLA isomer. Other FA standards were obtained from Sigma Chemical Co. (St. Louis, MO, USA). N.-J. Choi et al. / Animal Feed Science and Technology 123–124 (2005) 643–653 645 2.2. Animals and diets Three non-lactating ruminally fistulated dairy cows weighing 600 ± 50 kg were fed either a HFD or a HCD. Diet cows were fed about 9 kg/day at the HFD (770 g/kg DM) twice a day. The cows were then switched to the HCD (630 g/kg DM corn grain) with a 4 weeks interval and acclimated to this diet for 5 weeks. The feeding period was comprised of 3 weeks for adaptation to diets and 2 weeks for sampling. Feed composition of the HCD and the HFD diets are in the Table 1. Cows were fitted with ruminal cannulae (83 mm i.d.) by surgical procedures approved by the Cornell University Institutional Animal Care and Use Committee (protocol 95-1-00). The surgery was performed well before the rumen sampling so that cows could recover from the surgery. Table 1 Ingredients and chemical composition of bovine diet (DM basis) Diets (g/kg DM) HCDa HFDb Ingredient composition Corn grain 630 9.20 Alfalfa silage 135 17.0 Alfalfa hay 93.060.0 Soybean meal 12.012.0 Limestone 10.010.0 Calcium phosphate 5.05.0 Sodium bicarbonate 5.05.0 Calcium sulfate 0.50.5 Magnesium oxide 35.035.0 Protein supplementc 2.52.5 Mineral–vitamin supplementd 60.060.0 Chemical composition DM (g/kg) 605 440 ADF 124 205 NDF 245 355 CP 175 167 Fatty acid composition C16:0 6.87.6 C16:1 0.04 0.06 C18:0 1.52.4 C18:1 12.410.5 C18:2 25.031.0 C18:3 1.83.5 Other 0.40.6 a HCD: high concentrate diet. b HFD: high forage diet. c Commercial supplement containing blood meal, feather meal and corn and gluten meal (Taylor By-Products, Wyalusing, WA, USA). d Mineral–vitamin mix contained (mix) Mn, 3500 mg/kg; Zn, 3000 mg/kg; Cu, 750 mg/kg; Fe, 20 mg/kg; I, 85 mg/kg; Co, 15 mg/kg; Se, 35 mg/kg; retinyl acetate (2,100,000 IU/kg), cholecalciferol (390,000 IU/kg), ␣- tocopheryl acetate (7500 IU/kg). 646 N.-J. Choi et al. / Animal Feed Science and Technology 123–124 (2005) 643–653 2.3. Mixed rumen culture Rumen fluid was obtained from all three cows at 5 h after morning feeding. Rumen fluid from cows fed HCD was strained through cheese cloth into a flask. For the preparation of rumen fluid medium with different pH, rumen fluid was centrifuged twice at 5000 × g at 22 ◦C for 5 min to remove feed particles and diluted three-fold with N-free basal medium as described previously (Kim et al., 2003). Rumen bacteria from cows fed the different diets were inoculated into the clarified sterile rumen fluid medium (3 mL/10 mL). Mixed rumen bacterial cells (approximately ◦ 0.4 g protein/L) were incubated with LA (200 mg/L) at 39 C under O2-free CO2 in 15 mL rubber-stoppered glass tubes and the OD was measured at 600 nm with a spectrophotometer (UV-1201, Shimadzu, Kyoto, Japan). Bovine serum albumin was used to ensure that LA remained in suspension during incubation (Kim et al., 2002). Rumen fluid medium at five pHs between 5.6 and 6.8 was prepared by adding clarified sterile rumen fluid obtained from cows fed the HCD (pH 5.6 ± 0.32) or the HFD (pH 6.8 ± 0.25) in different ratios. The mixed rumen bacteria were inoculated to the rumen fluid medium with different pHs, and BH and CLA productions were determined after a 6 h incubation. Studies on the effect of aerobic incubation were performed with sterile aeration for 12 h. 2.4. Fatty acid analyses Fat and FA were extracted from cow’s diets and mixed rumen cultures with a mixture of organic solvents (one part hexane, three parts isopropanol, one part acetone). The sus- pensions were then centrifuged at 1000 × g for 3 min at 20 ◦C. The solvent (top) layer was removed, and flushed with N until dry. The FAs were dissolved in toluene and methylated (Kim and Liu, 1999). Fatty acid methyl esters were separated on a Supelcowax-10 fused silica capillary column (60 m × 0.32 mm, 0.5 ␮m film thickness; Supelco. Inc., Bellefonte, PA, USA) using a gas chromatograph (Hewlett-Packard, HP5890, Avondale, PA, USA) equipped with a flame ionization detector.