Effect of Ph and Oxygen on Conjugated Linoleic Acid (CLA) Production by Mixed Rumen Bacteria from Cows Fed High Concentrate and High Forage Diets

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).

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. The conditions were: 2.4 mL/min helium flow; in- jector 200 ◦C; detector 250 ◦C; the column chamber temperature was initially 40 ◦C (5 min), and was increased to 220 ◦Cat20◦C/min and held for 30 min. Samples (1 ␮L) containing 0.5–5 ␮g of FA were injected into the column. Heptadecanoic acid (C17:0) was used as an internal standard and all CLA isomers and other FAs were quantified using FA standards.

2.5. Statistical analyses

All data are presented as mean ± S.D. for at least three replications. Statistical analyses were conducted using SigmaStat program (Version 8.0; Jandel Corp., San Rafael, CA, USA). Differences among treatments were determined using the Student t-test (P < 0.05).

3. Results

When dietary effects on CLA production were determined at various pHs by incu- bating LA in pH-adjusted rumen ﬂuid with rumen bacteria from cows fed the HCD or N.-J. Choi et al. / Animal Feed Science and Technology 123–124 (2005) 643–653 647

Fig. 1. Effect of pH on (a) biohydrogenation products and (b) remaining LA in the incubation of rumen bacteria from the cows fed HFD (᭹) or HCD (). Mixed rumen cultures were obtained from HFD and HCD fed cows and were incubated with 200 mg/L of LA for 6 h in anaerobic conditions [asterisk (*) indicates difference between two groups (P < 0.05)].

the HFD duts, substantial internal variation was observed at low pH in the culture from both dietary groups, although the amounts of hydrogenated products (C18:0 and trans- C18:1) were higher with the HCD at pH 5.65, 6.4 and 6.8 after 6 h of incubation (P < 0.05, Fig. 1a). The remaining LA reﬂected the amount of hydrogenated products formed at each pH (Fig. 1b). Overall, BH was more active with rumen bacteria from cows on the HCD (75–170 mg/L) compared to the HFD (30–75 mg/L) from 200 mg/L LA at most of the tested pHs. 648 N.-J. Choi et al. / Animal Feed Science and Technology 123–124 (2005) 643–653

Fig. 2. The effect of pH on CLA isomer distribution (a) cis-9, trans-11 and (b) trans-10, cis-12: HFD (᭹) and HCD () [values on y-axis show portion of each CLA isomer in total fat].

For both diets, the level of trans-10, cis-12 CLA was higher than that of cis-9, trans-11 CLA at pH values lower than 6.0, whereas little trans-10, cis-12 CLA was detected at higher pHs. In contrast, the portion of cis-9, trans-11 isomer increased steadily with pH, and more than 90% of the detected CLA was cis-9, trans-11 isomer at pH higher than 6.3 (Fig. 2a). trans-10, cis-12 CLA production was inversely correlated with pH (r2 = 0.88), and little trans-10, cis-12 CLA was detected at pH 6.8 in the culture of cows fed both the HCD and HFD (Fig. 2b). Effects of aerobic incubation on CLA production of rumen bacteria obtained from HCD fed cows are shown in Fig. 3. Mixed rumen bacteria were incubated for 12 h in a rumen ﬂuid medium at pH 6.3, the pH at which the difference in the major isomer levels was N.-J. Choi et al. / Animal Feed Science and Technology 123–124 (2005) 643–653 649

Fig. 3. The effect of aerobic condition on (a) cis-9, trans-11 and (b) trans-10, cis-12 CLA production by rumen bacteria [mixed rumen cultures obtained from cows fed HCD were incubated with 200 mg/L of LA for 12 h in an aerobic condition (᭹) or an anaerobic condition (). Asterisk (*) indicates the difference between two groups (P < 0.05)]. greatest. The concentrations of cis-9, trans-11 CLA isomers were always higher (P < 0.05) in aerobic conditions than in anaerobic conditions throughout the incubation (Fig. 3a), but the difference was not signiﬁcant in trans-10, cis-12 CLA isomer production after 6 h of incubation (Fig. 3b). After 1 h of incubation, cis-9, trans-11 CLA rapidly accumulated aerobically, and was about three times higher than that accumulated anaerobically (Fig. 3a). A difference in cis-9, trans-11 CLA level (P < 0.05) occurred throughout the incubation between aerobic and anaerobic incubations. In both conditions, cis-9, trans-11 CLA isomer level did not increase after 6 h of incubation, and more (P < 0.05) trans-10, cis-12 CLA was accumulated than cis-9, trans-11 CLA in 12 h. 650 N.-J. Choi et al. / Animal Feed Science and Technology 123–124 (2005) 643–653

4. Discussion

Bovine diets were shown to impact ruminal BH and CLA production in previous studies (Kim et al., 2003). It has been suggested that factors such as oxygen, rumen pH, carbon sources and antibiotics can all affect CLA isomer production of rumen bacteria (Martin and Jenkins, 2002; Kim, 2003). The overall extent of BH is usually higher with mixed rumen bacteria obtained from cows fed the HCD versus those from cows fed HFD (Latham et al., 1972; Kim et al., 2003). When cows were fed the HCD, total lipid concentration in the rumen increased, and this could cause a surge of LA substrate for ruminal BH. Rumen fluid medium was used because unidentified compounds in rumen fluid were thought to be necessary for complete BH (Viviani et al., 1967). In fact, addition of rumen fluid to a pure culture of Butyrivibrio fibrisolvens A38 was shown to improve BH in inhibitory consitions (Kim, 2003). The pH had a marked influence on the ruminal BH. Ruminal pH depends strongly on diet, although rumen pH is relatively well maintained by buffering capacities of carbonate in saliva and volatile fatty acid absorption (Martin and Jenkins, 2002). However, active fermentation of excess carbohydrate in the rumen is typically accompanied by a decrease in ruminal pH (Latham et al., 1972; Komisarczuk et al., 1987). We observed very low CLA production occurred under non-physiological pH conditions, especially in rumen cultures from the HCD. Extreme pHs, either higher or lower than typical rumen pH (6.7 ± 0.2), may be unfavorable for CLA accumulation, because the physiology of the rumen bacteria, and the enzyme activity responsible for BH, could be affected (Kopecny et al., 1983). Overall, BH may be decreased if cell membrane integrity is disrupted at non-physiological pH conditions, because rumen bacterial enzymes responsible for BH are cell membrane associated (Kepler and Tove, 1967). Moreover, pH may also be an important factor in synthesis of CLA by non- ruminal bacteria, as pH above 5 during lactic acid fermentation improved CLA synthesis by a lactic acid bacterium, Lactococcus lactis (Kim and Liu, 2002). Even modest declines in pH interfere with adherence of rumen bacteria to cellulosic material and enzymatic cellulolysis (Kopecny et al., 1983). In fact, the major hydrogenating rumen bacteria, such as B. fibrisolvens sp., are known to be cellulolytic (Latham et al., 1972; Martin and Jenkins, 2002). In fact, the rate of cellulose digestion was optimal and the maximal growth of the most predominant cellulolytic rumen bacteria was observed when pH was neutral, whereas low pH was inhibitory to enzymatic cellulolysis (Van Gylswyk and Roche, 1970). Thus, rumen BH could be limited due to energy deficits in the cell when adherence of rumen bacteria to feed particles is loosened, or cellulolysis is inhibited. Higher trans-10, cis-12 CLA than cis-9, trans-11 CLA was observed at low pH conditions. Production of trans-10, cis-12 CLA was much higher (P < 0.05) than that of cis-9, trans-11 CLA at pH 5.65, and little trans-10, cis-12 CLA was found at pH higher than 6.5 in the culture of rumen bacteria from cows fed HFD (Fig. 2a). It was postulated that enzyme activity of trans-10, cis-12 CLA-producing bacteria may have been more active than that of cis-9, trans-11 CLA-producing bacteria at low pH or trans-10, cis-12 CLA-producing bacteria were selected in low pH. Some fermentation products, mainly volatile fatty acids, could affect the ruminal environment, which may benefit bacteria responsible for specific isomer production. Indeed, increases in lactogenic and propionogenic bacteria were observed when cows were fed HCD (Latham et al., 1972) and some lactic acid-utilizing bacteria produced N.-J. Choi et al. / Animal Feed Science and Technology 123–124 (2005) 643–653 651 more trans-10, cis-12 CLA than cis-9, trans-11 CLA (Kim et al., 2002). Indeed, some propionibacteria showed the unique activity of trans-10, cis-12 CLA production (Jiang et al., 1998). Thus, trans-10, cis-12 CLA-producing bacteria may be more acid-tolerant than cis-9, trans-11 CLA-producing bacteria. In the 6 h incubations, it is not clear if the higher portion of trans-10, cis-12 CLA in the culture from cows on the HFD was due to the altered bacterial population. Most cellulolytic bacteria in the rumen, including B. fibrisolvens sp., grow only in strictly anaerobic conditions making complete ruminal BH is possible only under anaerobic conditions. However, B. fibrisolvens A38 showed a rapid cis-9, trans-11 CLA accumulation in aerobic conditions leaving little hydrogenated product (Kim et al., 2000). Mixed rumen bacteria also produced cis-9, trans-11 CLA, which accumulated to a higher extent in aerobic conditions, but the decrease in a prolonged incubation was not as rapid as the pure culture of B. fibrisolvens A38 (Kim et al., 2000). Aerobic incubations with LA might be more favorable to production of CLA isomers by inactivating the CLA reduction steps, which are active only when energy metabolism is ‘normal’ (Kim, 2003). Indeed, less hydrogenated products of B. fibrisolvens A38 were produced in aerobic conditions versus anaerobic conditions (Kim et al., 2000). Aerobic incubations did not result in increase in trans-10, cis-12 CLA synthesis (Fig. 3b). Rumen fluid obtained from cows fed HCD was used for the aerobic incubations because no differences occurred in comparative preliminary studies with HFD (data not shown) and time-dependent changes in trans-10, cis-12 CLA both in aerobic and in anaerobic conditions. This indicates that trans-10, cis-12 CLA producing bacteria may be more aero-tolerant than the cis-9, trans-11 CLA-producing bacteria, and that each CLA-producing rumen bacterium differs in its sensitivity to conditions in the rumen, such as pH and oxygen, and thus some rumen bacteria could be enriched, and selected, from the rumen by modification of the ruminal environment (Jiang et al., 1998; Kim et al., 2002).

5. Conclusions

Results from these in vitro studies on rumen bacterial CLA production showed that rumen BH could give rise to large amounts of trans-10, cis-12 CLA when cows were fed a HCD which created a low pH. Under aerobic condition, more cis-9, trans-11 CLA accumulated compared to anaerobic conditions, whereas no difference was found in trans-10, cis-12 CLA in an aerobic condition. Thus, bacteria producing trans-10, cis-12 CLA may be less sensitive to low pH, as well as to the aerobic condition, both of which could be lethal to the cis-9, trans-11-producing anaerobes (e.g., B. ﬁbrisolvens A38) (Jiang et al., 1998; Kim et al., 2002). However, difference in net CLA production due to the cow’s diet obtained in vitro must be used to predict results in vivo with care.

Acknowledgements

The authors acknowledge the assistance of Dr. James B. Russell in the Ruminant Phys- iology laboratory in the section of Microbiology at Cornell University, who provided the 652 N.-J. Choi et al. / Animal Feed Science and Technology 123–124 (2005) 643–653 essential experimental tools, and Dr. Jean B. Hunter, associate professor in the Department of Biological and Environmental Engineering at Cornell University for valuable discussion and careful revision of the text. This work was supported by research grant (20050401-034-701-136-0300) from Bi- ogreen 21 of Rural Development Association.

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