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Factors affecting conjugated linoleic acid and trans-C_18:1 fatty acid production by mixed ruminal bacteria¹

S. A. Martin^*,2 and T. C. Jenkins

^* Department of Animal and Dairy Science, The University of Georgia, Athens 30602-2771 and and Department of Animal and Veterinary Sciences, Clemson University, Clemson, SC 30643

² Correspondence:
312 Animal and Dairy Science Complex (phone: 706-542-1065; fax: 706-583-0274; E-mail:
scottm{at}arches.uga.edu).

	Abstract

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The objective of this study was to identify environmental factors that influence conjugated linoleic acid (CLA) and trans-C_18:1 fatty acid production by mixed ruminal bacteria. Ruminal contents were collected from a 600-kg ruminally fistulated Hereford steer maintained on pasture. Mixed ruminal bacteria were obtained by differential centrifugation under anaerobic conditions and added to a basal medium that contained a commercial emulsified preparation of soybean oil and a mixture of soluble carbohydrates (cellobiose, glucose, maltose, and xylose). Culture samples were collected from batch culture incubations at 0, 2, 4, 6, 8, 12, 24, 26, 28, 30, 32, and 48 h. Continuous culture incubations were conducted at dilution rates of 0.05 and 0.10 h^-1 with extracellular pH values of 5.5 and 6.5, and 0.5 and 1.0 g/L of mixed soluble carbohydrates. Culture samples were obtained from the culture vessel once steady-state conditions had been achieved. In batch culture, trans-C_18:1 concentrations increased over time and reached a maximum at 48 h. Little CLA was produced during the first 8 h, but cis-9, trans-11 CLA concentrations remained high between 24 and 30 h. When mixed ruminal bacteria were maintained in continuous culture on 0.5 g/L of mixed soluble carbohydrates, concentrations of trans-C_18:1 and cis-9, trans-11 CLA were reduced (P < 0.05) at a dilution rate of 0.05 h^-1 and an extracellular pH of 5.5. Similar effects were also observed when 1.0 g/L of mixed soluble carbohydrates was used. When extracellular pH was lowered to 5.0, neither trans-C_18:1 or CLA isomers were detected. In conclusion, our results suggest that culture pH appears to have the most influence on the production of trans-C_18:1 and CLA isomers by mixed ruminal bacteria.

Key Words: Bacteria • Conjugated Linoleic Acid • Fermentation • Rumen

	Introduction

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Linoleic acid is an 18-carbon unsaturated fatty acid with two double bonds in positions 9 and 12, respectively, and both are in the cis configuration (Ip, 1994). Conjugated linoleic acid (CLA) contains cis and trans isomers at carbons 8 and 10, 9 and 11, 10 and 12, or 11 and 13 (Ip, 1994; Garcia et al., 1998). There are multiple potential isomers, but the cis-9, trans-11 and trans-10, cis-12 isomers are thought to be active as potential antioxidant, anticarcinogenic, antiobesity, and immune-modulating agents (Lin et al., 1995; Park et al., 1999). The details of how CLA mediates these effects have not been elucidated. However, based on the role of ruminal bacteria in the biohydrogenation of unsaturated fatty acids, there has been much interest over the past few years in maximizing CLA formation in the rumen with the goal of increasing CLA levels in milk and meat products.

In general, the dilution rate within the rumen is between 0.05 and 0.10 h^-1 (Hungate, 1966), and the concentrations of soluble sugars in ruminal fluid are usually very low (1.0 g/L or less), except immediately after feeding (Czerkawski, 1986). Ruminal pH can be as high as 7.0 on an all-forage diet, and as low as 5.0 with a diet high in rapidly fermentable carbohydrates (i.e., processed cereal grains) (Van Soest, 1982). It has been well documented that changes in the ruminal environment will lead to changes in microbial activity that correspond to altered end-product formation. Because the ruminal environment is subject to change, it is possible that these changes (pH, substrate types and concentrations, dilution rate) will have an effect on CLA formation by the ruminal microbial population. Therefore, the objectives of this study were to evaluate the effects of different soluble carbohydrate concentrations, extracellular pH, and dilution rates on long-chain fatty acid formation from an emulsified preparation of soybean oil by mixed ruminal bacteria.

	Materials and Methods

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Inoculum and Growth Medium.
Ruminal contents were collected from a 600-kg ruminally fistulated Hereford steer maintained on a mixed-forage pasture. Ruminal contents were squeezed through four layers of cheesecloth into an Erlenmeyer flask with an O₂-free headspace. The flask was not disturbed for 30 min while it was incubated in a 39°C water bath, permitting feed particles to rise to the top of the flask. Fluid was anaerobically transferred to centrifuge bottles (CO₂ gas phase) and centrifuged (150 x g, 4°C, 5 min) to sediment feed particles and protozoa. Particle-free fluid from the bottles that contained bacteria was anaerobically transferred (33% vol/vol) to a medium (pH 6.5) containing 292 mg of K₂HPO₄, 240 mg of KH₂PO₄, 480 mg of (NH₄)₂SO₄, 480 mg of NaCl, 100 mg of MgSO₄•7H₂O, 64 mg of CaCl₂•2H₂O, 4,000 mg of Na₂CO₃, and 600 mg of cysteine hydrochloride per liter. All animal procedures and protocols in this study were approved by the University of Georgia Animal Care and Use Committee, IACUC #A1997-10125-0.

Culture Conditions.
Particle-free fluid and media were mixed and 500 mL was anaerobically transferred to gas washing bottles (Fisher Scientific, Suwanee, GA) that were modified to remove samples through a butyl rubber stopper. The washing bottles contained 1 g/L of a mixture of soluble carbohydrates (cellobiose, glucose, maltose, xylose) and 0.56% (vol/vol) commercial emulsified soybean oil (Liposyn III 20%, Abbott Laboratories, North Chicago, IL). Incubations were also performed in the absence of the commercial emulsifed soybean oil. The bottles were constantly purged with O₂-free CO₂ and placed in a 39°C water bath and periodically mixed every few hours.

Continuous culture experiments were performed with a model F-1000 fermenter (New Brunswick Scientific Co., Edison, NJ) that had a modified 360-mL chemostat vessel for the culture of strictly anaerobic bacteria (Russell and Baldwin, 1979). The basal medium used in these incubations was identical to that described previously for use with the batch culture studies. Mixed soluble carbohydrate concentrations (cellobiose, glucose, maltose, and xylose) used in continuous culture were 0.5 g/L and 1.0 g/L, and 0.56% (vol/vol) of commercial emulsified soybean oil was used (Table 1). Dilution rates of 0.05 and 0.10 h^-1 were used. The extracellular pH was adjusted by adding concentrated hydrochloric acid to the medium reservoir. At least 98% turnover of medium in the culture vessel was allowed before samples were taken (Russell and Baldwin, 1979).

All culture samples were freeze-dried and methylated as described by King (1996). Fatty acids were analyzed by gas-liquid chromatography (Hewlett Packard 5890A, equipped with a flame ionization detector and a model 7673 auto injector, Palo Alto, CA) using a poly(alkeneglycol) fused-silica capillary column (30 m x 0.25 mm i.d., Supelco Inc., Bellefonte, PA). The temperature settings for the GLC were 260°C for the injector and 250°C for the detector, and the column oven was temperature-programmed with an initial temperature of 150°C for 2 min, raised 2°C per min for 35 min, and held for 11 min at a final temperature of 220°C. Flow rate for the carrier gas (He) was 20 cm/s. Peaks were quantified by peak area comparisons with a known amount of an internal standard (2 mg/mL of heptadecanoic acid; Sigma Chemical Co., St. Louis, MO). Peaks were identified by comparison with known commercially prepared standards. Protein from 0.2 N NaOH-hydrolyzed cells (100°C, 15 min) was determined by the method of Lowry et al. (1951), and bovine serum albumin was the standard that was treated similarly.

Design.
Batch culture incubations were performed in duplicate (n = 2) using two separate gas washing bottles, and one sample was taken from a single chemostat culture vessel on six separate days (n = 6). In the continuous culture experiments, data were analyzed by a least squares means ANOVA using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). The statistical model contained the fixed effects of soluble carbohydrate concentration, dilution rate, and culture pH, and these effects were tested by the residual mean squares.

	Results and Discussion

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Although pure cultures of ruminal bacteria have been identified that can hydrogenate unsaturated fatty acids, it is likely that many—rather than one or two individual—bacteria are involved in the hydrogenation process (Harfoot and Hazlewood, 1997; Kim et al., 2000). The involvement of many bacteria is supported by the scheme outlined by Harfoot and Hazlewood (1997). Because the bacteria seem to be more active in biohydrogenation than the protozoa or fungi, our experiments focused on the ability of mixed ruminal bacteria to produce long-chain fatty acids using in vitro batch and continuous culture techniques.

When mixed ruminal bacteria were grown in batch culture on mixed soluble carbohydrates and emulsified soybean oil, the proportion of trans-C_18:1 increased between 0 and 4 h of incubation and stayed constant up to 8 h (Table 2). There was little accumulation of cis-9, trans-11 CLA or trans-10, cis-12 CLA over this time period, and little change in C_16:0, cis-C_18:1, C_18:2, and C_18:3 was observed. Both CLA isomers began to accumulate at 12 h, and the highest proportions of cis-9, trans-11 CLA and trans-10, cis-12 CLA were observed between 24 and 30 h and 28 and 48 h, respectively. Bacterial protein synthesis peaked at 12 h and gradually decreased over the next 36 h, which is indicative of bacterial death or lysis. Because growing cultures of the ruminal bacterium Butyrivibrio fibrisolvens A38 did not produce significant amounts of CLA until the linoleic acid concentration was high, biohydrogenation was arrested, and the cell density (i.e., bacterial protein) had declined, Kim et al. (2000) suggested that the flow of CLA from the rumen may be due to linoleic acid-dependent bacterial inactivation, death, or lysis. Our results with mixed ruminal bacteria are consistent with this hypothesis. It should be noted that B. fibrisolvens is thought to be a key bacterium involved in ruminal biohydrogenation and CLA formation (Harfoot and Hazlewood, 1997).

Incomplete biohydrogenation in in vitro mixed ruminal microorganism fermentations is a common observation (Harfoot and Hazlewood, 1997). However, the reason for this incomplete biohydrogenation is not completely understood. The type of substrate used can also influence lipolysis and biohydrogenation. Diets low in roughage seem to result in decreased ruminal lipolysis and biohydrogenation (Latham et al., 1972; Gerson et al., 1985). Therefore, it has been suggested that the main ruminal biohydrogenating bacteria are cellulolytic (Latham et al., 1972; Harfoot and Hazlewood, 1997). Collectively, these factors may account for the lack of stearic acid (C_18:0) production in our batch culture incubations.

Most in vitro studies with ruminal microorganisms and long-chain fatty acids have utilized batch-culture techniques. Even though continuous culture of microorganisms mimics ruminal environmental conditions in the laboratory, few studies have evaluated biohydrogenation of long-chain fatty acids using this technique. Fellner et al. (1995) used continuous culture of strained ruminal fluid to demonstrate that artificial fermenters were a reliable predictor of fatty acid metabolism. These researchers evaluated the biohydrogenation of linoleic acid using a pelleted feed, one dilution rate, and a culture pH of 6.8 (Fellner et al., 1995). In a later study, Fellner et al. (1997) utilized their continuous culture technique and similar environmental conditions to evaluate the effects of several ionophores on the biohydrogenation of linoleic acid by strained ruminal fluid. They reported that monensin, nigericin, and tetronasin inhibited the rate of biohydrogenation of linoleic acid, but increased cis-9, trans-11-C_18:2 (Fellner et al., 1997). In our initial experiments, we tried using linoleic acid in our liquid medium as described by Fellner et al. (1995) without success. Therefore, we used emulsified soybean oil as our source of long-chain fatty acids because it remained evenly distributed in our medium.

Because the ruminal environment does not remain static, we conducted continuous culture incubations to determine the effects of different environmental conditions on the production of long-chain fatty acids by mixed ruminal bacteria. When mixed ruminal bacteria were grown in continuous culture in media that contained 0.5 g/L of mixed soluble carbohydrates and emulsified soybean oil, the highest (P < 0.05) proportions of C_18:0 and cis-9, trans-11 CLA were observed at a dilution rate of 0.05 h^-1 and culture pH of 6.7 (Table 3). However, when culture pH was 5.5 at a dilution rate of 0.05 h^-1, there was a decrease (P < 0.05) in C_16:0, C_18:0, trans-C_18:1, and cis-9, trans-11 CLA and an increase (P < 0.05) in C_18:2 and C_18:3. Increasing the dilution rate to 0.10 h^-1 at a culture pH of 6.7 resulted in higher (P < 0.05) C_16:0 and C_18:3 and lower (P < 0.05) C_18:0 and C_18:2 compared to the fermentations conducted at a dilution rate of 0.05 h^-1 and pH 6.7. Lowering the culture pH to 5.5 at a dilution rate of 0.10 h^-1 resulted in higher (P < 0.05) C_16:0 and trans-C_18:1 and lower (P < 0.05) C_18:2 compared to fermentations at a dilution rate of 0.05 h^-1 and pH 5.5. Little trans-10, cis-12 CLA accumulated, and the proportion of cis-C_18:1 did not change under any of the environmental conditions used in these 0.5 g/L mixed soluble carbohydrate incubations.

In order to examine the effect of a culture pH less than 5.5 on production of long-chain fatty acids, mixed ruminal bacteria were maintained at pH 5.0 on 1.0 g/L of mixed soluble carbohydrates and emulsified soybean oil at a dilution rate of 0.10 h^-1 (Table 5). Under these environmental conditions, there was no production of trans-C_18:1, cis-9, trans-11 CLA, or trans-10, cis-12 CLA by the mixed ruminal bacteria. Because the distribution of long-chain fatty acids in this low-pH culture was similar to the proportion of long-chain fatty acids in the medium (Table 5 vs Table 1), a culture pH of 5.0 seems to inhibit the ruminal bacteria involved in biohydrogenation. Van Nevel and Demeyer (1996) reported that biohydrogenation by ruminal contents in soybean oil batch cultures was reduced as culture pH decreased, but the effects of culture pH on CLA and trans-C_18:1 were not determined.

	Implications

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This research has shown that a culture pH less than 6.0 seems to have the most influence on the production of trans-C_18:1 and conjugated linoleic acid isomers by mixed ruminal bacteria grown in continuous culture. When mixed ruminal bacteria were maintained in continuous culture on mixed soluble carbohydrates at a dilution rate of 0.05 h^-1, concentrations of trans-C_18:1 were significantly reduced at a culture pH of 5.5. Because trans-C_18:1 monoenes serve as precursors for the synthesis of conjugated linoleic acid at the tissue level (i.e., mammary gland), feeding diets that lead to a reduction in ruminal pH may reduce the availability of trans-C_18:1 monoenes and consequently reduce synthesis of conjugated linoleic acid by ruminant tissues. Therefore, our results suggest that in order to maximize conjugated linoleic acid synthesis in ruminant tissues diets need to be formulated to maintain ruminal pH above 6.0.

	Footnotes

 
¹ Financial support provided by National Cattlemen's Beef Assoc., Chicago, IL; Northeast Dairy Foods Research Center, Ithaca, NY; and the University of Georgia Agricultural Experiment Station.

Received for publication April 8, 2002. Accepted for publication July 25, 2002.

	Literature Cited

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