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1 Department of Animal and Veterinary Sciences and
2 Department of Plant Pathology and Physiology, Clemson University, Clemson, SC 29634
Corresponding author: T. C. Jenkins; e-mail: TJNKNS{at}clemson.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Key Words: biohydrogenation • oleic acid • pH • dilution rate
Abbreviation key: APE = atom percent excess, CLA = conjugated linoleic acid, DMDS = dimethyl disulfide, FAME = fatty acid methyl ester, GC-MS = gas chromatography-mass spectroscopy.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Amounts of trans fatty acids and CLA produced in the rumen influence their concentrations in tissues and milk. Concentration of trans-11 C18:1 and cis-9, trans-11 CLA are greater in milk from cows fed fish oil and a linoleic acid fat source (AbuGhazaleh et al., 2002) or grazed on pasture (Loor et al., 2003). Trans-10 C18:1 and trans-10, cis-12 CLA concentration in milk fat increases when high grain diets are fed (Piperova et al., 2002). Other factors such as unsaturated fatty acid concentration and source, rumen pH, dilution rate, ionophores, and carbohydrate source have been shown to influence trans fatty acid production in the rumen (Bessa et al., 2000; Martin et al., 2002; Jenkins et al., 2003).
Oleic acid (C18:1 cis-9), a typical fatty acid in ruminant diets, is usually presented as being hydrogenated directly to stearic acid without the formation of trans fatty acid intermediates (Harfoot and Hazlewood, 1988). However, a recent in vitro batch culture study in our laboratory (Mosley et al., 2002) demonstrated that oleic acid is also a precursor for several trans C18:1 isomers, including trans-10 and trans-11 C18:1. A follow up study showed that once a trans-C18:1 isomer is formed, it is converted into other trans monoenes by microbial isomerases (Proell et al., 2002). This study was conducted to further confirm the conversion of oleic acid to trans monoenes by ruminal microorganisms and to get a better understanding of how two important environmental variables, namely rumen pH and dilution rate, affect the type and amount of trans monoenes produced during oleic acid biohydrogenation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Experimental Design
Four dual-flow continuous culture systems, as described by Teather and Sauer (1988), were used in a 4 x 4 Latin square with 4 periods of 10 d each (9 d for adaptation and d 10 for sampling). Treatments were arranged in a 2 x 2 factorial as follows: 1) 6.5 pH and 0.10/h dilution rate, 2) 6.5 pH and 0.05/h dilution rate, 3) 5.5 pH and 0.10/h dilution rate, and 4) 5.5 pH and 0.05/h dilution rate. A total of 22 g of feed (Table 1
) consisting of alfalfa pellets and concentrate mix (55:45; DM basis) were placed into each fermentor daily in 2 equal portions at 0830 and 1630 h. Because oleic acid was added to the concentrate mix, it accounted for 55% of the total fatty acids. Linoleic (C18:2 cis-9, cis-12) and linolenic (C18:3 cis-9, cis-12, cis-15) acids were also present in the diet, but at much lower proportions (9 and 4%, respectively).
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Whole ruminal contents were taken from a ruminally fisulated dry Holstein cow fed a predominantly forage diet. At each collection time, ruminal contents were strained through a double-layer of cheesecloth and transferred to the laboratory in a sealed container. Approximately 900 mL of the strained ruminal fluid were added to each of the 4 fermentors. Anaerobic conditions were maintained by infusion of CO2 at a rate of 20 mL/min. Using a precision pump with 2 different tubing sizes, buffer (Slyter et al., 1966) was delivered continuously at a flow rate of 1.5 and 0.75 mL/min resulting in a fractional dilution rate of 0.10 and 0.05/h, respectively. Buffer pH was titrated each day with sufficient 3 N NaOH or 3 N HCl to maintain treatment pH values (6.5 or 5.5). The pH was measured daily at 0830, 1200, and 1640 h using a portable pH meter. The temperature of ruminal cultures was maintained at 39°C by a circulating water bath, and cultures were stirred continuously at 25 rpm.
Sample Collection and Analysis
On d 10 of each period, two 5-mL samples were taken from each fermentor at 0 (prior to addition of feed or oleic acid-1-13C) and 24 h after adding the oleic acid-1-13C. Prior to sampling, the contents of the fermentor were thoroughly mixed by increasing agitation to 250 rpm, and samples were withdrawn with a pipette into 16- x 125-mm glass culture tubes and immediately placed in an ice bath. Samples were freeze-dried and fatty acid methyl esters (FAME) were prepared according to Kramer et al. (1997). The FAME were then separated into saturated, trans monoene, cis monoene, and diene fractions using solid phase extraction as described by Mosley et al. (2002). The FAME in the trans and cis monoene fractions were converted to DMDS adducts as described by Mosley et al. (2002) to identify double bond position from mass spectral data.
The FAME in the saturated fraction were analyzed on a gas chromatograph equipped with a Saturn II ion trap mass spectrometer (Varian Instruments Inc., Walnut Creek, CA) using a 30-m x 0.25-mm with 0.2-µm film Supelco 2380 column (Supelco, Inc., Bellefonte, PA). Column temperature was programmed at 140°C for 3 min, then increased to 220°C at 3°C/min, and finally held at 220°C for 5.5 min. The carrier gas was He (20 cm/s) with an injector split of 100:1. The gas chromatography-mass spectroscopy (GC-MS) analysis of FAME-DMDS adducts were performed using the same GC-MS conditions described previously except that the column temperature was programmed at 185°C for 31.6 min, then increased to 189°C at 0.2°C/min, and finally increased to 215°C at 4°C/min and held at this temperature for 6.8 min.
Additional FAME samples were analyzed by gas chromatography (Shimadzu GC-14A, Columbia, MD) to determine fatty acid profile. The gas chromatograph was equipped with a flame ionization detector and a 100-m x 0.25-mm (0.2-µm film) capillary column coated with CP-Sil 88 (Chrompack, Raritan, NJ). The injector and detector temperatures were held at 250°C. The carrier gas was H2 (33 cm/s) with an inlet pressure of 250 kPa. The column temperature was programmed for 140 °C for 3 min, then increased to 220°C at 2°C/min, and held at 220°C for 2 min.
Calculations and Statistics
The DMDS derivatives of FAME produce 2 distinctive spectral fragments that are indicative of the double bond position in monoenes when analyzed by GC-MS. The F fragment is the methyl thio adduct of the methyl end of the FAME. The G fragment is the methyl thio adduct of the carboxyl end of the FAME. The atom percent excess (APE) is calculated from the mass abundance of the G and G + 1 fragments using the following equation {APE = [(G + 1)/G (G + 1)]}. To eliminate the natural levels of 13C, the APE for unlabelled cultures (0-h sample; before adding oleic acid-1-13C) was subtracted from the APE of labeled cultures (24-h sample). Therefore, fatty acid enrichments were expressed on a percentage basis and calculated as (APE24h – APE0h) x 100. Zero enrichment values indicate that an isomer was detected, but its (APE24h – APE0h) x 100 did not differ from 0 by t-test (P < 0.05). The select ion mode in GC-MS was used to measure 13C enrichment in all fatty acids.
Data were analyzed as a Latin square with a 2 x 2 factorial arrangement of treatments using the mixed procedure of SAS (2000). Fixed effects were period, pH, dilution, and the pH x dilution interaction, while random effects were fermentors. The only significant interactions detected were for enrichments in the trans-12 through trans-15 C18:1 isomers. Interactions for these isomers were caused by the failure to observe enrichment for any treatment except for the pH 6.5 and 0.10/h dilution rate treatment. Therefore, only the main effects of pH and dilution rate are presented in tables. Main effects were considered significant if P 
0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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and 2). Culture pH was adjusted by varying buffer pH, but without affecting its composition. Other researchers have altered culture pH by changing buffer composition (Yang et al., 2002) or by adding concentrated acids and bases directly into fermenters (De Veth and Kolver, 2001). It is unknown whether the abrupt changes in pH brought about by the direct addition of acids (e.g., from 6.4 to 5.5 in 4 min) have a direct effect on the microbial population. Some may argue that constant ruminal pH and dilution rate are not biologically meaningful because ruminal pH and dilution rate show diurnal variation in vivo. A valid counterargument is that when the main objectives are to study pH and dilution rate effects, these factors should be isolated and kept as constant as possible.
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). We did not find any significant 13C enrichment in palmitic, linoleic, and linolenic acids (data not shown). A closer examination of GC-MS results revealed 13C enrichment in 4 unknown peaks. Two of these unknown 13C enriched peaks appeared after C24:0 and were relatively large peaks, representing 4.3 to 14.3% of total fatty acids in fermentor contents; the other 2 unknown 13C enriched peaks appeared between C18:1 and C18:2 and were very small (<0.1% of total fatty acids in culture; data not shown). Although the identity of the 4 unknown peaks was not determined in this study, their enrichment indicates that biohydrogenation of oleic acid by ruminal microorganisms leads to formation of intermediates other than just stearic acid or trans-C18:1 isomers.
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0.03) or completely eliminated enrichment in most of the trans-C18:1 isomers examined. Reducing dilution rate from 0.10 to 0.05/h also affected enrichment (Table 2
), causing a decline in the enrichment of stearic acid and several trans-C18:1 isomers (with double bond positions at C6, C10, C14, and C16). Among all trans-C18:1 isomers examined, only enrichment in the trans-7 positional isomer was not affected in some way by lowering pH or dilution rate. The presence of a trans-8 C18:1 isomer could not be verified; therefore, no determination of enrichment was possible.
Whether the inhibitory effect of low pH and dilution rate on 13C enrichment of trans monoenes was due to a shift on the bacterial population or due to direct interference with enzyme activity could not be deduced from this experiment. Low rumen pH and dilution rate were shown to have a negative effect on microbial growth (Hoover et al., 1984; Martin et al., 2002), particularly on growth of cellulolytic bacteria (Latham et al., 1972). The acidification of ruminal contents and low dilution rate may give rise to a modification in the distribution (number, species) of cellulolytic bacteria colonizing the plant material. Cellulolytic bacteria, such as Butyrivibrio fibrisolvens, are indeed the main ruminal bacteria responsible for biohydrogenation (Harfoot and Hazlewood, 1988). When high concentrate/low forage (80:20) diets were fed to cows, promoting low pH conditions, the ratios of cellulolytic (primarily Butyrivibrio fibrisolvens) to propionogenic, lactogenic, or amylolytic bacteria in the rumen were severely reduced (Latham et al., 1972). Using continuous cultures, Hoover et al. (1984) showed that, at a 0.04/h dilution rate, lactic acid accounted for 53% of all organic acids, but as dilution rate increased (0.08/h), lactic acid was reduced to 28%. A further increase in dilution rate decreased lactic acid to 8%. The growth yield of Butyrivibrio fibrisolvens was 75% of its maximum yield when pH was 5.75; at pH 5.5, the organism was washed out in continuous cultures (Russell and Dombrowski, 1980).
In addition to affecting bacterial growth, low pH and dilution rate could also have influenced microbial attachment to feed particles. Low pH conditions alters microbial attachment to feed particles and binding affinity of fatty acids onto both bacterial membranes and feed particles (Martin et al., 2002). At low pH and dilution rate, the "balance" of microorganisms capable of carrying out various steps of biohydrogenation may be altered, leading to changes in production of biohydrogenation intermediates.
The reduction in 13C enrichment of trans monoenes at low pH and dilution rate may also indicate a significant inhibition of oleic acid isomerization to trans monoenes. Previous work in our laboratory showed that during the biohydrogenation of oleic acid, trans monoenes are formed as transient intermediates via enzymatic isomerization (Mosley et al., 2002; Proell et al., 2002). Low rumen pH may negatively affect the physiology of ruminal bacteria, including the enzyme activity responsible for biohydrogenation (Kopecny et al., 1983). Biohydrogenation may be decreased if cell membrane integrity is disrupted at non-physiological pH, as the enzymes responsible for biohydrogenation are membrane-associated (Kepler and Tove, 1967).
It is not clear whether the same bacterial species can isomerize both oleic acid and trans monoenes (possess a multitude of cis/trans isomerizes) or if different species are involved. Van de Vossenberg and Joblin (2003) investigated the monounsaturated fatty acid specificities of Butyrivibrio hungatei Su6. They showed that B. hungatei Su6 isomerized and hydrogenated cis-6-, cis-9-, and cis-11-octadecenoic acids yielding a mixture of trans monoenes and stearic acid. However, B. hungatei Su6 did not isomerize trans-6, trans-9, and trans-11-octadecenoic acids, but only hydrogenated these trans monoenes to stearic acid.
We still cannot rule out the possibility that other ruminal bacteria have the ability to isomerize both cis-and trans-octadecenoic acids at the same time. Using pure cultures, Kemp et al. (1975) showed the ability of a single rumen isolate to isomerize and hydrogenate linoleic and linolenic acids. It is also not clear if these series of trans monoenes are substrate for one or a multitude of isomerases. The fact that pH 5.5 did not reduce the 13C enrichment in some trans monoenes (trans-6 and trans-7) may indicate that more than one isomerase is involved in trans monoene production from oleic acid. Isomerization of linoleic acid to cis-9, trans-11 CLA and trans-10, cis-12 CLA are thought to be carried out by 2 different enzymes (linoleate-cis-12, trans-11-isomerase and linoleate-cis-9, trans-10-isomerase, respectively) (Kepler and Tove, 1967).
Among the trans monoenes, 2 specific isomers (trans-11 and trans-10) are of particular interest. Under most dietary conditions, trans-11 is the major trans isomer produced during the biohydrogenation of linoleic and linolenic acids (Griinari et al., 1998). Trans-11 can be used as a substrate for endogenous synthesis of cis-9, trans-11 CLA (anticarcinogenic) via 
9 desaturase (Bauman et al., 2003). It was estimated that at least 93% of cis-9, trans-11 CLA in cows milk was produced postruminally via 9 desaturase (Bauman et al., 2003). Trans-10 replaced trans-11 as the predominant trans monoene isomer in the rumen when high concentrate-low fiber diets were fed to cows (Griinari et al., 1998) and steers (Beaulieu et al., 2002). Under such conditions, Bauman et al. (2003) proposed a putative pathway for the production of trans-10 where the trans-10, cis-12 CLA-producing bacteria become the predominant bacteria in the rumen, resulting in formation of trans-10, cis-12 CLA as the first intermediate during linoleic acid biohydrogenation. Hydrogenation of the cis-12 bond would then result in formation of trans-10, analogous to the production of trans-11 from cis-9, trans-11 CLA. Trans-10 has been associated with reduced mammary gland lipogenic enzyme activity and milk fat depression (Piperova et al., 2002). The presence of 13C enrichment in trans-11 and trans-10 in this study adds to previous evidence that these 2 isomers are also produced during isomerization of oleic acid (Mosley et al., 2002). In a follow up study, Proell et al. (2002) showed that once a single trans monoene isomer formed, it can be converted to many other positional isomers. Therefore, an alternate pathway for trans-10 synthesis, other than directly from oleic, linoleic, and linolenic acid bio-hydrogenation, might be from isomerization of their trans monoene intermediates.
In this study, the concentration (g/100 g of total fatty acids) of trans-10 in fermentors was higher than trans-11 at both pH levels (Figure 3). The presence of trans-10 as the predominant trans monoene at pH 6.5 may indicate that acidic conditions are not necessary for trans-10 formation from oleic acid and that trans-11 is not the major trans monoene intermediate during oleic acid biohydrogenation. After 24 h of incubation, the percentage of carbons in trans-10 and trans-11 derived from 13C oleic acid was 89 and 37%, respectively (Mosley et al., 2002). Additionally, when dairy cows were fed a 50:50 forage:concentrate diet high in oleic acid, the proportion of trans-10 and trans-11 in milk fat increased by 114 and 52%, respectively, compared with a control diet (AbuGhazaleh et al., 2003). As expected, lowering culture pH to 5.5 reduced the concentration of trans-11, but increased trans-10 concentration (Figure 3). The fact that 13C enrichment of trans-10 was lower at pH 5.5 compared with pH 6.5 (Table 2) indicates that more of the trans-10 at low pH originates from sources other than oleic acid.
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). The percentage of trans monoenes originating from oleic acid biohydrogenation ranged from 24 to 72% at pH 6.5 and 0.10/h dilution rate. Among the individual trans monoene isomers, only double bond positions at C7, C9, and C14 were primarily (>50%) derived from oleic acid. The trans-10 and trans-11 isomers of greater interest and importance were 25% derived from oleic acid in this study. The percentage of trans monoenes originating from oleic acid were all affected by either pH or dilution rate, except for the isomers with double bond positions at carbons C7, C12, and C15. The trans-6 isomer was the only isomer affected by both pH and dilution rate. When significant, lowering pH and dilution rate reduced the percentage of trans monoenes originating from oleic acid, except for the trans-16 isomer. The percentage of stearic acid produced from oleic acid was not affected by dilution rate, and it was higher at pH 5.5 compared with pH 6.5.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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Received for publication June 21, 2005. Accepted for publication August 21, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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9-cis, 11-trans-isomerase from Butyrivibrio fibrisolvens. J. Biol. Chem. 242:5686–5692.This article has been cited by other articles:
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  L. Liu, J. Q. Wang, D. P. Bu, S. J. Liu, S. Liang, H. Y. Wei, L. Y. Zhou, and K. L. Liu Short Communication: Malic Acid Does Not Promote Vaccenic Acid Accumulation in Mixed Ruminal Fluid with Fractionated Fish Oil by a Rumen-Simulation Technique J Dairy Sci, October 1, 2008; 91(10): 3993 - 3997. [Abstract] [Full Text] [PDF]
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P. J. Moate, R. C. Boston, T. C. Jenkins, and I. J. Lean Kinetics of Ruminal Lipolysis of Triacylglycerol and Biohydrogenation of Long-Chain Fatty Acids: New Insights from Old Data J Dairy Sci, February 1, 2008; 91(2): 731 - 742. [Abstract] [Full Text] [PDF]
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A. A. AbuGhazaleh, D. O. Felton, and S. A. Ibrahim Milk Conjugated Linoleic Acid Response to Fish Oil and Sunflower Oil Supplementation to Dairy Cows Managed Under Two Feeding Systems J Dairy Sci, October 1, 2007; 90(10): 4763 - 4769. [Abstract] [Full Text] [PDF]
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S. K. R. Karnati, J. T. Sylvester, S. M. Noftsger, Z. Yu, N. R. St-Pierre, and J. L. Firkins Assessment of Ruminal Bacterial Populations and Protozoal Generation Time in Cows Fed Different Methionine Sources J Dairy Sci, February 1, 2007; 90(2): 798 - 809. [Abstract] [Full Text] [PDF]
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B. Vlaeminck, V. Fievez, D. Demeyer, and R. J. Dewhurst Effect of forage:concentrate ratio on fatty acid composition of rumen bacteria isolated from ruminal and duodenal digesta. J Dairy Sci, July 1, 2006; 89(7): 2668 - 2678. [Abstract] [Full Text] [PDF]
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K. J. Shingfield, C. K. Reynolds, G. Hervas, J. M. Griinari, A. S. Grandison, and D. E. Beever Examination of the Persistency of Milk Fatty Acid Composition Responses to Fish Oil and Sunflower Oil in the Diet of Dairy Cows J Dairy Sci, February 1, 2006; 89(2): 714 - 732. [Abstract] [Full Text] [PDF]
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), pH 6.5 and 0.05/h dilution rate (
), pH 5.5 and 0.10/h dilution rate (•), and pH 5.5 and 0.05/h dilution rate (
). Standard errors of the means were 0.09, 0.07, 0.08, and 0.08, respectively.
