Effect of caprylic , capric and oleic acid on growth of rumen and rabbit caecal bacteria *

In a search for alternatives o f in-feed antibiotics, the antimicrobial activity o f caprylic (C8:0), capric ( C I 0:0) and oleic (C 18:1) acid was investigated in pure cultures o f 19 strains o f rumen and rabbit caecal bacteria, and in incubations o f the rumen and rabbit caecal contents. In glucose-grown bacterial cultures the min imum inhibitory concentration ( M I C ) o f caprylic acid ranged from 1 to 3 u T m l 1 . Two strains o f Bacteroides ovatus were less susceptible to capric than to caprylic acid. In other strains, the M I C o f capric acid was 0.25-0.50 uTml~ . The growth o f most strains was not much affected by oleic acid. A n exception to this were rumen bacteria Butyrivibrio fibrisolvens ( M I C from < 0.05 to 1 uTml" ) and Lachnospira multiparus ( M I C o f 0.25 to 1 uTml" ). In incubations o f the rumen and caecal contents caprylic and capric acid decreased the production o f volatile fatty acids and gas, and increased production o f lactate. In latter incubations the inhibitory effects o f caprylic and capric acid were similar. In incubations o f the rumen contents, capric acid was more efficient than caprylic acid when supplied at low concentrations (<1.25 u T m l 1 ) , but less efficient when supplied at 2.5 and 5 uTml" 1 . Effects o f oleic acid in rumen and caecal cultures were not significant, except the increase in production o f lactate by rumen microorganisms. It can be concluded that microorganisms o f the animal digestive tract are susceptible to inhibit ion by caprylic and capric acid added to microbial cultures at fairly low concentrations. Oleic acid was far less effective. K E Y W O R D S : fatty acids, rumen, rabbit caecum, bacteria, inhibit ion, fermentation * Supported by Grant No. 523/02/0460 o f the Grant Agency o f the Czech Republic. The authors wish to thank the donors o f the cultures 3 Corresponding author 508 GROWTH OF RUMEN AND RABBIT CAECAL BACTERIA


INTRODUCTION
Antimicrobial activity of fatty acids is well known.In the presence of fatty acids, the transport of protons through microbial membranes becomes uncontrolled.Consequently, fatty acids interfere with the metabolism of energy within the cell and disturb energy-dependent processes, e.g., active transport of nutrients into bacterial cells, maintenance of concentration gradients, etc. (Galbraith and Miller, 1973).Results of various investigations of the antimicrobial activity of fatty acids showed that (/) Gram-positive bacteria were more susceptible to the action of fatty acids in minute concentrations than Gram-negative bacteria, (//) inhibitory properties of unsaturated fatty acids were more pronounced than those of saturated acids, and (Hi) antibacterial activity of saturated fatty acids was optimal for a chain length around C12 (reviewed by Nieman, 1954).Several authors examined effects of fatty acids on rumen bacteria.Henderson (1973) found the that fatty acids containing 10 to 18 carbon atoms inhibited ruminococci, butyrivibrios and production of methane in cultures of Methanobacterium ruminantium.No effect on growth of Gram-negative bacteria Anaerovibrio lipo/ytica, Peptostreptococcus (now Megasphaera) elsdenii, Selenomonas ruminantium and Bacteroides (now Prevotella) ruminicola was observed.Oleic acid was the most inhibitory of the series of acids.In experiments of Maczulak et al. (1981) the growth of Ruminococcus albus and Ruminococcus flavefaciens was almost completely inhibited at oleic acid concentration as low as 5 mgT 1 .This concentration of oleic acid inhibited also the growth of one out of five strains of Butyrivibrio fibrisolvens.The growth of other butyrivibrios was not much affected by oleic acid.Low concentrations of oleic acid enhanced growth of S. ruminantium and B. ruminicola.In most strains, palmitic and stearic acid had little effect on growth.In mixed cultures of rumen microorganisms lauric acid (C12:0) decreased production of volatile fatty acids to a greater extent than myristic, palmitic, stearic, oleic, linoleic and arachidic acid (Chalupa et al., 1984).Lauric acid increased, but other acids decreased the acetate to propionate molar ratio.
Few studies deal with bacteriocidal effects of medium-chain fatty acids.Smith (1965) reported that suckling rabbits were unique among young of seven animal species tested in that the contents of their stomach and small intestine were almost completely sterile.To explain this finding, Canas-Rodriguez and Smith (1966) suggested that the rabbit milk fat contained antimicrobial compounds, identified as eight-and ten-carbon saturated fatty acids (caprylic and capric acid, respectively).These acids are the principal fatty acids in the rabbit milk.Typically, caprylic and capric acid represent from one third to one half of the total fatty acids in the rabbit milk fat (Christ et al., 1996;Lebas et al, 1996).Both acids are practically absent from the feed, thus, rabbits synthesize them in the mammary gland.The antimicrobial activity of rabbit milk was confirmed by Marounek et al. (1999).Rabbit milk added at 4.8% (v/v) significantly decreased production of microbial metabolites in cultures of rabbit caecal contents, whereas no inhibitory effect of a corresponding mixture of cow milk fat, casein and lactose was observed.Capric acid at a concentration higher than 1.2 to 1.6 gT 1 virtually completely killed the acetogenic and methanogenic population of methanogenic sludge (Rinzema et al., 1994).Matsumoto et al. (1991) found that capric acid was the most toxic for rumen protozoa among six fatty acids tested.Progressively less inhibition was displayed with either an increase or a decrease in the carbon chain length.An adverse effect of caprylic and capric acid on rumen protozoa was observed also by Dohme et al. (2001).Some authors have reported the inactivation of lipid-enveloped viruses by caprylic and capric acid (Lundblad and Seng, 1991;Isaacs et al, 1995).
Antimicrobial substances are often used to stimulate growth of farm animals and for the control of enteritis infections.However, most of antimicrobial performance promoters were banned recently, and the use of antibiotics for enteritis prevention has been viewed critically.Thus, there is a need for alternatives to antibiotics, usable in animal production and acceptable by consumers.The purpose of this study was to evaluate the antibacterial activity of caprylic, capric and oleic acid using pure strains of rumen and rabbit caecal bacteria, and in incubations of rumen and rabbit caecal contents.
Media were dispensed to gas-tight glass flasks, and caprylic, capric and oleic acid (Sigma) added at 0, 0.05, 0.1, 0.25, 0.5, 1, 2, 3 and 5 fil-ml" 1 , together with an equivalent amount of 5 M NaOH.Capric acid was heated to 40°C before pipetting.Media were supplemented with 0.05% cysteine-HCl.Flask were filled with C0 2 , closed with rubber stoppers and autoclaved at 110°C for 45 min.Inoculated cultures were incubated in triplicate at 39°C for 24 h.Then the cultures were examined for visible growth and pH was measured.The minimum inhibitory concentration (MIC) was the lowest concentration of caprylic and capric acid that prevented the visible growth and pH drop in treated cultures (typically from 6.6 to 5.8 in controls).Media with oleic acid were turbid, thus residual glucose was determined using a commercial kit from Lachema (Brno, Czech Republic).The MIC was the lowest concentration of oleic acid that prevented glucose utilization in treated cultures.

Cultivation of rumen and caecal contents
Rumen contents were obtained from two dry fistulated cows fed 1 kg of a commercial concentrate per day, 2 kg lucerne hay and maize silage ad libitum.The rumen content was homogenized in a laboratory blender under C0 2 atmosphere for 2 min.Ten ml of the homogenate were added to 30 ml of warm (39°C) Burroughs buffer (Burroughs et al., 1950) in 100 ml incubation flasks (serum bottles) containing wheat bran (0.8 g), yeast extract (40 mg), urea (20 mg) and sodium sulphide (20 mg) as a reducing agent.The NaHC0 3 concentration in the buffer was increased to 0.07 M, to prevent a pH drop below 6.0 and 5.5 in rumen and caecal cultures, respectively.In the first experiment, caprylic and capric acid were added at 0, 0.625, 1.25,2.5 and 5 (il-ml 1 , together with an equivalent amount of NaOH.In the second experiment, oleic acid was added at 0, 2 and 5 (il-ml" 1 .The flasks were flushed with C0 2 , hermetically closed with rubber stoppers and incubated at 39°C for 8h in a shaking water bath.Each control or experimental arrangement was incubated in four replicates. The same experimental procedure was used for incubation of the rabbit caecal content.Three-month-old rabbits (Hyla 2000 genotype) were fed ad libitum a granulated concentrate containing dehydrated lucerne, wheat bran, extracted sunflower meal, barley, oat and a vitamins-minerals supplement.Four rabbits were slaughtered, their caeca emptied and pooled caecal contents were used for inoculation of cultures as described above.
At the end of incubations, manometric pressure in the incubation vessels was measured.Samples of the incubation fluid from the beginning and the end of incubation were analysed.Total volatile fatty acids (VFA) were estimated by titra-tion, after steam distillation.Lactic acid was assayed by the microdiffusion method (Conway, 1957).

RESULTS
Minimum inhibitory concentrations of caprylic and capric acid in 19 bacterial strains tested are shown in Table 1.In 17 strains, the MIC of caprylic acid was higher than that of capric acid.Concentrations around 2.0 jal-ml" 1 inhibited bacterial growth in caprylic acid-treated cultures.B. ovatus was the only bacterium less susceptible to capric than to caprylic acid.In other strains the MIC of capric acid ranged from 0.25 to 0.5 jil-ml" 1 .Gram-positive and Gram-negative bacteria did not differ in sensitivity to caprylic acid.Similarly, there was no significant difference Caprylic and capric acid, ml ml" Effects of caprylic and capric acid in mixed cultures of rumen and caecal microorganisms are shown in Figure 1.Both caprylic and capric acid decreased the VFA and gas production and increased production of lactate.In rumen cultures, caprylic acid was less efficient than capric acid when supplied at low concentrations (0.625 and 1.25 (il-ml" 1 ).The high caprylic acid concentration (5 jil-ml" 1 ), however, completely inhibited production of microbial metabolites, whereas capric acid at 5 jal-ml" 1 apparently allowed a moderate microbial growth.In rabbit caecal cultures effects of both acids were similar.Oleic acid had a negligible effect on VFA and gas production, but increased production of lactate in rumen incubations from 0 to 12.9 ^mol-ml" 1 (Figure 2).

DISCUSSION
Antibacterial effects of caprylic and capric acid have generally received less attention than those of long-chain fatty acids.In the present study, caprylic and capric acid were shown to inhibit growth of various bacteria isolated from the ani-mal digestive tract.In pure cultures of 17 out of 19 bacterial strains capric acid inhibited growth at lower concentrations than caprylic acid.Similarly, Galbraith et al. (1971) found that capric acid was more inhibitory than caprylic acid in cultures of Bacillus megaterium and Pseudomonas phaseolicola (now Pseudomonas syringae pv.phaseolicola).In incubations of the rabbit caecal contents, however, the inhibitory effect of both acids was similar.In incubations of the rumen contents, capric acid supplied at 0.625 jilml* 1 decreased the VFA production by 50% (caprylic acid by 7%).High concentration of capric acid (5 jil-ml" 1 ) decreased the VFA production by 71%, whereas the same concentration of caprylic acid prevented the VFA formation.Thus, capric acid was more efficient at low concentrations than caprylic acid but less efficient at high concentrations.The following facts have to be taken into account to explain this contradiction.Firstly, capric acid is less soluble than caprylic acid.Stearic acid, which is virtually insoluble in a water environment, had no effect on production of VFA in rumen incubations (Chalupa et al., 1984).Secondly, in these incubations fatty acids are adsorbed competitively onto feed particles and bacteria (Harfoot et al., 1974).The adsorption of fatty acids decreases with increasing unsaturation (Harfoot et al., 1974), and increases with the chain length (Neys and Joos, 1998).Preferential binding to feed particles decreases toxicity of fatty acids towards rumen and caecal bacteria.Inclusion of powdered cellulose in the medium reversed the inhibitory effect of added fatty acids (Maczulak et al., 1981).Oleic acid, which was inhibitory in pure cultures of some rumen bacteria, influenced production of microbial metabolites in our rumen and caecal incubations only marginally.
As far as we know, no previous study examined effect of fatty acids on lactate production.In the present experiments, in rumen and caecal cultures fatty acids tested increased production of lactate.This may be caused either by proliferation of lactate producers or by inhibition of lactilytic microflora.

TABLE 1
Minimum inhibitory concentrations of caprylic, capric and oleic acid in cultures of fourteen rumen