Effects of Essential Oils on Ruminal Microorganisms and Their Protein Metabolism

A commercial blend of essential oil (EO) compounds was addedto a grass, maize silage, and concentrate diet fed to dairycattle in order to determine their influence on protein metabolismby ruminal microorganisms. EO inhibited (P < 0.05) the rateof deamination of amino acids. Pure-culture studies indicatedthat the species most sensitive to EO were ammonia-hyperproducingbacteria and anaerobic fungi.

INTRODUCTION

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Introduction

The drive toward a decrease in the use of antibiotics in animalproduction has intensified the search for natural products thatenhance production, in the case of ruminants, by modulatingruminal fermentation. Essential oils (EO) are natural productsthat give plants and spices their characteristic odor and color.Oh et al. (12) found that EO from the Douglas fir pine needlehave an antibacterial effect in the rumens of sheep and deer.Fernandez et al. (6) used a commercial blend of EO compoundsto manipulate rumen fermentation, inhibiting the breakdown ofprotein, thus potentially increasing the dietary protein availableto the ruminant. The aim of the present study was to investigatethe influence of EO on protein metabolism by mixed ruminal microorganismsand on different ruminal bacteria, protozoa, and fungi.

In order to identify the mechanisms and microbial species affectedby EO, the effects of EO were measured in ruminal microorganismsobtained from four Holstein-Friesian cows, each fitted witha ruminal cannula and fed ad libitum a diet consisting of grass,maize silage, and concentrate mix (330, 220, and 450 g kg^-1on a dry-matter basis). The concentrate contained soybean meal,rapeseed meal, sunflower meal, maize gluten feed, maize distillersgrain, cottonseed meal, ground wheat, palm kernel meal, molassedsugar beet pulp, wheat feed, and minerals (106, 106, 45, 22,89, 89, 171, 122, 111, 111, and 28 g of dry matter kg^-1, respectively).Cattle received the diet with or without 1,000 mg of a blendof EO (Crina Ruminants; Akzo Nobel Surface Chemistry Ltd., Hertfordshire,United Kingdom) day^-1 as part of a two-by-two factorial designwith 4-week periods. Crina Ruminants is a defined, patentedmixture of natural and nature-identical EO compounds that includesthymol, eugenol, vanillin, and limonene on an organic carrier(14). Measurements were made on 2 consecutive days during thelast week of each period.

Samples of ruminal fluid were removed 2 h after morning feedingand strained (strained ruminal fluid [SRF]) though two layersof cheese cloth before use in experimental measurements. Ruminaldegradation of proteins was investigated by using casein, lactoglobulin,hide powder azure, albumin, and elastin Congo red (Sigma ChemicalCo., Poole, United Kingdom) reductively methylated with [¹⁴C]formaldehyde(18). Peptidase activities were measured with Ala₂, Ala₅, AspLysArgValTyr,GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA), and Ala₂-p-nitroanilide(Ala₂-pNA) by measuring peptide breakdown by reverse-phase high-performanceliquid chromatography (20), fluorimetrically by the appearanceof MNA, or spectrophotometrically by the appearance of pNA (20,21). Deaminase activity was determined with casein acid hydrolysateas the substrate (11). Monensin, which is known to inhibit someof the major species of deaminative organisms (13), was addedto samples incubated with casein acid hydrolysate at a finalconcentration of 5 µM.

The effect of EO on bacterial growth was measured by inoculatingstock cultures grown in the liquid version of Hobson's M2 medium(8) into the same medium containing serial twofold increasesin EO concentration (10). EO was added aseptically after themedium was autoclaved to give final concentrations ranging from5 to 1,000 ppm. Bacterial growth was measured by reading theoptical density at 650 nm hourly. To investigate possible adaptationof bacteria to EO, bacteria were inoculated into tubes containingthe lowest concentration of EO and incubated at 39°C anaerobicallyfor 48 h. This tube was used to inoculate (5%, vol vol^-1) mediumcontaining twice the EO concentration and so on until a concentrationthat inhibited growth totally was reached. The results wererecorded as the EO concentration at which the optical densitywas half of that measured in the absence of EO (IC₅₀). The influenceof EO on rumen ciliate protozoa was determined by the bacteriolyticactivity of protozoa in ruminal fluid (22). The effect of EOon the activity of Methanobrevibacter smithii PS (ATCC 35061)and Neocallimastix frontalis RE1 was determined from gas productionover 10 days in the presence of increasing concentrations ofEO (10). The microbial species used are all maintained in theculture collection of the Rowett Research Institute; their originswere described previously (10, 21).

Data were analyzed as a two-by-two factorial design with Genstat5 (7).

Influence of dietary EO on protein metabolism.

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Influence of dietary eo...

The different proteins incubated with SRF in vitro were hydrolyzedat rates that spanned 2 orders of magnitude (Table 1). EO hadno effect on the breakdown of any of the proteins tested (Table1), even with their different secondary and tertiary structures.Casein and lactoglobulin are soluble proteins with little orderedstructure; albumin is also soluble but contains abundant disulfidecross-links that make it more resistant to degradation (18);hide powder azure and elastin are both insoluble, and the latteris also heavily cross-linked (18).

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TABLE 1. Effect of feeding essential oils to cattle on the subsequent breakdown of proteins, peptides, and amino acids in rumen fluid in vitro

The peptides selected also covered a range of potential substrates,including di- and oligopeptides and substrates for dipeptidylaminopeptidases. Once again, EO had no effect (Table 1). Therewas, however, a significant decrease (P < 0.05) in the rateof ammonia production from casein acid hydrolysate, which comprisesfree amino acids, when casein acid hydrolysate was incubatedwith SRF from cattle receiving EO (Table 1). A similar studywith sheep indicated a 24% decrease in deamination of aminoacids (our unpublished results). The decrease in ammonia productioncaused by EO was abolished in the presence of monensin (Table1), and the deamination rate was reduced to similar values inboth treatments. Taken together, these results suggest thatEO suppresses some of the species that are inhibited by monensinbut has little effect on other deaminating species.

Influence of EO on pure cultures of ruminal microorganisms.

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Influence of eo on...

EO, when added to the culture medium, inhibited the growth ofmost pure cultures of ruminal bacteria at concentrations ofless than 100 ppm; Streptococcus bovis was the most resistantspecies, and Prevotella ruminicola, Clostridium sticklandii,and Peptostreptococcus anaerobius were the most sensitive species(Table 2). Some species, including P. ruminicola and P. bryantii,adapted and were able to grow in higher concentrations of EO,while others, including C. sticklandii and P. anaerobius, remainedsensitive. The last two species are among those identified byRussell and his colleagues (2, 3, 13, 15, 16) as "hyper-ammoniaproducing" (HAP) species detrimental to the efficiency of nitrogenretention in ruminants. Any adaptation to EO would diminishthe effect in vivo. A further consideration may be the survivalof bacteria in stationary phase: the loss of viability of Ruminobacteramylophilus was greatly increased by EO (Fig. 1).

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TABLE 2. Effect of essential oils on growth of pure cultures of rumen bacteria after direct inoculation or after adaptation

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FIG. 1. Effect of EO on the growth of R. amylophilus WP109. Growth was determined with the following additions of EO: 0 ppm ( ${diamondsuit}$ ), 50 ppm ( ${blacksquare}$ ), 100 ppm ( ${blacktriangleup}$ ), 200 ppm ( ${square}$ ), 400 ppm ( ${circ}$ ), 800 ppm (•), and 1,600 ppm ( ${triangleup}$ ). Results are mean values and standard errors of triplicates.

The bacteriolytic activity of ruminal protozoa was unaffected(data not shown). However, EO affected the activity of the ruminalfungus N. frontalis, with H₂ production almost totally inhibitedat a concentration of 40 ppm (Fig. 2). The sensitivity of N.frontalis to EO after adaptation was not determined, as themeasurements of H₂ production were made after 10 days of incubation,sufficient time to allow the fungi to adapt. Growth of the methanogenM. smithii was unaffected by EO concentrations of up to 160ppm, although inhibition occurred at 1,000 ppm (Fig. 3).

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FIG. 2. Effect of EO on the growth of N. frontalis RE1. Growth was measured indirectly by the production of hydrogen in the headspace gas after 10 days of incubation. The results are mean values and standard errors of the means from three cultures.

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FIG. 3. Effect of EO on growth of M. smithii. Growth was measured indirectly by the production of methane in the headspace gas after 10 days of incubation. The results are mean values and standard errors of the means from three cultures.

Mode of action of EO.

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Mode of action of...

These results suggest that the range of sensitivity to EO israther small for most species and that none is particularlysensitive. As the rumen volume of the animals used here is about60 liters and 1,000 mg of EO per day was fed, the likely maximumliquid concentration would be 17 ppm, assuming that no absorptionor metabolism takes place. This is equivalent to half of theIC₅₀ for the most sensitive species (Table 2). Nevertheless,local concentrations of EO, some of which are sparingly soluble,may be higher in vivo, particularly on surfaces of ingestedplant materials, which may increase the potency of EO in vivo.The relatively narrow range of sensitivity contrasts with thesensitivity to ionophores such as monensin and tetronasin, forwhich the range of sensitivities between the most and leastsensitive organisms may be 1,000-fold (4, 10). Nevertheless,the influence of EO may be greater on microorganisms associatedwith solids than on liquid-associated species, adaptation willundoubtedly be a major factor, and the influence of EO on survival,rather than growth, may be a potent selective characteristic.It is also possible that EO elicits an antimicrobial effecton species of ruminal bacteria that have not yet been isolatedin pure culture. Cultivated species do not represent the truebacterial diversity of the rumen ecosystem. PCR-based analysisof the 16S rRNA gene of the whole population revealed that themajority of bacteria are not represented as cultivated species,and some species associated with solids are quite differentfrom those in fluid (17).

The present results indicate that the main effect on proteincatabolism is at the final stage, deamination of amino acids.Ammonia production in the rumen is predominantly performed bytwo distinct groups of organisms: species present in high numbersbut with low specific activity, as well as those present inlow numbers but with very high specific activity (16). The latterare the group of HAP species, which are characterized as beingmonensin sensitive and nonsaccharolytic (13). The finding thatmonensin-insensitive deaminating activity was similar in bothtreatments suggests that EO does not affect deaminating speciesunaffected by monensin. Furthermore, while HAP species all seemto be sensitive to monensin (1-3, 5, 15, 16), EO inhibited C.sticklandii and P. anaerobius while the other HAP species tested,Clostridium aminophilum, was not affected. This may explainwhy EO was less effective than monensin at inhibiting ammoniaproduction. It would be of interest to determine the effectsof EO on the wider range of HAP species now available (1, 5,9). HAP species vary from diet to diet and perhaps geographically.It would also be informative to determine which oil in the mixtureis most effective at inhibiting deamination, as well as themechanisms of its toxicity.

EO may also affect proteolysis in vivo, however. Experimentsin which protein supplements were incubated in the sheep rumensuggested that their rate of degradation was suppressed by EO(our unpublished results). The absence of an effect in vitrowith any of the proteins tested here suggests that the colonizationand/or hydrolysis of nonprotein components of the supplementsmay be affected. Furthermore, the toxicity of EO to the proteolyticand amylolytic species R. amylophilus in stationary phase mayslow the breakdown of starchy and/or high-protein supplements,which may be beneficial to the animal. In the present experiments,proteolysis was measured in SRF. Similar measurements shouldbe made in large particulate matter, where the sensitivity ofruminal fungi, which are proteolytic as well as fibrolytic (19,23), to EO may be of greater significance.

Any beneficial effects of EO on protein metabolism may haveto be balanced against possible detrimental effects on fiberbreakdown. Here, both cellulolytic ruminal bacteria and fungiwere affected by EO, although the bacteria showed adaptationto EO. Fernandez et al. (6) found that a 20-fold increase inthe amount of EO given to sheep, from 50 to 1,000 mg day^-1,caused a marked inhibition in fiber digestion in the rumen,which may be consistent with these antimicrobial effects.

Implications.

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Implications.

These experiments with EO demonstrate that it is possible touse natural products, particularly plant secondary metabolites,to manipulate ruminal fermentation by selective suppressionof certain microbial species. EO were considered valuable asantimicrobial agents until the discovery of antibiotics overshadowedthem. As microbiologists strive to retain the usefulness ofantibiotics for human therapy, reexamination of the potentialof EO for a variety of purposes, including growth promotionin animal production, is timely.

ACKNOWLEDGMENTS

The Rowett Research Institute receives financial support fromthe Scottish Executive Environment and Rural Affairs Department.

FOOTNOTES

* Corresponding author. Mailing address: Rowett Research Institute, Greenburn Rd., Bucksburn, Aberdeen AB21 9SB, United Kingdom. Phone: 44 (0) 1224-712751. Fax: 44 (0) 1224-716687. E-mail: FMM{at}ROWETT.AC.UK.

REFERENCES

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