APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2003, p. 50115014 Vol. 69, No.

8
0099-2240/03/$08.000 DOI: 10.1128/AEM.69.8.50115014.2003
Copyright 2003, American Society for Microbiology. All Rights Reserved.
Effects of Essential Oils on Ruminal Microorganisms and Their
Protein Metabolism
F. M. McIntosh,
1
* P. Williams,
2
R. Losa,
3
R. J. Wallace,
1
D. A. Beever,
4
and C. J. Newbold
1
Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB,
1
Akzo Nobel Surface Chemistry Ltd., St. Albans AL1 3AW,
2
and CEDAR, Department of Agriculture, University of Reading, Reading RG6 6AJ,
4
United Kingdom,
and Crina SA, 1196 Gland, Switzerland
3
Received 4 March 2003/Accepted 2 June 2003
A commercial blend of essential oil (EO) compounds was added to a grass, maize silage, and concentrate diet
fed to dairy cattle in order to determine their inuence on protein metabolism by ruminal microorganisms. EO
inhibited (P < 0.05) the rate of deamination of amino acids. Pure-culture studies indicated that the species
most sensitive to EO were ammonia-hyperproducing bacteria and anaerobic fungi.
The drive toward a decrease in the use of antibiotics in
animal production has intensied the search for natural prod-
ucts that enhance production, in the case of ruminants, by
modulating ruminal fermentation. Essential oils (EO) are nat-
ural products that give plants and spices their characteristic
odor and color. Oh et al. (12) found that EO from the Douglas
r pine needle have an antibacterial effect in the rumens of
sheep and deer. Fernandez et al. (6) used a commercial blend
of EO compounds to manipulate rumen fermentation, inhib-
iting the breakdown of protein, thus potentially increasing the
dietary protein available to the ruminant. The aim of the
present study was to investigate the inuence of EO on protein
metabolism by mixed ruminal microorganisms and on different
ruminal bacteria, protozoa, and fungi.
In order to identify the mechanisms and microbial species
affected by EO, the effects of EO were measured in ruminal
microorganisms obtained from four Holstein-Friesian cows,
each tted with a ruminal cannula and fed ad libitum a diet
consisting of grass, maize silage, and concentrate mix (330, 220,
and 450 g kg
1
on a dry-matter basis). The concentrate con-
tained soybean meal, rapeseed meal, sunower meal, maize
gluten feed, maize distillers grain, cottonseed meal, ground
wheat, palm kernel meal, molassed sugar 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 blend of EO (Crina Rumi-
nants; Akzo Nobel Surface Chemistry Ltd., Hertfordshire,
United Kingdom) day
1
as part of a two-by-two factorial de-
sign with 4-week periods. Crina Ruminants is a dened, pat-
ented mixture of natural and nature-identical EO compounds
that includes thymol, eugenol, vanillin, and limonene on an
organic carrier (14). Measurements were made on 2 consecu-
tive days during the last week of each period.
Samples of ruminal uid were removed 2 h after morning
feeding and strained (strained ruminal uid [SRF]) though two
layers of cheese cloth before use in experimental measure-
ments. Ruminal degradation of proteins was investigated by
using casein, lactoglobulin, hide powder azure, albumin, and
elastin Congo red (Sigma Chemical Co., Poole, United King-
dom) reductively methylated with [
14
C]formaldehyde (18).
Peptidase activities were measured with Ala
2
, Ala
5
, Asp-
LysArgValTyr, GlyArg4-methoxy-2-naphthylamide (GlyArg-
MNA), and Ala
2
p-nitroanilide (Ala
2
-pNA) by measuring
peptide breakdown by reverse-phase high-performance liquid
chromatography (20), uorimetrically by the appearance of
MNA, or spectrophotometrically by the appearance of pNA
(20, 21). Deaminase activity was determined with casein acid
hydrolysate as the substrate (11). Monensin, which is known to
inhibit some of the major species of deaminative organisms
(13), was added to samples incubated with casein acid hydro-
lysate at a nal concentration of 5 M.
The effect of EO on bacterial growth was measured by in-
oculating stock cultures grown in the liquid version of Hob-
sons M2 medium (8) into the same medium containing serial
twofold increases in EO concentration (10). EO was added
aseptically after the medium was autoclaved to give nal con-
centrations ranging from 5 to 1,000 ppm. Bacterial growth was
measured by reading the optical density at 650 nm hourly. To
investigate possible adaptation of bacteria to EO, bacteria
were inoculated into tubes containing the lowest concentration
of EO and incubated at 39C anaerobically for 48 h. This tube
was used to inoculate (5%, vol vol
1
) medium containing twice
the EO concentration and so on until a concentration that
inhibited growth totally was reached. The results were re-
corded as the EO concentration at which the optical density
was half of that measured in the absence of EO (IC
50
). The
inuence of EO on rumen ciliate protozoa was determined by
the bacteriolytic activity of protozoa in ruminal uid (22). The
effect of EO on the activity of Methanobrevibacter smithii PS
(ATCC 35061) and Neocallimastix frontalis RE1 was deter-
mined from gas production over 10 days in the presence of
increasing concentrations of EO (10). The microbial species
used are all maintained in the culture collection of the Rowett
Research Institute; their origins were described previously (10,
21).
* Corresponding author. Mailing address: Rowett Research Insti-
tute, Greenburn Rd., Bucksburn, Aberdeen AB21 9SB, United King-
dom. Phone: 44 (0) 1224-712751. Fax: 44 (0) 1224-716687. E-mail:
FMM@ROWETT.AC.UK.
5011
Data were analyzed as a two-by-two factorial design with
Genstat 5 (7).
Inuence of dietary EO on protein metabolism. The differ-
ent proteins incubated with SRF in vitro were hydrolyzed at
rates that spanned 2 orders of magnitude (Table 1). EO had no
effect on the breakdown of any of the proteins tested (Table 1),
even with their different secondary and tertiary structures. Ca-
sein and lactoglobulin are soluble proteins with little ordered
structure; albumin is also soluble but contains abundant disul-
de cross-links that make it more resistant to degradation (18);
hide powder azure and elastin are both insoluble, and the latter
is also heavily cross-linked (18).
The peptides selected also covered a range of potential sub-
strates, including di- and oligopeptides and substrates for
dipeptidyl aminopeptidases. Once again, EO had no effect
(Table 1). There was, however, a signicant decrease (P
0.05) in the rate of ammonia production from casein acid
hydrolysate, which comprises free amino acids, when casein
acid hydrolysate was incubated with SRF from cattle receiving
EO (Table 1). A similar study with sheep indicated a 24%
decrease in deamination of amino acids (our unpublished re-
sults). The decrease in ammonia production caused by EO was
abolished in the presence of monensin (Table 1), and the
deamination rate was reduced to similar values in both treat-
ments. Taken together, these results suggest that EO sup-
presses some of the species that are inhibited by monensin but
has little effect on other deaminating species.
Inuence of EO on pure cultures of ruminal microorgan-
isms. EO, when added to the culture medium, inhibited the
growth of most pure cultures of ruminal bacteria at concen-
trations of less than 100 ppm; Streptococcus bovis was the most
resistant species, and Prevotella ruminicola, Clostridium stick-
landii, and Peptostreptococcus anaerobius were the most sensi-
tive species (Table 2). Some species, including P. ruminicola
and P. bryantii, adapted and were able to grow in higher con-
centrations of EO, while others, including C. sticklandii and P.
anaerobius, remained sensitive. The last two species are among
those identied by Russell and his colleagues (2, 3, 13, 15, 16)
as hyper-ammonia producing (HAP) species detrimental to
the efciency of nitrogen retention in ruminants. Any adapta-
tion to EO would diminish the effect in vivo. A further con-
sideration may be the survival of bacteria in stationary phase:
the loss of viability of Ruminobacter amylophilus was greatly
increased by EO (Fig. 1).
The bacteriolytic activity of ruminal protozoa was unaffected
(data not shown). However, EO affected the activity of the
ruminal fungus N. frontalis, with H
2
production almost totally
inhibited at a concentration of 40 ppm (Fig. 2). The sensitivity
of N. frontalis to EO after adaptation was not determined, as
the measurements of H
2
production were made after 10 days
of incubation, sufcient time to allow the fungi to adapt.
Growth of the methanogen M. smithii was unaffected by EO
concentrations of up to 160 ppm, although inhibition occurred
at 1,000 ppm (Fig. 3).
Mode of action of EO. These results suggest that the range
of sensitivity to EO is rather small for most species and that
none is particularly sensitive. As the rumen volume of the
animals used here is about 60 liters and 1,000 mg of EO per
day was fed, the likely maximum liquid concentration would be
17 ppm, assuming that no absorption or metabolism takes
place. This is equivalent to half of the IC
50
for the most sen-
sitive species (Table 2). Nevertheless, local concentrations of
EO, some of which are sparingly soluble, may be higher in vivo,
particularly on surfaces of ingested plant materials, which may
TABLE 1. Effect of feeding essential oils to cattle on the subsequent breakdown of proteins, peptides, and amino acids
in rumen uid in vitro
Treatment or
parameter
Amt (mg) of substrate
degraded mg of protein
1
h
1
Amt (nmol) of peptide
degraded mg of protein
1
h
1
Amt (nmol) of NH
3
produced mg of
protein
1
h
1
Casein
Lacto-
globulin
Hide powder
azure
Albumin
Elastin
Congo red
Ala
2
Ala
5
Arg Lys Asp
Val Tyr
Ala
2
-
pNA
GlyArg-
MNA
No
monensin
5 M
monensin
Control 0.46 0.21 0.048 0.033 0.0056 0.60 1.03 0.33 1.64 0.36 410 280
Essential oils 0.49 0.24 0.049 0.035 0.0054 0.69 1.22 0.35 1.58 0.40 372 287
SED
b
0.038 0.032 0.0035 0.0016 0.00092 0.088 0.230 0.035 0.056 0.060 9.8
a
10.9
a
P 0.05.
b
SED, standard error of the difference.
TABLE 2. Effect of essential oils on growth of pure cultures of
rumen bacteria after direct inoculation or after adaptation
Strain
IC
50
of essential oils (ppm)
a
Direct
inoculation
Adaptation
Clostridium sticklandii 12662 36.0 35.0
Peptostreptococcus anaerobius 27337 42.5 52.0
Selenomonas ruminantium Z108 57.0 58.0
Ruminococcus avefaciens FD1 60.0 60.0
Prevotella brevis GA33 57.5 61.0
Prevotella albensis M384 50.0 65.0
Eubacterium ruminantium 2377 70.0 76.0
Anaerovibrio lipolytica 5S 73.8 80.0
Veillonella parvula L59 88.0 86.0
Prevotella ruminicola 23 33.8 94.3
Fibrobacter succinogenes S85 95.0 110.0
Butyrivibrio brisolvens SH13 56.2 112.5
Prevotella bryantii B14 54.0 127.5
Lachnospira multipara D15d 112.5 120.0
Ruminococcus albus SY3 49.0 132.5
Ruminobacter amylophilus WP109 42.6 150.0
Mitsuokella multiacidas 46/5 113.5 160.0
Megasphaera elsdenii J1 113.0 190.0
Lactobacillus casei LB17 56.2 221.2
Streptococcus bovis ES1 127.5 240.0
Clostridium aminophilum 49906 94.2 262.5
a
IC
50
is the concentration of essential oils that led to a 50% decrease in cell
density at 24 h of incubation. The results shown are means of three culture tubes
at each concentration.
5012 MCINTOSH ET AL. APPL. ENVIRON. MICROBIOL.
increase the potency of EO in vivo. The relatively narrow range
of sensitivity contrasts with the sensitivity to ionophores such
as monensin and tetronasin, for which the range of sensitivities
between the most and least sensitive organisms may be 1,000-
fold (4, 10). Nevertheless, the inuence of EO may be greater
on microorganisms associated with solids than on liquid-asso-
ciated species, adaptation will undoubtedly be a major factor,
and the inuence of EO on survival, rather than growth, may
be a potent selective characteristic. It is also possible that EO
elicits an antimicrobial effect on species of ruminal bacteria
that have not yet been isolated in pure culture. Cultivated
species do not represent the true bacterial diversity of the
rumen ecosystem. PCR-based analysis of the 16S rRNA gene
of the whole population revealed that the majority of bacteria
are not represented as cultivated species, and some species
associated with solids are quite different from those in uid
(17).
The present results indicate that the main effect on protein
catabolism is at the nal stage, deamination of amino acids.
Ammonia production in the rumen is predominantly per-
formed by two distinct groups of organisms: species present in
high numbers but with low specic activity, as well as those
present in low numbers but with very high specic activity (16).
The latter are the group of HAP species, which are character-
ized as being monensin sensitive and nonsaccharolytic (13).
The nding that monensin-insensitive deaminating activity was
similar in both treatments suggests that EO does not affect
deaminating species unaffected by monensin. Furthermore,
while HAP species all seem to be sensitive to monensin (13,
5, 15, 16), EO inhibited C. sticklandii and P. anaerobius while
the other HAP species tested, Clostridium aminophilum, was
not affected. This may explain why EO was less effective than
monensin at inhibiting ammonia production. It would be of
interest to determine the effects of 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 in-
formative to determine which oil in the mixture is most effec-
tive at inhibiting deamination, as well as the mechanisms of its
toxicity.
EO may also affect proteolysis in vivo, however. Experi-
ments in which protein supplements were incubated in the
sheep rumen suggested that their rate of degradation was sup-
pressed by EO (our unpublished results). The absence of an
effect in vitro with any of the proteins tested here suggests that
the colonization and/or hydrolysis of nonprotein components
of the supplements may be affected. Furthermore, the toxicity
of EO to the proteolytic and amylolytic species R. amylophilus
in stationary phase may slow the breakdown of starchy and/or
high-protein supplements, which may be benecial to the an-
imal. In the present experiments, proteolysis was measured in
SRF. Similar measurements should be made in large particu-
FIG. 1. Effect of EO on the growth of R. amylophilus WP109.
Growth was determined with the following additions of EO: 0 ppm
(), 50 ppm (I), 100 ppm (), 200 ppm (), 400 ppm (E), 800 ppm
(F), and 1,600 ppm (). Results are mean values and standard errors
of triplicates.
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.
FIG. 3. Effect of EO on growth of M. smithii. Growth was mea-
sured 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.
VOL. 69, 2003 ESSENTIAL OILS IN THE RUMEN 5013
late matter, where the sensitivity of ruminal fungi, which are
proteolytic as well as brolytic (19, 23), to EO may be of
greater signicance.
Any benecial effects of EO on protein metabolism may
have to be balanced against possible detrimental effects on
ber breakdown. Here, both cellulolytic ruminal bacteria and
fungi were affected by EO, although the bacteria showed ad-
aptation to EO. Fernandez et al. (6) found that a 20-fold
increase in the amount of EO given to sheep, from 50 to 1,000
mg day
1
, caused a marked inhibition in ber digestion in the
rumen, which may be consistent with these antimicrobial ef-
fects.
Implications. These experiments with EO demonstrate that
it is possible to use natural products, particularly plant second-
ary metabolites, to manipulate ruminal fermentation by selec-
tive suppression of certain microbial species. EO were consid-
ered valuable as antimicrobial agents until the discovery of
antibiotics overshadowed them. As microbiologists strive to
retain the usefulness of antibiotics for human therapy, reex-
amination of the potential of EO for a variety of purposes,
including growth promotion in animal production, is timely.
The Rowett Research Institute receives nancial support from the
Scottish Executive Environment and Rural Affairs Department.
REFERENCES
1. Attwood, G. T., A. V. Klieve, D. Ouwerkerk, and B. K. Patel. 1998. Ammonia-
hyperproducing bacteria from New Zealand ruminants. Appl. Environ. Mi-
crobiol. 64:17961804.
2. Chen, G., and J. B. Russell. 1988. Fermentation of peptides and amino acids
by a monensin-sensitive ruminal peptostreptococcus. Appl. Environ. Micro-
biol. 54:27422749.
3. Chen, G., and J. B. Russell. 1989. More monensin-sensitive, ammonia-
producing bacteria from the rumen. Appl. Environ. Microbiol. 55:1052
1057.
4. Chen, M., and M. J. Wolin. 1979. Effect of monensin and lasalocid-sodium
on the growth of methanogenic and rumen saccharolytic bacteria. Appl.
Environ. Microbiol. 38:7277.
5. Eschenlauer, S. C. P., N. McKain, N. D. Walker, N. R. McEwan, C. J.
Newbold, and R. J. Wallace. 2002. Ammonia production by rumen microor-
ganisms and enumeration, isolation, and characterization of bacteria capable
of growth on peptides and amino acids from the sheep rumen. Appl. Envi-
ron. Microbiol. 68:49254931.
6. Fernandez, M., E. Serrano, P. Frutos, F. J. Giraldez, A. R. Mantecon, and
J. R. Llach. 1997. Effect of Crina HC supplement upon the rumen degra-
dative activity in sheep. Inf. Tech. Econ. Agraria 18(Vol. Extra):160162.
7. Genstat 5 Committee. 1987. Genstat 5 users manual. Oxford University
Press, Oxford, United Kingdom.
8. Hobson, P. N. 1969. Rumen bacteria. Methods Microbiol. 3B:133149.
9. McSweeney, C. S., B. Palmer, R. Bunch, and D. O. Krause. 1999. Isolation
and characterization of proteolytic ruminal bacteria from sheep and goats
fed the tannin-containing shrub legume Calliandra calothyrsus. Appl. Envi-
ron. Microbiol. 65:30753083.
10. Newbold, C. J., R. J. Wallace, N. D. Watt, and A. J. Richardson. 1988. The
effect of the novel ionophore tetronasin (ICI 139603) on ruminal microor-
ganisms. Appl. Environ. Microbiol. 54:544547.
11. Newbold, C. J., R. J. Wallace, and N. McKain. 1990. Effects of the ionophore
tetronasin on nitrogen metabolism by ruminal microorganisms in vitro. J.
Anim. Sci. 68:11031109.
12. Oh, H. K., T. Sakai, M. B. Jones, and W. M. Longhurst. 1967. Effect of
various essential oils isolated from Douglas r needles upon sheep and deer
rumen microbial activity. Appl. Microbiol. 68:777784.
13. Paster, B. J., J. B. Russell, C. M. J. Yang, J. M. Chow, C. R. Woese, and R.
Tanner. 1993. Phylogeny of the ammonia-producing ruminal bacteria Pep-
tostreptococcus anaerobius, Clostridium sticklandii, and Clostridium aminophi-
lum sp. nov. Int. J. Syst. Bacteriol. 43:107110.
14. Rossi, J. December 1999. Additives for animal nutrition and technique for
their preparation. European patent EP 0646321 B1.
15. Russell, J. B., H. J. Strobel, and G. Chen. 1988. Enrichment and isolation of
a ruminal bacterium with a very high specic activity of ammonia production.
Appl. Environ. Microbiol. 54:872877.
16. Russell, J. B., R. Onodera, and T. Hino. 1991. Ruminal protein fermenta-
tion: new perspectives on previous contradictions, p. 681697. In T. Tsuda,
Y. Sasaki, and R. Kawashima (ed.), Physiological aspects of digestion, me-
tabolism in ruminants: proceedings of the Seventh International Symposium
on Ruminant Physiology. Academic Press, London, England.
17. Tajima, K., R. I. Aminov, T. Nagamine, K. Ogata, M. Nakamura, H. Matsui,
and Y. Benno. 1999. Rumen bacterial diversity as determined by sequence
analysis of 16S rDNA libraries. FEMS Microbiol. Lett. 29:159169.
18. Wallace, R. J. 1983. Hydrolysis of
14
C-labelled proteins by rumen micro-
organisms and by proteolytic enzymes prepared from rumen bacteria. Br. J.
Nutr. 50:345355.
19. Wallace, R. J., and K. N. Joblin. 1985. Proteolytic activity of a rumen
anaerobic fungus. FEMS Microbiol. Lett. 29:1925.
20. Wallace, R. J., and N. McKain. 1989. Analysis of peptide metabolism by
ruminal microorganisms. Appl. Environ. Microbiol. 55:23722376.
21. Wallace, R. J., and N. McKain. 1991. A survey of peptidase activity in rumen
bacteria. J. Gen. Microbiol. 137:22592264.
22. Wallace, R. J., and C. A. McPherson. 1987. Factors affecting the rate of
breakdown of bacterial protein in rumen uid. Br. J. Nutr. 58:313323.
23. Yanke, L. J., Y. Dong, T. A. McAllister, and K.-J. Cheng. 1993. Comparison
of amylolytic and proteolytic activities of ruminal fungi grown on cereal
grains. Can. J. Microbiol. 39:817820.
5014 MCINTOSH ET AL. APPL. ENVIRON. MICROBIOL.