Journal of Applied Microbiology 1998, 84, 847–851

Influence of dietary acetylated peptides on fermentation andpeptidase activities in the sheep rumen

M.W. Witt, C.J. Newbold and R.J. WallaceRowett Research Institute, Aberdeen, UK

6381/09/97: received 5 September 1997 and accepted 30 September 1997

M.W. WITT, C.J. NEWBOLD AND R.J. WALLACE. 1998. The predominant mechanism of peptidebreakdown by rumen micro-organisms is aminopeptidase. Thus acetylation of the N-terminusof peptides inhibits their degradation by rumen micro-organisms in short-term incubationswith rumen fluid in vitro. An experiment was undertaken to determine if adaptation of therumen microbial population would take place when acetylated peptides were fed for aprolonged period, which would enable the microbial population to break down the protectedpeptides and thus decrease their nutritive value. Three adult sheep, fitted with permanentrumen cannulae, received a maintenance hay/concentrate diet to which was added, at eachmeal, 20 g of casein enzymic hydrolysate (‘peptides’) or 20 g of peptides previously treatedwith acetic anhydride. The diets were fed for 28 d in a 3 × 3 latin square and samples weretaken during the last 7 d. Fermentation products and NH3 concentrations indicated thatacetylated peptides remained less degradable than untreated peptides. There was a trendtowards increased proteolytic activity with acetylated peptides, and dipeptidase activityincreased by 18% and 28%, respectively, compared with untreated peptides and controltreatments. Activity against N-acetyl-Ala2 also increased when acetylated peptides were fed,but it remained only 13% of the rate of Ala2 hydrolysis. No increase was found in the rate ofammonia production from acetylated peptides in animals receiving acetylated peptides–thisrate was 26% of that found with untreated peptides–and acetylated peptides continued topersist for longer in the rumen than untreated peptides after feeding. Thus it was concludedthat the rumen microbial population did not adapt to utilize acetylated peptides.

INTRODUCTION

Excessive breakdown of protein to ammonia in the rumen canlead to inefficient utilization of dietary protein in ruminants(Tamminga 1979; Leng and Nolan 1984). Rumen microbialactivities also mean that amino acids added to the diet arebroken down before they reach the abomasum, and someprotection, either physical or chemical, is necessary if theyare to survive and correct amino acid imbalances in intestinaldigesta (Kaufmann and Lupping 1982; Broderick et al. 1991).When it was discovered that microbial peptidase activity inthe mixed rumen microbial population acts almost exclusivelyfrom the N-terminus of small peptides (Wallace and McKain1989; Wallace et al. 1990a), the possibility that peptides mightbe protected by chemical modification of N-terminal amino

Correspondence to: Dr R.J. Wallace, Rowett Research Institute, Bucksburn,Aberdeen AB21 9SB, UK (e-mail: RJW@RRI.sari.ac.uk).

© 1998 The Society for Applied Microbiology

groups was suggested and tested (Wallace 1992a; Wallaceet al. 1993). Small peptides were protected very effectivelyfrom degradation in rumen fluid in vitro when they had beentreated with various anhydrides. It was therefore proposedthat N-terminal modification of peptides might be a meansof supplying rumen non-degradable amino acids to rumi-nants, or of protecting otherwise rapidly degraded peptidespresent in foodstuffs (Wallace 1992a; Wallace et al. 1993).

If chemically modified peptides are to be of nutritionalvalue to ruminants, the ability of N-terminal modification toprotect peptides from microbial attack must be sustained fora significant period of time. Carboxypeptidases are present inmany species of bacteria (Webb 1992). Although none hasbeen reported in any rumen species, it seemed possible thatadaptation of the rumen microbial population might takeplace over time, resulting in the enrichment of such species,or of species able to remove the modified N-terminal aminoacid from protected peptides. Such adaptation would result

848 M.W. WITT ET AL.

in protection of the constituent amino acids being lost. Theexperiments described in this paper were carried out to inves-tigate this possibility.

MATERIALS AND METHODS

Animals, diets and sampling

Three adult Suffolk cross sheep, fitted with permanent rumencannulae, received a maintenance diet of hay, barley,molasses, fish meal and a vitamins-minerals mix (500, 299·5,100, 91 and 9·5 g kg−1 of dry matter, respectively). Meals(500 g) were given at 08·00 and 16·00 h. The sheep receivedthe basal diet either alone or with 20 g of peptides, or 20 g ofpeptides, previously treated with acetic anhydride, wereadded at each meal. The diets were fed for 28 d in a 3× 3 latinsquare experiment. Samples of rumen fluid were removed on4 days during the last week at 0, 1, 2, 4 and 6 h after themorning meal. Large particulate material was removed bystraining through two layers of muslin.

Preparation of acetylated peptides

A pancreatic casein hydrolysate (Peptone 140, Gibco BRL,Life Technologies, Paisley, UK) was used either unmodified(‘peptides’) or treated with acetic anhydride by a modificationof the method used previously (‘acetylated peptides’; Wallace1992a). Casein hydrolysate (400 g) was dissolved in 1 l ofdistilled water; the mixture was chilled on ice, then 200ml ofacetic anhydride was added. The mixture was stirred for 1 h,then freeze-dried. The degree of modification was determinedby reaction with ninhydrin (Moore and Stein 1954). Themolecular weight distribution of the peptides was determinedby gel filtration (Wallace 1992b).

Chemical analysis of rumen fluid

Samples of rumen fluid were processed and analysed forvolatile fatty acids (VFA), pH and ammonia concentrationsas described by Frumholtz et al. (1989). Peptide and aminoacid concentrations in extracellular rumen fluid were deter-mined by centrifugation, removal of protein using perchloricacid, hydrolysis of the supernatant fluid, and analysis of aminoacids by ion-exchange chromatography as described by Wal-lace et al. (1993). The protein content of strained rumen fluidwas analysed by mixing 1·0ml of strained rumen fluid with0·25ml of 25% (w/v) trichloroacetic acid, centrifuging themixture at 12 600 g for 5min, and analysing the pellet usingthe Folin reagent (Herbert et al. 1971).

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 847–851

Analysis of enzyme activities

Proteinase activity was determined on samples of rumen fluidtaken 2 h after feeding using 14C-labelled casein as substrate(Wallace 1983). Peptidase activity was determined on thesame samples, using either mixed peptides or dialanine (Ala2)and their corresponding acetylated products, as follows.Strained rumen fluid (10ml) was added to bottles containing1mg of N-acetyl Ala2 or unmodified Ala2, and the bottleswere incubated at 39 °C. Samples (1·0ml) were removed at 0and 1 h into 0·25ml of 1·25mol l−1 H3PO4, the precipitatewas removed by centrifugation, and the remaining peptidewas measured by reverse-phase HPLC (Wallace and McKain1989). Breakdown of mixed peptides was measured by adding0·6ml of strained rumen fluid to 0·2ml of 5 g l−1 peptides oracetylated peptides. Incubations were terminated at 0, 2, 4and 6 h by adding 0·2ml of 25% (w/v) trichloroacetic acid,and ammonia concentrations were determined as before.Rates of ammonia production were calculated by linearregression.

Statistical analysis

Data were analysed by ANOVA procedures as a single 3 x 3latin square design using Genstat 5 (Lawes Agricultural Trust1990). Sampling time was considered as a subplot. Treatmentmeans were compared by least significant differences, pro-tected by a significant F-value (Snedecor and Cochran 1980).

RESULTS

The gel filtration profile (data not shown) of the peptidepreparation used in the present experiment was very similarto those of other pancreatic hydrolysates of casein, which hadan average molecular mass of about 520 kDa (Wallace 1992b).A simplified procedure was used to make acetylated peptidesto avoid high salt concentrations in the resulting material.This resulted in 71·5% modification of peptides as deter-mined using ninhydrin.

Dietary peptides or acetylated peptides had no effect onrumen pH, but total VFA concentrations were higher withuntreated peptides (Table 1). The concentrations of theminor, long- and branched-chain VFA were increased withdietary peptides, and the concentrations of these acids withthe acetylated peptides diet were intermediate between sheepon the peptides diet and the unsupplemented control diet(Table 1). Average rumen ammonia concentration showed asimilar pattern (Table 1).

The proteolytic activity of rumen contents was not changedsignificantly by peptides or acetylated peptides in the diet(Table 2). Dipeptidase activity towards Ala2 was 18% and28% higher with the acetylated peptides diet, compared tountreated peptides and control treatments, respectively.

PEPTIDASE ACTIVITY IN SHEEP RUMEN 849

Table 1 Influence of dietaryuntreated peptides and acetylated peptideson fermentation productconcentrations in the sheeprumen

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Diet—––––––––––––––––––––––––––––––––––––No added Untreated Acetylatedpeptides peptides peptides s.e.d.

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––pH 6·4 6·4 6·4 0·08Total VFA (mmol l−1) 102·1a 112·3b 103·2a 1·79Acetic acid (mmol mol−1) 662 656 648 1·8Propionic acid (mmol mol−1) 188 190 194 13·1Butyric acid (mmol mol−1) 118 114 121 8·9Isobutyric acid (mmol mol−1) 11·0 12·3 11·5 1·64Isovaleric acid (mmol mol−1) 10·8 13·5 13·0 0·65Valeric acid (mmol mol−1) 10·3a 12·9b 11·3a 0·37Caproic acid (mmol mol−1) 1·3a 2·1b 1·4a 0·14NH3 (mg l−1) 192 242 215 19·3—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––a,b Results are means of estimations carried out on samples taken from three sheep at 0,1, 2, 4 and 6 h after feeding. Means with different superscripts are significantly different(P ³ 0·05). d.f. � 2.

Table 2 Enzyme activities in rumen fluid of sheep receiving dietary untreated peptides or acetylated peptides—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Diet—––––––––––––––––––––––––––––––––––––No added Untreated Acetylated

Enzyme activity peptides peptides peptides s.e.d.—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Proteinase (mg 14C casein hydrolysed h−1 mg protein−1) 0·91 0·96 1·14 0·074Ala2 hydrolysis (nmol h−1 mg protein−1) 31·7 34·3 40·5 2·27N-Acetyl-Ala2 hydrolysis (nmol h−1 mg protein−1) 2·4 3·6 5·3 5·9Breakdown of mixed peptides (nmol NH3 produced h−1 mg 315 289 260 94·9protein−1)Breakdown of acetylated peptides (nmol NH3 produced h−1 mg 83 103 70 20·7protein−1)—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Results are means of estimations carried out on samples taken from three sheep 2 h after feeding.

Activity against N-acetyl-Ala2 was not increased significantlyand it was still degraded much less rapidly than Ala2 (Table 2).No significant change occurred in the rate of ammonia for-mation from peptides in vitro, and ammonia was formedfrom acetylated peptides at 36% and 27% of the rate fromuntreated peptides with the peptides- and acetylated pep-tides-containing diets, respectively (Table 2).

The pattern of ammonia concentrations after feeding wasconsistent with peptides being broken down within the first2 h, with acetylated peptides causing a slower release ofammonia (Fig. 1). No difference occurred in peptide con-centrations in rumen fluid with the peptides-containing dietcompared to controls (Fig. 2). In contrast, peptide con-centrations more than doubled to 121mg N l−1 1 h afterfeeding the diet containing acetylated peptides. The amino

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 847–851

acid composition of the peptides which accumulated in rumenfluid was not significantly different with the different diets(data not shown).

DISCUSSION

Mixed rumen micro-organisms degrade protein partly to pro-vide amino acids for microbial growth, but also to provideenergy via deamination and fermentative metabolism of thecarbon skeletons of the amino acids (Russell et al. 1991; Wal-lace et al. 1997a). The latter process prevents much of theamino acids consumed by the animal from reaching the smallintestine (Leng and Nolan 1984). Inhibition of the degra-dation process at any of the degradation steps, proteolysis,peptide hydrolysis or amino acid breakdown, would be

850 M.W. WITT ET AL.

Fig. 1 Ammonia concentrations in rumen fluid of sheep afterreceiving a meal containing no added peptides (T), untreatedpeptides (R), or acetylated peptides (Ž)

Fig. 2 Peptide concentrations in rumen fluid of sheep afterreceiving a meal containing no added peptides (T), untreatedpeptides (R), or acetylated peptides (Ž)

beneficial to the nutrition of the animal. In particular, preventionof peptide breakdown could lead to alternative methods forsupplying defined mixtures of amino acids to the small intes-tine, or for upgrading diets in which the protein has beenextensively degraded during processing, or for the deliveryof bioactive peptides to the abomasum.

Peptide hydrolysis by mixed rumen micro-organismsappears to occur principally from the N-terminus; the pre-dominant peptidases present are dipeptidyl peptidases, whichrelease dipeptides from longer oligopeptides (Wallace andMcKain 1989; Wallace et al. 1990a; Wallace 1996). The mixedpopulation has low carboxypeptidase activity (Prins et al.1983; Wallace and Kopecny 1983). Ciliate protozoa possess

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 847–851

high dipeptidase activity, but the breakdown of larger pep-tides is mediated mainly by bacteria (Wallace et al. 1990b).The main species possessing dipeptidyl peptidase activitiesare members of the genus Prevotella (Wallace and McKain1991; McKain et al. 1992; Avgustin et al. 1997; Wallace et al.1997a). Other species of rumen bacteria possess amino-peptidases which cleave single amino acids from peptides(Wallace et al. 1997a,b), notably Streptococcus bovis (Russelland Robinson 1984; Wallace and McKain 1991). Anaerobicfungi also possess aminopeptidase but not carboxypeptidaseactivity (Michel et al. 1993). N-Terminal modification wasshown to result in protection of peptides from degradationby rumen micro-organisms in in vitro experiments, par-ticularly for peptides of low molecular weight (Wallace 1992a;Wallace et al. 1993). Despite this apparent preponderanceof aminopeptidases rather than carboxypeptidases in rumenmicroorganisms, there were grounds for concern that thepopulation might adapt to use acetylated peptides by enrich-ing for organisms with carboxypeptidase.

All of the indications from the present experiments werethat, if adaptation takes place, it occurs slowly. Fluctuationsin the rumen microbial population can occur extremelyrapidly, as with lactic acidosis when a change in diet can leadto a catastrophic overgrowth of lactobacilli, and death of theanimal may occur within 24 h (Slyter 1976; Russell and Stro-bel 1988). Other dietary adaptations occur more slowly, butmost would be expected within about 2 weeks. For example,resistance to a synthetic methanogenesis inhibitor occurredwithin 2–3 weeks (Clapperton 1977). In the present experi-ment, where measurements were made between 21 and 28 dafter introduction of acetylated peptides, the concentrationsof ammonia and higher and branched chain VFA, which areall products of amino acid breakdown, were all lower thanwith the peptides diet. Rates of ammonia formation andpeptide concentrations in vivo also reflected that acetylatedpeptides were broken down substantially more slowly thanunmodified peptides, in spite of only 71·5% modificationbeing achieved by the acetic anyhydride treatment used here.Peptidase and protease activities showed small increases, butnot sufficient to affect the resistance of modified peptides.Even though aminopeptidases are much more abundant thancarboxypeptidases in rumen and other micro-organisms (Laz-dunski 1989; Webb 1992; Kok and de Vos 1994; Wallace et al.1997a), it is remarkable that adaptation did not take place.Peptidases of rumen bacteria are intracellular enzymes, so themetabolism of peptides also depends on their transport, whichmight also be inhibited by acetylation. Clearly, micro-organ-isms rely heavily on a free N-terminus in their metabolismof peptides.

It can therefore be concluded that adaptation by the rumenmicrobial population will not compromise any of the sug-gested applications of N-terminally modified peptides inruminants for at least short- to medium-term exposure.

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ACKNOWLEDGEMENT

This work was supported by the Scottish Office Agriculture,Environment and Fisheries Department.

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