J Sci Food Agric 1990,50, 191-199

Metabolism of Small Peptides in Rumen Fluid. Accumulation of Intermediates during Hydrolysis of Alanine Oligomers, and Comparison of Peptidolytic

Activities of Bacteria and Protozoa

R John Wallace, Nest McKain and C James Newbold

Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK

(Received 28 October 1988; revised version received 17 March 1989; accepted 19 April 1989)

ABSTRACT

Oligopeptides of L-alanine up to ala, were incubated in vitro in either strained rumen fluid or suspensions of mixed rumen bacteria. The disappearance of substrates and formation of products were measured for 40 min, by which time ala,, ala, and ala, were almost totally hydrolysed. Ala, was more slowly hydrolysed, and accumulated in incubations with the other peptides. The pattern of formation of ala, but not ala, @om ala,, and ala, and ala, but not ala, @om ala,, suggested that the peptides were being hydrolysed by a dipeptidyl peptidase mechanism. Three diffirent sheep receiving different diets gave similar results. Neither substrates nor products appeared to be accumulated intracellularly, except for ala,, and then only if protozoa were present. Protozoa were more active than bacteria in ala, hydrolysis, whereas bacteria had greater activities with higher homologues. Similar preferences were observed with glycine peptides, although unlike alanine peptides gly, and sly, were more slowly degraded than the dimer. These experiments suggest that protozoa are of importance in the accumulation and hydrolysis of dipeptides, whereas bacteria are responsible for the breakdown of larger molecules by a dipeptidyl peptidase mechanism that does not appear to involve accumulation within the cell.

Key words: Rumen, peptides, protozoa, bacteria.

191

J Sci Food Agric 0022-5142/89/$03.50 0 1989 Society of Chemical Industry. Printed in Great Britain

192 R J Wallace, N McKain, C J Newbold

INTRODUCTION

The breakdown of dietary protein to ammonia in the rumen can be one of the most inefficient features of ruminant nutrition because ammonia is often produced in excess of microbial requirements for protein synthesis and much of the nitrogen is therefore excreted (Tamminga 1979; Leng and Nolan 1984). Peptides are intermediates in the catabolic process. They can accumulate in rumen contents (Russell et al1983; Chen et al1987a; Broderick and Wallace 1988), sometimes to an extent that apparently results in significant amounts of amino acids passing from the rumen in the form of peptides (Chen el a1 1987a). Inhibition of peptide hydrolysis could enhance the quantity of amino acids escaping degradation by this route.

Some selectivity occurs in the metabolism of peptides by rumen microorganisms. Hydrophilic molecules are utilised more rapidly than hydrophobic compounds (Chen et a1 1987b), and prolinecontaining peptides are hydrolysed more slowly than other neutral peptides (Broderick et a1 1988). Fluorimetric determination of peptides by fluorescamine indicated that ala, was hydrolysed more rapidly than other alanine oligomers (Broderick et a1 1988). The present paper describes the pattern of hydrolysis of alanine peptides by rumen bacteria and protozoa, and the sequence and location of appearance of intermediates and products. A preliminary account of some of this work has already been presented (Newbold et al 1989).

EXPERIMENTAL

Animals, diets and sample preparation

All sheep were mature animals fitted with a permanent rumen cannula, and were fed twice daily at 0800 and 1600 h. At each feed, sheep A received 700g grass hay, supplemented with 1 % urea, and minerals and vitamins according to ARC (1980) recommendations, sheep B received 450g of a diet of 67% grass hay and 33% concentrate (Whitelaw et al 1983), sheep C and D were fed the same diet as sheep B except that grass nuts replaced half of the hay, and sheep E received 500 g of a 50% hay/50% concentrate diet (Stewart and Duncan 1985).

Strained rumen fluid (SRF) was prepared about 2 h after the morning feeding by straining rumen contents through four layers of muslin. Portions of SRF were fixed for protozoal counts by adding to an equal volume of 4% formaldehyde. Mixed rumen bacteria (MRB) were prepared by sedimenting protozoa from SRF at 30 x g at 5°C for 15 min. This very low speed was to ensure that loss of bacteria was minimal. Fewer than 0.4 % of the original number of protozoa in SRF remained in MRB. Resuspended bacteria (RMRB) were prepared by centrifuging SRF at 650 x g at 5°C for 15 min. This higher force ensured that all protozoa were removed. The resultant supernate was centrifuged at 49 000 x g at 5°C for 30 min to pellet the bacteria. The bacterial pellet was then resuspended in Coleman’s salts solution D (Coleman 1978). Protozoa were recovered from SRF by sedimentation followed by washing on 10-pm-aperture cloth (Newbold et al1987). Washed protozoa were also suspended in Coleman’s salts solution D.

Peptide metabolism by rumen microorganisms 193

Incubation of SRF and MRB with alanine peptides: determination of fate of peptide substrates and products

SRF or MRB was diluted in anaerobic phosphate buffer as described previously (Broderick et al 1988). Portions (0.6 ml) of diluted samples were then mixed with 0.2 ml of 1.25 mM peptide and incubated at 39°C under CO,. Tubes were mixed gently at c 5-min intervals. For the determination of total peptides, the reaction was stopped by adding 0-2 ml of 250 g litre-' TCA, then the mixture was centrifuged in a microcentrifuge (12000xg for 3 min, 20°C). For the determination of extracellular peptides, the incubation mixture was chilled in an ice-water slurry, then centrifuged in the same way. The supernate was immediately decanted into 0.2 ml of 250 g litre-' TCA and centrifuged again. The supernates from the TCA extracts were then filtered through glass fibre filters (type A/E; Gelman Sciences) before analysing for peptides by high-performance liquid chromatography (HPLC). All incubations were done in duplicate.

Peptide hydrolysis in bacterial and protozoal fractions from rumen fluid

Suspensions of rumen protozoa and RMRB were prepared from sheep C and D. Portions (0.6 ml) were incubated with 0-2 ml of 1.25 mM peptide under CO, as before, and the reaction was terminated by adding 0.2 ml of 300 g litre- perchloric acid (PCA) at 0, 20, 40 and 60 min. Samples were stored and analysed as before.

Peptide, amino acid and protein analysis

Peptides were estimated by hydrophobic ion-pairing HPLC (Newbold et al 1989). L-Alanine was measured enzymically using alanine dehydrogenase (Williamson 1985), NADH oxidation being measured fluorimetrically by excitation at 340 nm and emission at 457 nm. Protein was determined by a modified Lowry method and dry matter was estimated as described previously (Broderick et a1 1988).

Materials

Peptides contained amino acids entirely of the L-configuration and were obtained from Sigma Chemical Co. Antibiotics were also from Sigma. Other chemicals were analytical-reagent grade from British Drug Houses.

RESULTS Fate of substrates and products during the hydrolysis of alanine peptides in rumen fluid in vitro

Samples of rumen fluid from sheep B were removed on different days for the analysis of the metabolism of ala,, ala,, ala, and ala, by rumen microorganisms in vitro. The rate of hydrolysis of ala, in SRF (3.5 nmol ml-' min-'; Fig 1) was considerably slower than the initial rates of hydrolysis of the other oligomers. Ala, (Fig 2), ala, (Fig 3) and ala, (Fig 4) were removed at approximate rates of 10-6, 18.9 and 12.0 nmol ml-' min-' respectively during the first 5-min incubation. Ala, concentrations were very low in incubations with ala, (Fig 3), yet accumulation

I94

300

200

- z 100

E . - 0

v

C 0 .- I

300 C m 0 C

0

m 200 .- C m m 0

- .-

100

15 30 45 Incubation time (mid

Fig 1. Metabolism of ala, in (a) SRF, and (b) MRB prepared from rumen fluid taken from sheep B, receiving 2:1 liay:concentrate. 0, Total ala, in incubation mixture; 0, extracellular ala,. The dry matter contents of the incubation mixtures with SRF and MRB were 6.06 and 2.43mgml-' respectively, with corresponding protein contents of 2.67 and 1.19mgml-'. The protozoal population in SRF incubations was

7.32 x lo5 m1-l.

R J Wallace, N McKain, C J Newbold

300

200

e E z 100 E - c 0 .- e

2 - c

c 0 0 W P

W

300

.- ; 200 a

100

15 30 45 Incubation t ime (rnin)

Fig 2. Metabolism of ala, in (a) SRF, and (b) MRB prepared from rumen fluid taken from sheep B, receiving 2:l hay:concentrate. Open symbols represent the total concentration of the peptide in the incubation mixture, and closed symbols the extracellular concentrations. A, ala,; 0, ala,. The dry matter contents of the incubation mixtures with SRF and MRB were 5-55 and 2.81 mg ml- respectively, with corresponding protein contents of 2.76 and 1.35 mg ml-'. The protozoal population in ,SRF incubations was

5.54 x lo5 d-'.

occurred in incubations with ala, which was hydrolysed more slowly (Fig 4). Similarly, ala, was undetectable during ala, metabolism (Fig 4).

In order to determine if the substrates and products were located intra- or extracellularly, TCA was added to the incubation mixture before or after centrifugation. Ala, differed from the others in that it was the only peptide that gave substantial differences between extracellular and total concentrations. The difference was most obvious when it was the peptide added initially (Fig la), but a similar phenomenon can be seen when ala, concentrations increased as the result of hydrolysis of other peptides in SRF (Figs 2 4 ) . The concentration difference, implying intracellular accumulation of the peptide, did not appear to occur when MRB alone were used. The concentration of other peptides tended to be slightly greater in extracts when TCA was added immediately, rather than the sample being centrifuged before the addition of TCA. This might be taken to indicate some degree

Peptide metabolism by rumen microorganisms 195

A E > 0

E Y

c 0 .- I

2 - C a, 0 C 0 V

Q .- '0

n n

- 0

300

200

100

300

200

100

15 30 45 Incubation time (rnin)

Fig 3. Metabolism ofala, in (a) SRF, and (b) MRB prepared from rumen fluid taken from sheep B, receiving 2:l hay:concentrate. Open symbols represent the total concentration of the peptide in the incubation mixture, and closed symbols the extracellular concentrations. 0, ala,; A, ala,; 0, ala,. The dry matter contents of the incubation mixtures with SRF and MRB were 6.57 and 3.35 mg ml-' respectively, with corresponding protein contents of 3.36 and 1.68 mg m1-l. The protozoal population in SRF incubations was

3.72 x 10s ml-'.

300

200

h

100 1 0 E C Y

C 0 .- - 2 300 * c Q V C 0 V Q 200 rr n

c. P 0

100

15 30 45 Incubation time (rnin)

Fig 4. Metabolism ofala, in (a) SRF, and (b) MRB prepared from rumen fluid taken from sheep B, receiving 2:l hay:concentrate. Open symbols represent the total concentration of the peptide in the incubation mixture, and closed symbols the extracellular concentrations. V, ala,; 0, ala,; A, ala,; 0, ala,. The dry matter contents of the incubation mixtures with SRF and MRB were 6.65 and 3.14 mg ml-' respectively, with corresponding protein contents of 3.33 and 1.68 mg ml- '. The protozoal population in SRF

incubations was 3 . 1 4 ~ lo5 ml-'.

of accumulation of these molecules. However, the chilling and subsequent centrifugation that was required before TCA was added to these samples more probably failed to stop the reaction as rapidly as immediate TCA addition.

The peptidolytic activity of rumen bacteria was determined in SRF that had been centrifuged at 30 x g to remove protozoa. This procedure had a greater influence on the rate of breakdown of ala, than the other peptides. Ala, hydrolysis was 34% less after 40 min incubation in MRB compared with SRF (Fig 1). The initial rates of hydrolysis of the other peptides were decreased by 9,19 and 12 % for ala,, ala, and alas (Figs 2-4).

Influence of diet on peptide metabolism

Ala,, ala, and alas were incubated with SRF from sheep fed grass hay diets with different amounts of concentrate. The extent of peptide hydrolysis was higher with

196 R J Wallace, N McKain, C J Newhold

concentrate in the diet, but this was probably caused by the increased concentration of microbial protein in these incubations (Table 1). The pattern of product formation was the same in all three sheep (Table 1). Ala, continued to be the main intermediate accumulating during hydrolysis, and again ala, was not formed from ala, and alas hydrolysis did not result in the accumulation of ala,. Alanine also accumulated, but almost always to a concentration considerably lower than that of ala, . Role of protozoa and bacteria in hydrolysis of different peptides

Buffered suspensions of protozoa and bacteria were incubated with ala,, ala, and ala,, and analysed for the rate of disappearance of original peptide, enabling calculations of the specific activity of protozoal and bacterial peptidases against the different substrates to bemade (Tab!e 2). Protozoa had a specific activity against the dimer almost three times that of bacteria. The specific activity of both preparations was higher with ala,, with the protozoal suspension being slightly less active than bacteria. Ala, hydrolysis was three times higher with bacteria, and was the highest activity observed in these experiments. The addition of antibiotics to inhibit bacterial growth in the protozoal suspensions had little influence on the specific activities observed.

Incubations with glycine peptides were included in the same experiment, to determine if a similar pattern emerged. The specific activities were lower, particularly with gly, and gly, (Table 2), but again the activity of bacteria relative to protozoa increased as chain length increased. Protozoa had twice the activity of bacteria against gly,, but only half their activity against gly,.

TABLE 1 Hydrolysis of alanine peptides in strained rumen fluid from sheep receiving different diets"

Sheep Peptide Peptide concentration (nmol mi- ') added

Ala, Ala, Ala, Ala, Ala,

A Ala, - - 154 38 47 Ala, - 69 14 155 20 Alas 84 ND 74 96 30

- 84 I10 40 Ala, - 10 ND 235 30 Alas 42 ND 59 136 50

- 100 131 14 Ala, - 35 ND 174 29

B Ala, -

E Ala, -

Ala, 74 ND 69 144 49

a Sheep A, B and E received hay diets containing 0, 33 and 50% concentrate respectively. Results are means obtained from duplicate incubations for 20 min at 39°C. Initial peptide concentration was 313 nmol m1-I. SRF from the three sheep contained 2.75, 3.65 and 3.24 mg protein ml- ' respectively. ND, Not detectable.

Peptide metabolism by rumen microorganisms I97

TABLE 2 Peptide metabolism by suspensions of rumen bacteria and protozoa

Specific activity (nmol mg - protein rnin - )"

Ala, Ala, Ala, GlY* GlY, GlYs

Bacteria 1.4 7.5 12.8 0.8 0.4 0.3 Protozoa 3.8 1.0 4.4 1.7 0 5 0.2 Protozoa + antibiotics' 3.8 6.2 3.7

a Mean of initial rate of peptide hydrolysis measured in duplicate in suspensions prepared from four samples of rumen fluid for alanine peptides and three samples of rumen fluid for glycine peptides.

' Protozoa were incubated with a mixture of antibacterial antibiotics, comprising neomycin, streptomycin and penicillin, each at 500 pg ml-', for 30 min before peptides were added.

b - - -

Not determined.

DISCUSSION

Several main conclusions can be drawn from these experiments about the way in which peptides are metabolised by rumen microorganisms. First, the products of hydrolysis of higher peptides of alanine gave the appearance that they had arisen by a dipeptidyl peptidase mechanism, whereby dipeptides are cleaved from the peptide chain. This was not an isolated phenomenon, as it occurred with different sheep receiving different diets. The mechanism is by no means proven, however. Many more peptides require investigation. Analysis of the products formed from glycine peptides failed to provide additional evidence of a dipeptidyl peptidase mechanism, because the shorter peptides were metabolised more rapidly than the longer ones, in contrast to the alanine peptides.

Direct measurement of individual peptides by HPLC, as compared with the fluorimetric method used previously (Broderick et a1 1988), has caused the present authors to revise some of their earlier conclusions. Measurement of total peptides with fluorescamine suggested that ala, was the most rapidly metabolised alanine peptide. It now appears that ala, is most rapidly removed and that earlier conclusions were confounded by the accumulation of ala,. The major difference between gly, and ala, in rates of hydrolysis was also not previously apparent (Broderick et al 1988), presumably because of the build-up of ala, but not gly,. Similarly, the marked decline in the rate of breakdown with gly, and gly,, in contrast to the alanine peptides, was not apparent. The present authors are currently analysing the metabolism of a number of other peptides in order to shed more light on the nature of the hydrolytic mechanism and on how the amino acid composition of the peptide substrate affects its rate of breakdown.

Dialanine hydrolysis differed from that of the higher peptides, in that the enzyme activity was partly extracellular (Broderick et al 1988) and that cell-associated activity was higher in ciliate protozoa than bacteria. Protozoa appeared to accumulate the peptide, presumably in intracellular organelles. Bacteria also had dipeptidase activity, however. The relative importance of each type of microbe will

198 R J Wallace, N McKoin, C J Newbold

therefore depend on the population sizes of the protozoa. Dipeptidase activity was similar in faunated and ciliate-free sheep, which prompted the conclusion that protozoa were unimportant in this activity (Wallace et al 1987). It would now appear that the likely reason for this observation was that bacteria with higher dipeptidase activity proliferated in ciliate-free sheep to fill the niche previously occupied by the protozoa.

A decreasing role of ciliate protozoa in peptide metabolism as chain length increases was evident from the present experiments, again contrasting with results from ciliate-free sheep, in which ala, hydrolysis was significantly less than in faunated animals (Wallace et a1 1987). Clearly the bacterial population in protozoa- free animals must be quite different in its properties from the normal flora that occurs in faunated animals. The metabolic consequences of defamation, which is a means of manipulating the fermentation that is sometimes advocated to improve the efficiency of rumen fermentation (Leng and Nolan 1984), are therefore impossible to predict simply from studies on the protozoa alone.

Peptide substrates and products, other than dialanine, appeared to occur only extracellularly. Peptides in the extracellular medium were measured in suspensions which had been chilled to prevent further peptide metabolism then centrifuged. Although it was not confirmed that this procedure did not lead to emux of intracellular pools of peptides, similar methods are routinely used to measure solute uptake in bacteria, including rumen bacteria (Martin and Russell 1988). When this probable lack of intracellular peptide accumulation is considered in conjunction with the finding that peptidase activity is cell associated (Broderick et al1988), it can be deduced either that peptidolytic activity is located in the cell envelope, perhaps in the periplasmic space of Gram-negative bacteria, or that transport to intracellular enzymes occurs but the products then leave the cell rapidly following hydrolysis of the transported peptide. At present there is no evidence to favour either possibility.

The authors are now attempting to identify the bacteria responsible for peptide hydrolysis, and to clarify whether transport across the cytoplasmic membrane of bacteria is an integral part of the process.

ACKNOWLEDGEMENTS

Margaret Falconer and Nicola Watt are thanked for their technical assistance.

REFERENCES

Agricultural Research Council 1980 The Nutrient Requirements of Ruminant Livestock. Commonwealth Agricultural Bureaux, Slough.

Broderick G A, Wallace R J 1988 Effect of dietary nitrogen source on concentrations of ammonia, total free amino acids and peptides in the rumen of sheep. J Anim Sci 66

Broderick G A, Wallace R J, McKain N 1988 Uptake of small neutral peptides by mixed 2233-2238.

rumen microorganisms in vitro. J Sci Food Agric 42 109-1 18.

Peptide metabolism by rumen microorganisms 199

Chen G, Russell J B, Sniffen C J 1987a A procedure for measuring peptides in rumen fluid and evidence that peptide uptake can be a rate-limiting step in ruminal protein degradation. J Dairy Sci 70 1211-1219.

Chen G, Strobe1 H J, Russell J B, Sniffen C J 1987b Effect of hydrophobicity on utilization of peptides by ruminal bacteria in vitro. Appl Environ MicrobiolS3 2021-2025.

Coleman G S 1978 Rumen entodiniomorphid protozoa. In: Methods of Cultivating Parasites I n Vitro, eds Taylor A C K & Baker J R. Academic Press, London, pp 39-54.

Leng R A, Nolan J V 1984 Nitrogen metabolism in the rumen. J Dairy Sci 67 1072-1089. Martin S A, Russell J B 1988 Mechanisms of sugar transport in the rumen bacterium

Selenomonas ruminantium. J Gen Microbiol 134 819-827. Newbold C J, Williams A G, Chamberlain D G 1987 The in vitro metabolism of D,L-lactic

acid by rumen microorganisms. J Sci Food Agric 38 9-18. Newbold C J, McKain N, Wallace R J 1989 The role of protozoa in ruminal peptide

metabolism. In: Biochemistry and Molecular Biology of ‘Anaerobic’ Protozoa, eds Lloyd D, Coombs G H & Paget T A. Harwood Academic Press, London, pp 42-55.

Russell J B, Sniffen C J, Van Soest P J 1983 Effect ofcarbohydrate limitation on degradation and utilisation of casein by mixed rumen bacteria. J Dairy Sci 66 763-775.

Stewart C S, Duncan S H 1985 The effect of avoparcin on cellulolytic bacteria of the ovine rumen. J Gen Microbwl 131 427435.

Tamminga S 1979 Protein degradation in the forestomachs of ruminants. J Anim Sci 49

Wallace R J, Broderick G A, Brammall M L 1987 Microbial protein and peptide metabolism in rumen fluid from faunated and ciliate-free sheep. Br J Nutr 58 87-93.

Whitelaw F G, Bruce L A, Eadie J M, Shand W J 1983 2-Aminoethylphosphonic acid concentrations in some rumen ciliate protozoa. Appl Enuiron Microbiol46 95 1-953.

Williamson D H 1985 L-Alanine: determination with alanine dehydrogenase. In: Methods of Enzymatic Analysis (3rd edn), Vol 8, ed Bergmeyer H U. Verlag Chemie, Weinheim,

1615-1630.

pp 341-344.