J Sci Food Agric 1992, 58, 177-184

Gel Filtration Studies of Peptide Metabolism bv J

Rumen Microorganisms R John Wallace Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK (Received 4 July 1991 ; revised version received 6 September 1991 ; accepted 29 September 1991)

Abstract: Peptide metabolism by rumen microorganisms was investigated by gel filtration using Sephadex G-25 with water as eluant. Protein hydrolysates containing a mixture of peptides were used to calibrate the column. Total peptide in each fraction was estimated from its A,,,, and free peptide amino groups were analysed by fluorescamine, thus enabling the average peptide M , to be calculated. Three different protein hydrolysates produced similar, nearly linear, relations between log M , and elution volume for peptides between 300 and 2000 Da. Trypticase, a pancreatic hydrolysate of casein, was metabolised rapidly in rumen fluid in uitro. Hydrolysis appeared to be complete after 6 h, leaving a small residual peptide concentration which persisted up to 24 h, equivalent to about 15 % of the original peptide concentration of 2 g litre-'. Residual peptides from casein hydrolysis were 0.05 g litre-' at 24 h. Peptides accumulated in rumen fluid of sheep receiving dietary ionophores. Two hours after feeding, the accumulation with monensin appeared to be of peptides of a wide M, range, while tetronasin caused an accumulation mainly of smaller, < 570 Da, peptides. Treatment of Trypticase with acetic anhydride afforded 76 % protection of its peptides from degradation to ammonia in a 6-h incubation. When Trypticase was fractionated by gel filtration then acetylated, none of the fractions was protected significantly better than others.

Key words : Gel filtration, peptides, rumen microorganisms, Sephadex G-25, ionophores.

INTRODUCTION larger peptides (Newbold et a1 1989; Wallace et a1 1990b).

Microbial proteolytic, peptidolytic and deaminative Gel filtration has been used previously to study peptide activities often lead to excessive ammonia production in metabolism by rumen microorganisms. Wright (1 967) the rumen, leading to inefficient utilisation of dietary made a tryptic hydrolysate of ['4C]-Chlorella protein and protein by ruminants (Leng and Nolan 1984; Wallace analysed its fate in rumen fluid using Sephadex G-25. At 1988). The breakdown of peptides to amino acids is an the low concentrations of 1-2 mg litre-' used, much of integral part of the degradation sequence, and is a site at the label was incorporated into microorganisms, par- which the rate of breakdown might be controlled ticularly from larger peptides. Nugent and Mangan (Wallace et a1 1990a). (1981) followed the hydrolysis of [14C]-fraction 1 protein

Peptide size is one factor that determines how rapidly with a variety of Sephadex sizes from G-200 to G-10, and different compounds in a homologous series of peptides demonstrated that no intermediate peptides accumulated are broken down by rumen microorganisms (Newbold during its hydrolysis. An accumulation of peptides and Wallace 1989; Newbold et a1 1989; Wallace et a1 resulting from casein hydrolysis was observed using 1990b). Peptide size also determines which category of Sephadex G-10 when monensin was added to a semi- microorganism is likely to hydrolyse a peptide. Rumen continuous culture in uitro (Whetstone et a1 1981). ciliate protozoa have a much higher dipeptidase activity The aim of the present experiments was to evaluate gel than bacteria, whereas bacteria are more active with filtration as a means of analysing how M , affects the

J Sci Food Agric 0022-5142/92/$05.00 0 1992 SCI. Printed in Great Britain 177

178 R J Wallace

breakdown of unlabelled peptides by rumen micro- organisms. Two specific objectives arising from previous experiments were, first, to determine the M , of the peptides which accumulate in vivo when ionophores are fed (Wallace et a1 1990c) and secondly, to assess how the M , of peptides affects the efficiency of their protection by ace tylation (Wallace 1992).

EXPERIMENTAL

Experiments with rumen fluid

Three separate experiments were done to investigate peptide metabolism by mixed rumen microorganisms. Two of the experiments have been reported elsewhere, and only the gel filtration analysis is described here. The first, in which the degradation of Trypticase peptides and of casein in rumen fluid in vi/ro was monitored for times up to 24 h, was described by Wallace (1992). The second experiment was the ionophore feeding trial reported by Wallace et a1 (1990~). Experiment 3 was designed to determine how treating peptides of different M, ranges with acetic anhydride affects their rate of degradation by rumen microorganisms.

previously (Wallace 1992). Tubes were chilled at 4 "C and centrifuged at 12600 x g for 5 min, and the super- natant fluid was analysed by gel filtration.

In experiment 3, acetylation of peptides was carried out by a method based on that described by Means and Feeney (1964). Trypticase and fractions derived from Trypticase by gel filtration were dissolved in saturated sodium acetate solution at a concentration of 40 g litre-'. The solutions were chilled on ice, and five lots of 8 ml of acetic anhydride were added litre-' at 10 min intervals. The solutions were incubated at 4 "C for 24 h. Control incubations were done in which sodium acetate was present but water rather than acetic anhydride was added. Aliquots (52 pl, containing 2 mg of peptides) were added to microcentrifuge tubes and the tubes were dried by incubating at 60 "C for 18 h. Duplicate 1.0 ml samples of strained rumen fluid from four sheep were added to the tubes and they were incubated at 39 "C. Similar incubations were done in which no peptides were added: saturated sodium acetate was dried in the tubes and strained rumen fluid was added. After 6 h in- cubation, 0.25 ml of 25 YO TCA was added to each tube. Ammonia analysis was done on the supernatant fluid after centrifuging at 12600 x g for 5 min.

Gel filtration Animals, diets and sampling procedures

In experiments 1 and 3, where casein, Trypticase or acetylated Trypticase peptides were added to rumen microorganisms in vitro, the rumen fluid was obtained from four mature sheep receiving a maintenance diet of hay, barley, molasses, fish meal and a vitamins/minerals mix (500, 299.5, 100, 91 and 9.5 g kg-' DM respectively). Samples of rumen fluid were removed via the rumen cannulae between 1.5 and 2.5 h after feeding. These samples were strained through four layers of muslin and used immediately.

In experiment 2 with ionophores, three mature sheep were fed the same diet containing 33 mg kg-' monensin or 10 mg kg-' tetronasin in a 3 x 3 Latin square design (Wallace et al 1990~). Samples of rumen fluid were removed via the rumen cannulae 2 h after feeding, and extracellular fluid was obtained by centrifuging at 48000 x g for 15 min at 4 "C. The cell-free fluid was then stored at - 70 "C. Immediately before analysis, the samples were thawed and trichloroacetic acid (TCA) was added to a final concentration of 5 % . Protein was then precipitated by centrifuging at 12600 x g for 5 min. Analyses were done on the supernatant fluid.

Incubations with peptides in vitro

In experiment 1, Trypticase peptides or casein were added to strained rumen fluid to a final concentration of 2 g litre-', the mixture was incubated under CO, at 39 "C, and samples were removed into TCA as described

A column 82.0cm by 1.0cm ID of Sephadex G-25 Superfine was eluted with distilled water at a flow rate of approximately 0.9 ml min-' and fractions were collected for 3 min. Samples were applied to the column by pumping for 1 min. The precise flow rate was determined by weighing five random fractions from each run, and sample volume taken on to the column was calculated accordingly. Samples from experiment 1 were diluted fourfold in distilled water before being applied to the column.

The same column was used to separate Trypticase into fractions containing different M , ranges. One millilitre of a 500 g litre-' solution was applied to the column and fractions were collected as before. Fractions 9-13, 14 and 15, 16 and 17, and 18-36 were pooled and dried at 60 "C, yielding 69, 96, I 1 1 and 84 mg dry weight respectively.

Analysis

Calibration Absorption spectra were done in a Cecil CE595 UV- visible double-beam spectrophotometer. Fractions from gel filtration were analysed for total peptides by determining their A,,, in the same instrument, using an absorption coefficient of 20.2 for a 1 g litre-' solution of Trypticase. Aliquots (0.2 ml) of each fraction were analysed for free peptide amino groups by fluorescamine, using the procedure described previously (Broderick and Wallace 1988) except that the fluorescamine was dis- solved in methanol rather than acetone.

Peptide metabolism by rumen microorganisms 179

Experiment 1 : Metabolism of Trypticase and casein in vitro The AZo6 of the different fractions was used to calculate the concentration of peptides present using Trypticase as standard.

Experiment 2 : effects of ionophores in vivo Samples were analysed by the modified fluorescamine procedure. Clarified rumen fluid gave high blanks with this method, so the measurements made were (1) sample + fluorescamine, (2) sample alone, (3) water fluorescamine, and (4) water. The relative fluorescence due to peptides in the sample was calculated as (1 - 2) - (3 - 4), and this value was converted to a molar concentration as described for the calibration of the column.

Experiment 3 : Breakdown of acetylated peptides Ammonia was determined in duplicate in each sample by an automated phenol-hypochlorite method (Whitehead et a1 1967).

Materials

Sephadex G-25 Superfine was from Pharmacia. Casein pancreatic hydrolysate was Trypticase from BBL Micro- biology Systems, Cockeysville, MD 21030; lactalbumin hydrolysate was also a pancreatic hydrolysate (Oxoid L48, Oxoid Ltd, Basingstoke, Hampshire), and Special Peptone (Oxoid L72) was a mixture of peptides derived from meat, plant and yeast digests. Casein acid hydro- lysate was Oxoid L41. LysGlyTrpLys, ValGlySerGlu, and GluAlaAla were from Bachem AG, Bubendorf, Switzerland. Tetronasin was a gift from Mr S James, Pitman-Moore Ltd, Harefield, Middlesex. Other peptides and fine chemicals were from Sigma.

RESULTS AND DISCUSSION

Detection of peptides

Ultraviolet absorption was used to measure total peptides present in fractions eluted from Sephadex G-25. Several components of the samples, including rumen fluid itself, free amino acids, volatile fatty acids (VFA) and TCA, as well as peptides, absorb in the UV region (Fig. 1). A wavelength of just below 200 nm would maximise the absorbance of peptides relative to rumen fluid and amino acids, but 206 nm was chosen as the lowest available value at which the spectrophotometer functioned without problems associated with low energy.

This procedure was selected as a means of maximising sensitivity when analysing small volumes of sample, and it is a convenient, simple estimate to make. It suffers from two main limitations, however. First, the extinction coefficient is different for different peptides (Table 1).

0.4 0'5 1

a 2 0.3 rn

0 f 2 0.2 a

0.1

- TCA --- Clarified rumen fluid --.__ Trypticase . . . . . . , , . Casein acid hydrolysate - - VFA

0 180 190 200 210 220 230 240 250 260 270 280

Wavelength (nm) Fig 1. Absorption spectra of TCA (16.7 mg litre-l), clarified rumen fluid (1/600 dilution), Trypticase (3.33 mg litre-l), casein acid hydrolysate (3.33 mg litre-I), and VFA (]/I800 dilution of Caldwell and Bryant (1966) concentrated VFA solution, containing 5.2 mM acetic, 1.4 mM propionic, 0.8 mM butyric, 0.2 mM isobutyric, 0.2 mM n-valeric, 0.2 mM isovaleric and

0.2 mM methylbutyric acids).

The average extinction coefficient of 12 peptides was 28.2 f 12.9 compared with 20.2 for Trypticase. Secondly, the A,,, of a sample derived from rumen fluid does not determine the absolute quantity of peptides present. Nevertheless, the difference in A,,, between samples to which peptides have been added and control incubations with rumen fluid, when combined with gel filtration, might be expected to give a reasonable estimate of how much peptide of average composition remains.

Gel filtration of peptides and other materials

The void volume of the Sephadex G-25 column used in these experiments was 21 ml, and most of the Trypticase applied to the column eluted between 25 and 65 ml (Fig 2). Lactalbumin hydrolysate and especially Special Peptone contained a higher proportion of peptides which eluted earlier than Trypticase (Fig 2). This was also reflected in the relative fluorescence of the unfractionated mixtures. Lactalbumin hydrolysate and Special Peptone had a specific fluorescence relative to Trypticase (1.0) of 0.67 and 067 respectively. Casein acid hydrolysate, which contains only free amino acids, and likely fermentation products did not elute until about 45 ml. Thus the metabolism of the majority of the peptides contained in Trypticase can be analysed by gel filtration without interference from the products of peptide hydrolysis.

Calibration of the column

Individual peptides applied to the column gave sym- metrical peaks, but the relation between their elution volume and M , was very poor (Fig 3). ValGlySerGlu, GluAla, and Ala, eluted at positions close to a curve

14 JFA 58

180

1.2

1.0

0.8

0.6

0.4

0.2

0

R J Wallace

- - TrypI~casa ---- Lactalbumin hydrolyiale S p m d peplone

-

-

-

-

-

I I I I I I I I

A206

Elution volume (ml) Fig 2. Elution profiles of peptides and other compounds. Peptide solutions (0.85 ml) were applied to a column of Sephadex G-25 at a concentration of 0.5 g litre-' and fractions were diluted threefold for determination of A,,,. The values have been multiplied by 3 to account for this dilution. Casein acid hydrolysate (1.0 g litre-l) and a fermentation product mixture (10 mM formic acid, 30 mM acetic acid, 20 mM propionic acid, 20 mM butyric acid, 5 mM isobutyric acid, 5 mM isovaleric acid, 5 mM n-valeric acid, 10 mM lactic acid, 10 mM succinic acid) were applied as 09-ml aliqudts and the A,,, of

fractions was measured without dilution.

0 Trypticase

0 Special Peptone Lactalbumin

100 t 25 30 35 40 45 50 55 60

Eluant volume (ml)

Fig 3. Calibration of Sephadex G-25 column. Individual peptides were added as 0.25 g litre-' solutions. Peaks were determined by triangulation. 1, Insulin; 2, bradykinin; 3, angiotensin I11 ; 4, leucine enkephalinamide ; 5, ValGlySerGlu ; 6, Ala,; 7, GluAla,. The Trypticase fractions from Fig 2 were analysed by fluorescamine and the average M , was calculated as described in Experimental. Similar filtrations, analyses and calculations were done for lactalbumin hydrolysate and Special

Peptone.

generated using mixed peptides (see below), but insulin, bradykinin, angiotensin I11 and Des-Tyrl-leucine en- kephalinamide eluted much later. The latter group contain aromatic amino acid residues whereas the smaller peptides did not, and this may be the reason for their anomalous behaviour. Peptides containing aromatic

TABLE 1 Absorption coefficients and fluorescamine reactivities of pep-

tides

Peptide A,,, ( I g litre-' soh tion)

Relative molar Jluorescence

(Ala, = I)

Ala, Ala, Ala, Ala, GluAla, PheGly, Gly HisLys GlyPheLeu ValGlySerGlu L ysGlyTrpLys ArgLy s AspValTyr Des-Tyrl-Leu enkephalinamide

Trypticase

15.6 17.4 17.3 16.7 17.2 45.1 37.1 39.9 17.9 32.6 29.3 51.7

20.2

0.58 1 .oo 0.99 0.9 1 1.24 2.13 1.16 1.39 1.33 1.44 292 1.98

2.72"

a Fluorescence of I g litre-' Trypticase relative to 1 mM Ala,

residues, especially tryptophan, tend to be retained on the column matrix (Porath 1960), and ionic binding also occurs (Porath 1960; Glazer and Wellner 1962; Miranda et a1 1962).

Fractions generated by the gel filtration of Trypticase, lactalbumin hydrolysate and Special Peptone were analysed by Azos for total peptides and by fluorescamine for peptide amino-N. The ratio of the two enabled calculation of the average M , of peptides present in each fraction. Fluorescamine was chosen to detect peptide N- terminal amino groups partly because of its sensitivity and also because of its discrimination between peptides and free amino acids (Broderick and Wallace 1988). Different peptides react with fluorescamine to different extents (Table 1). For the purposes of subsequent calculations, it was assumed that the fluorescence of the amino groups of mixed peptides was the same as the average of the pure peptides described in Table 1, namely 1.42 times that of Ala,. Thus, for example, the average M , of Trypticase can be calculated:

Fluorescence of 1 mM average peptide= 1.42

Fluorescence of 1 g Trypticase litre-' = 2.72

Therefore concentration of peptides

in Trypticase = 2.72/1.42

= 1.92 mmol g-'

and average M, = 1000/1.92

= 521 Da

If the average M , of an amino acid in Trypticase is 137

Peptide metabolism by rumen microorganisms 181

(Chen et a1 1987), the average peptide length would be 3.80, close to the values obtained for Trypticase using ninhydrin (Chen et a1 1987) and Casitone (another pancreatic hydrolysate of casein) with fluorescamine (Broderick and Wallace 1988).

Using the M , values calculated by this method, a calibration curve of elution volume vs M , was drawn up (Fig 3) for the three hydrolysates. Trypticase, lac- talbumin hydrolysate and Special Peptone gave cali- bration curves that were almost identical and fairly close to a negative linear relation between log M , and volume, at least for peptides of 300-2000 Da.

The fact that peptides of different origins eluted from Sephadex G-25 with a similar M , profile, without the need for chelating reagents (Fazakerley and Best 1965) or organic solvents (Carnegie 1965) and using water alone as eluant, is remarkable. It may simply be a consequence of the similar content of hydrophobic amino acid residues in the hydrolysates. The aromatic amino acid contents of lactalbumin hydrolysate, Special Peptone and an enzymic casein hydrolysate are 5.1, 4.9 and 6.0 % respectively (Anon 1982). Alternatively, it may be that the influences which result in anomalous behaviour with pure peptides are of much less importance when mixed peptides are analysed. This is illustrated by comparing the A,,, (total peptides) with A,,, (peptides containing aromatic residues) of Trypticase (Fig 4). The elution of aromatic residues was delayed, but the effect was relatively minor. Whereas 43 YO of the A,,, eluted in the first 45 ml, 58 Yo of the A,,, eluted in the same volume.

Analysis of peptide metabolism by rumen microorganisms

Rumen fluid caused a high background absorbance when samples were analysed by gel filtration (Fig 5). Nevertheless, it was possible to discriminate the peptide elution pattern of Trypticase when it was added to rumen fluid (Fig 5). The high background A,,, of rumen fluid was due mainly to materials other than peptides. When the 24 h control samples from experiment 1 were analysed by A,,, and by fluorescamine, approximately one-third of the background absorbance was attributable to peptides and this was predominantly peptides of high M , eluting close to the void volume (Table 2).

In experiment 1, where Trypticase was added to rumen fluid from sheep not receiving ionophores, most of the smaller peptides were degraded rapidly (Fig 6). A small amount of residual material remained, however, even after 24 h incubation, and this material covered a range of M , including molecules of M , > 1000 (Fig 6). It can be estimated from the area below the elution profiles that 0.21 and 030 g litre-' peptides remained at 12 and 24 h respectively. It seems unlikely that these compounds were not peptides, since they must have been derived from Trypticase and did not elute with amino acids and

2.0 r r 0.20

1.6

1.2 0 0

0.8 2

0.4

0.0

0.16

0.12 P N 0 0

0.08

0.04

0.00 0 30 60 90 120 150

Elution volume (rnl) Fig 4. Analysis of G-25 gel filtration of Trypticase ( 2 g litre-I) by AZoe and AZg0. Conditions were as described in Fig 2. Samples were diluted 10-fold in water before determining A,,,.

0.6 - Rumen fluid - Rumen fluid + T r ~ p I ~ c a s e Difference

---_ / . . . . . . . . , .

0.4 -

A206 /

01"""' 20 25 30 35 40 45 50 55

Elution volume (ml]

Fig 5. Gel filtration of rumen fluid and rumen fluid to which Trypticase (2 g litre-l) was added. A sample (1.0 ml) was immediately extracted with 0.25 ml of 25 'YO TCA, centrifuged, and diluted fourfold in water before loading on to the column.

other fermentation products (Fig 1). Incomplete hy- drolysis of Trypticase by rumen microorganisms in vitro was implied by unidentified peaks persisting in HPLC elution profiles (Chen et a1 1987) and in the study from which the present samples were derived (Wallace 1992). No attempt was made to determine the composition of the residual material. Some peptides, such as those with Pro or GlyGly at their N-terminus, are more slowly degraded than others (Wallace and McKain 1989a; Wallace et a1 1990a); they would not be expected to escape degradation for 24 h, however.

When casein replaced Trypticase in the incubation mixture, a lower residual concentration of peptides was seen after 24 h, equivalent to 0.05 g litre-' (Fig 6). Chen and Russell (1991), using a higher initial concentration of casein (670 mg N litre-' or - 4.8 g litre-') and a ninhydrin assay, also found that a small proportion of the N (60-100 mg N litre-') appeared to survive as non-

14-2

182

TABLE 2 Contribution of peptides to background UV absorbance of TCA-extracted rumen fluid

R J Wallace

Fractiona Peptide free M, (au) Peptide concn Estimated A,,, Total A,,, no amino-N (mg litre-') due to peptides'

9 10 11 12 13 14 15 16 17

(pmol litre-l)b

Meand SD

17.7 3.6 17.7 3.9 16.2 3.9 16.2 3.4 15.1 6.9 21.8 3.7 24.0 3.8 28.1 4.0 30.0 4.0

2902 1715 1214 794 596 492 392 306 288

Meand SD Meand SD Meand SD

51.4 10.4 1.04 021 1.00 0.19 30.4 6.7 0.6 1 014 1.09 0.20 19.7 4.7 0.40 0.09 0.82 0.14 19.7 2.7 0.40 0.05 0.77 0.13 9.0 4.1 0.18 0.08 0.79 0.13

10.7 1.8 0.22 0.04 0.86 0.14 9.4 1.5 0.19 0.03 0.98 0.17 8.6 1.2 0.17 002 1.16 0.20 8.6 1.2 0.17 002 1.56 0.32

aAverage fraction volume = 2.85 ml. Estimated using fluorescamine, and expressed per litre of rumen fluid.

<Assuming the extinction coefficient is the same as for Trypticase, 20.2. Samples of rumen fluid from four sheep were incubated in vitro for 24 h (experiment I), extracted with 25 YO TCA and applied to

the column as a fourfold dilution.

0 h Trypticase 3 h Trypticase 6 h Trypticase

12 h Trypticase

40 c c /

\

L -

20 25 30 35 40 45 50

Elution volume (ml) Fig 6. Degradation of Trypticase and casein (2 g litre-l) incubated in vitro with rumen fluid from four sheep not receiving dietary ionophores (experiment 1). The results were calculated from the difference between the AZo6 of rumen fluid and the A,,, of rumen fluid to which Trypticase was added and converted to units of mg peptide litre-' as described in the text.

ammonia, non-protein N at 100 h. Neither of these studies distinguished peptides derived from dietary protein and peptides formed as a result of microbial breakdown, which could be extensive in long incubations in uitro. When [14C]-lucerne fraction I protein was incubated with rumen microorganisms and the products were run through Sephadex columns, no labelled peptides were observed to accumulate (Nugent and Mangan 198 1). Little fluorescamine-reactive peptide

survives except transiently after feeding in the rumen of sheep fed similar diets (Broderick and Wallace 1988 ; Wallace and McKain 1990).

Influence of dietary ionophores on peptide accumulation in vivo

Ionophores such as monensin and tetronasin improve the feed efficiency of ruminants, partly by improving their nitrogen retention (Newbold et a1 1990). To some extent this effect may be mediated by an inhibition of rumen peptide breakdown. Early in-vitro batch culture experiments (Van Nevel and Demeyer 1977) suggested that the breakdown of protein was inhibited by mon- ensin. It was subsequently shown (Newbold et a1 1990) that protein was hydrolysed normally but peptides accumulated in similar incubations to which tetronasin was added. Chen and Russell (1 99 1) also found that non- ammonia non-protein N accumulated when monensin was added to short-term batch incubations in v i m .

Evidence that the inhibition of peptide breakdown persists beyond acute experiments was obtained by Whetstone et al (1981), who observed that peptides, apparently of large M,, accumulated when monensin was added to a semi-continuous culture of rumen micro- organisms. Wallace et a1 (1990~) found that monensin and tetronasin both caused rumen peptide concen- trations to rise in uiuo, and speculated on the basis of the Whetstone et a1 (1981) data and on changes in the permeability of the cell envelope of Bacteroides rum- inicola in the presence of ionophores (Newbold and

Peptide metabolism by rumen microorganisms 183

TABLE 3 Characterisation of the M , ranges of peptides present in rumen

fluid 2 h after feeding

M, range Peptide concentration CUM)

Control Tetronasin Monensin (10 mg kg-') (33 mg kg-')

Mean" SD Meana SD Mean" SD

1050-2700 39.8 7.5 34.5 12.0 45.0 20.3 570-1050 26.3 17.3 25.3 21.0 45.0 18.8 360-570 33.0 14.3 735 23.3 45.8 15.8 220-360 45.0 18.0 123.8 9.0 58.5 23.3

"Mean of three sheep, experiment 2

TABLE 4 Ammonia production by rumen microorganisms in uitro from different fractions of Trypticase before and after treatment with

acetic anhydride ~~~~~ ~ ~~

Fraction M M b Ammonia produced"

No treatment After ~ acetylation

Mean' SD Meanc SD

Trypticase 52 1 116 15 30 14 Fractions 77 1 116 15 35 9

Fractions 499 121 22 43 16

Fractions 373 129 10 54 27

Fractions 147 111 19 52 23

9-13

14 & 15

16 8~ 17

18-36 ~~~ ~ ~~ ~ ~ ~

a Ammonia produced from peptide (2 g litre-l) during 6 h incubation in vitro, mg N litre-' Calculated from the total peptide concentration (from A2,,J

and the peptide amino-N concentration (fluorescamine). Mean of duplicate determinations using rumen fluid from four

sheep.

Wallace 1992) that the accumulation would be of large peptides. The samples taken in the Wallace et a1 (1990~) study were analysed here for their M , distribution.

Both ionophores caused peptide concentrations to increase, but only tetronasin caused the M, distribution to change significantly (Table 3). The accumulation with tetronasin was of smaller molecules, however. An exclusion mechanism of the type proposed previously (Newbold and Wallace 1992) must therefore be of less

importance than other adaptive changes that occur in the rumen ecosystem in response to ionophores.

Acetylation as a means of protecting peptides from degradation in the rumen

Since most of the peptidase activity in the rumen has aminopeptidase specificity (Wallace and McKain 1989b), it has proved possible to protect peptides from ruminal degradation by treating them with acetic anhydride, which blocks the N-terminus with an acetyl group (Wallace 1992). Larger peptides were less effectively protected than smaller molecules in a small study with pure peptides (Wallace 1992). The protection of Try- pticase was incomplete for reasons which were not established. Gel filtration was used here to prepare fractions from Trypticase with different average M,, to determine if the degree of protection of Trypticase depended on the M , of its component peptides.

Ammonia production from all of the fractions was similar, and no different from that of the unfractionated Trypticase (Table 4). Acetylation decreased ammonia production from Trypticase, but this was again incomplete (Table 4). None of the fractions was protected as well as the unfractionated Trypticase, and, although the variation was high, peptides of higher M, did not appear to be less protected.

Conclusions

Gel filtration with Sephadex G-25 appears to be a valid method for determining the M , range of mixtures of peptides, despite its anomalous performance with many pure peptides. Although clearly a laborious procedure for determining the peptide content of rumen fluid, gel filtration can be used to resolve mixtures of peptides in rumen fluid in order to provide information, for example on M, distribution, that is not available by other methods or to confirm measurements made by other methods, such as the total concentration of peptides and acetylated peptides present in rumen fluid.

The present studies suggest that the breakdown of peptides to smaller molecules is almost complete in rumen fluid in uitro, and that the same is true in uiuo unless ionophores are present in the diet. Thus ion- ophores already achieve one of the main nutritional objectives of the work, namely to slow the conversion of dietary protein to ammonia by rumen microorganisms. Protection of peptides added to rumen fluid can be achieved by blocking their N-terminus. The modification is not fully effective for protecting mixtures of peptides, but M, does not seem to be as important as was indicated previously by the study of pure peptides. It will be important to determine the chemical features of peptides that enable some to be protected by N-terminal acety- lation and others to be degraded. It may then be Dossible

R J Wallace

t o design and synthesise peptides that will pass from the rumen intact. If the peptides can then be digested, either before or after absorption, their constituent amino acids will then become available as supplementary nutrients for the host animal.

ACKNOWLEDGEMENTS

I gratefully acknowledge the contribution of Dr C J Newbold in the ionophore experiments and I thank Dr Newbold and Dr C I Harris for their critical assessment of the manuscript.

REFERENCES

Anonymous 1982 The Oxoid Manual. Oxoid Ltd, Basingstoke,

Broderick G A, Wallace R J 1988 Effects of dietary nitrogen source on concentrations of ammonia, free amino acids and fluorescamine-reactive peptides in the sheep rumen. J Anim Sci 66 2233-2238.

Caldwell D R, Bryant M P 1966 Medium without rumen fluid for nonselective enumeration and isolation of rumen bac- teria. Appl Microbiol 14 794-801.

Carnegie P R 1965 Estimation of molecular size of peptides by gel filtration. Biochem J 65 9P.

Chen G, Russell J B 1991 Effect of monensin and a pro- tonophore on protein degradation, peptide accumulation, and deamination by mixed ruminal microorganisms in vitro. J Anim Sci 69 2196-2203.

Chen G, Russell J B, Sniffen C J 1987 A procedure for measuring peptides in rumen fluid and data suggesting that peptide uptake is a rate-limiting step in ruminal protein degradation. J Dairy Sci 70 121 1-1219.

Fazakerley S, Best D R 1965 Separation of amino acids, as copper chelates, from amino acid, protein, and peptide mixtures. Anal Biochem 12 29&295.

Glazer A N, Wellner D 1962 Adsorption of proteins on ‘Sephadex’. Nature 194 862-863.

Leng R A, Nolan J V 1984 Nitrogen metabolism in the rumen. J Anim Sci 67 1072-1089.

Means G E, Feeney R E 1964 Chemical Modification of Proteins. Holden-Day, San Francisco.

Miranda F, Rochat H, Lissitsky S 1962 Proprietes echangeuses d’ions du gel de dextrane (Sephadex). Application a la mise au point d’une technique de rttention rtversible des proteines basiques de faible poids moleculaire. J Chromatogr 7

Newbold C J, Wallace R J 1992 Properties of ionophore-

pp 242-243.

142- 1 54.

resistant Bacteroides ruminicola enriched by cultivation in the presence of tetronasin. J . Appl Bacteriol 12 65-70.

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.

Newbold C J, Wallace R J, McKain N 1990 Effect of the ionophore tetronasin on nitrogen metabolism of rumen microorganisms in vitro. J Anim Sci 68 1103-1 109.

Nugent J H A, Mangan J L 1981 Characteristics of the rumen proteolysis of fraction 1 (18s) leaf protein from lucerne (Medicago sativa L.). Br J Nutr 46 39-58.

Porath J 1960 Gel filtration of proteins, peptides and amino acids. Siochim Siophys Acta 39 193-207.

Van Nevel C J, Demeyer, D I 1977 Effect of monensin on rumen metabolism in vitro. Appl Environ Microbiol 34

Wallace R J 1988 Ecology of rumen microorganisms: protein use. In: Aspects of Digestive Physiology in Ruminants, eds Dobson A & Dobson M J. Cornell University Press, Ithaca,

Wallace R J 1992 Acetylation of peptides inhibits their degradation by rumen microorganisms. Br J Nutr 68 in press.

Wallace R J, McKain N 1989a Some observations on the susceptibility of peptides to degradation by rumen micro- organisms. Asian-Austral J h i m Sci 2 333-335.

Wallace R J, McKain N 1989b Analysis of peptide metabolism by ruminal microorganisms. Appl Environ Microbiol 55

Wallace R J, McKain N 1990 A comparison of methods for determining the concentration of peptides in rumen fluid of sheep. J Agric Sci Camb 114 131-148.

Wallace R J, Newbold C J, McKain N 1990a Patterns of peptide metabolism by rumen microorganisms. In : The Rumen Ecosystem. The Microbial Metabolism and Its Regu- lation, eds Hoshino S, Onodera R, Minato H & Itabashi H. Japan Scientific Societies Press, Tokyo, pp 4349.

Wallace R J, McKain N, Newbold C J 1990b Metabolism of small peptides in rumen fluid. Accumulation of intermediates during hydrolysis of alanine oligomers, and comparison of peptidolytic activities of bacteria and protozoa. J Sci Food Agric 50 191-199.

Wallace R J, Newbold C J, McKain N 1990c Influence of ionophores and energy inhibitors on peptide metabolism by rumen bacteria. J Agric Sci Camb 115, 285-290.

Whetstone H D, Davis C L, Bryant M P 1981 Effect of monensin on the breakdown of protein by ruminal micro- organisms in vitro. J Anim Sci 53 803-809.

Whitehead R, Cooke G H, Chapman B T 1967 Problems associated with the continuous monitoring of ammoniacal nitrogen in river water. Autom Anal Chem 2 377-380.

Wright D E 1967 Metabolism of peptides by rumen micro- organisms. Appl Microbiol 15 547-550.

251-257.

NY, pp 99-122.

2372-2376.