Dynamic study of the release and the utilisation of
15
N-labeled pea globulin peptides by mixed
ruminal bacteria in vitro
A. Lambert
*, F. Lucas, G. Blanchart
Laboratoire de Sciences Animales, E.N.S.A.I.A. 2, avenue de la foreÃt de Haye, 54500 Vandoeuvre-leÁs-Nancy, France
Received 19 January 1999; received in revised form 15 June 1999; accepted 20 July 1999
Abstract
There is no consensus about the effects of the size of peptides on their extracellular breakdown and their utilisation by rumen bacteria. This study was done to describe these effects for the peptides released during the first steps of the hydrolysis of a plant protein.
The fates of five peptide fractions, characterised by their molecular weights (a> 10 000 Da, 5000 <b< 10 000 Da, 2000 <g< 5000 Da, 1000 <d< 2000 and e< 1000 Da) were monitored. These fractions were obtained by hydrolysing 15N-labelled pea globulins with pronase E and separation by HPLC. The utilisation of each of them as a part of a complex mixture of unlabelled globulin peptides by a goat mixed rumen bacteria inoculum was individually followed for 5 h. The excess15N in each of the initially labelled fractions gradually decreased and labelled compounds were found in smaller peptides. Bacteria were labelled with15N only after at least 30 min. This delay increased with the length of the incubated labelled peptide. Large peptides (aandb) were hydrolysed most rapidly and extensively. About 80% (SE 1.9) of the excess 15N coming from
fractionawas found in smaller peptides in only 30 min. During the same time, only 45% (SE 3.3) of the excess 15N provided by fraction
g was recovered in smaller fractions. Dipeptidyl-aminopeptidase type 1 and Dipeptidyl-aminopeptidase activities combined with endopeptidase activities to produce nitrogenous compounds that could be absorbed by bacteria. The monitoring of15N enabled us to obtain information on the effect of globulins peptides size on their extracellular degradation.
#1999 Elsevier Science B.V. All rights reserved.
Keywords: Proteolysis; Rumen bacteria; Pea globulin; Peptide; Molecular weight 82 (1999) 75±89
*Corresponding author. Tel.:33-383-595-889; fax:33-383-595-804.
1. Introduction
The hydrolysis of proteins by rumen micro-organisms releases peptides which are, in turn, themselves degraded into oligopeptides and free amino acids. Many studies suggest that the utilisation of these peptides, especially by rumen bacteria, is a key step in the regulation of nitrogen flow in the gastrointestinal tract of ruminants (Chen et al., 1987; Wallace and Cotta, 1988). The utilisation of these peptides by bacteria can be divided into extracellular hydrolysis, product transport and intracellular metabolism. Permeases involved in the transport of peptides into the cells limit the size of the peptides that can cross the bacterial envelopes to less than 1000 Da (Alves et al., 1985; Westlake and Mackie, 1990).
The effects of peptide size on their extracellular degradation are also somewhat controversial (Wright, 1967; Chen et al., 1987; Wallace, 1992; Armstead and Ling, 1993; Depardon et al., 1995, 1996).
This work, therefore, monitors the fate of peptides of various molecular weights in the presence of an inoculum of mixed rumen bacteria. The protocol used in this study was adapted from that used with casein peptides by Lambert et al. (1998). Pea globulins were chosen because these plant proteins are likely to be part of ruminant diets.
2. Material and methods
The 15N-labelled pea globulins peptide were incubated in vitro with mixed rumen bacteria. A labelled peptide fraction of known molecular weight was added to a whole unlabelled hydrolysate of pea globulins. We thus followed the fate of this fraction within a complex mixture of peptides. This protocol was repeated successively for each different labelled fraction.
2.1. Preparation of labelled and unlabelled globulins peptides
Pea proteins were extracted by the method developed by Gueguen and Barbot (1988) with 3 extraction steps in phosphate buffer 0.1 M pH 7, K2SO4(5% w/v).
The separation of globulins and albumins were based on the difference between their solubilities in solvents of different ionic strengths (Gueguen and Barbot, 1988). The resulting supernatants were dialysed against water to reduce their salt concentration and, hence, enable the precipitation of globulins, poorly soluble in low salt solutions. Dialysis was carried out in membranes with a molecular weight cut-off of 6000±8000 Da (Spectra/ Por) for 96 h at 48C.
freeze-dried. 15N-labelled peptides were separated in accordance with their molecular weights by semi-preparative gel filtration HPLC (BIOSEP-S-2000, Phenomenex, Torrance, USA) and freeze-dried. Four peptide fractions were isolated: >10 000 Da (fraction a), 5000±10 000 Da (fraction b), 2000±5000 Da (fraction g), 1000±2000 Da (fraction d). A fifth fraction containing peptides weighing <1000 Da (fraction e) was assayed when following the distribution of labelling after incubation.
2.2. Animals, diet and sample preparation
Rumen contents were obtained from three goats with rumen fistulae. They were fed 250 g of dried alfalfa and grass hay twice daily ad libitum. Rumen fluid was taken 1 h after feeding. Large particles and protozoa were removed by slow speed centrifugation (150g, 20 min, 158C); The supernatant was centrifuged at a higher speed to harvest bacteria (6 000g, 20 min, 158C) (Depardon et al., 1995). The bacterial pellet corresponding to 500 ml rumen fluid was resuspended in 50 ml anaerobic culture medium without peptide.
2.3. Incubations
The anaerobic culture techniques were similar to those described by Hungate (1969). The medium was that of Russell et al. (1983), containing glucose (2.5 g/l), cellobiose (2.5 g/l) and soluble starch (0.5 g/l).
Each tube of Hungate contained:
8 ml medium, previously reduced with Na2S, 9H2O (0.5 g/l);
0.5 ml unlabelled peptide solution (5 mg peptides /ml, final concentration); 0.5 ml solution of one of the labelled peptide fractions (0.4 mg peptides/ml); 1 ml rumen inoculum.
This technique enabled us to follow the fate of each fraction in turn when they were incubated with the whole hydrolysate.
For each labelled fraction, six incubation times were chosen: 0, 0.5, 1, 1.5, 2 and 5 h. Bacterial growth was measured for each incubation time and, afterwards, the bacteria were collected by centrifugation (9000g, 20 min, 48C). The supernatant was stored at ÿ208C before analysis. The corresponding bacterial pellet was washed twice in 10 ml 0.9% NaCl (w/v) and stored atÿ208C. Each incubation was repeated three times.
2.4. Analysis
Bacterial growth was measured by absorbency at 660 nm (Depardon et al., 1995). For each culture, a standard curve for Dry Matter was established with a sample of the mixed rumen bacteria used for the incubations. Viable bacteria were counted on MGA medium (Hobson, 1969) for incubation times of 0, 0.5, 1, 2 and 5 h.
Total nitrogen concentration in the supernatants was assayed using an automatic Kjeldahl apparatus (Gehrart, Les Essarts Le Roi, France). Ammonia concentration was determined with an ammonia gas sensing electrode (model 95±12, Orion, Boston, MA).
The dipeptidylaminopeptidase activity type 1 (DAP-1) was determined using Gly-Arg-4-methoxy-2-naphtylamid (MNA, Sigma, L'isle d'Abeau Chenes, France) as fluorogen-ous substrate (Wallace and McKain, 1989) with incubation for 6 h under anaerobic conditions at 398C. MNA was determined by its fluorescence (excitation350 nm, emission420 nm). The activity is given in enzymatic units (EU,mmol MNA/6 h).
The leucine- and alanine exoaminopeptidase activities were measured on Ala-pNa and Leu-pNA (Sigma, L'isle d'Abeau Chenes, France) as chromogenous substrates (Wallace and McKain, 1989) with incubation for 4 h at 398C. The pNA released was assayed by diazotation (Appel, 1974). Activities are given in enzymatic units (EU,mmol pNA/4 h). The 15N-labellings of bacteria, ammonia and the various peptide fractions, isolated with HPLC, were determined by mass spectrometry on aliquots of lyophilised samples. Total nitrogen concentration of these samples were estimated in the same way.
3. Results
3.1. Bacterial growth and fermentation conditions
Data shown for bacterial growth, total nitrogen and ammonia are average across treatments and repetitions.
OD660, which is proportional to the total number of bacterial cells, increased during the first 2 h of incubation to reach a plateau (Fig. 1(A)). After 5 h, the medium contained 8.5 mg microbial dry matter per ml, which represented a gain of 1.4 mg/ml. The number of viable bacteria increased steadily from 108cells/ml (SE 2108) to 5108cells/ml (SE 6105) at 5 h (Fig. 1(A)). This number increased much more rapidly than the total bacterial dry matter. The gap between total bacterial matter estimated by OD660 and viable bacteria counts was probably due to the culture conditions which caused selection of the microflora, that was initially highly heterogeneous.
The total nitrogen concentration of the cell-free culture medium decreased throughout the incubation from 359 to 302 mg nitrogen per ml (Fig. 1(B)). Assuming that all the nitrogen that disappeared (57 mg/ml) was used for bacterial synthesis, the nitrogen content of the new bacterial dry matter was estimated at about 4%. This percentage was rather low; It was however confirmed by direct measurements on bacterial pellets (3± 6%). This was probably due to a low ammonia availability associated with a high carbohydrate storage in the bacteria. During the same time, the ammonia concentrations increased steadily from 0.76 to 2.48 mM (Fig. 1(B)). Ammonia nitrogen made up only 0.29% of the initial total nitrogen. This percentage increased to 1.15% after 5 h of incubation, which represented 34.7 mg/ml.
3.2. Enzymatic activities
The leucine- and alanine-aminopeptidase activities are shown in Fig. 2(A). They both increased steadily throughout the incubation period. The leucine-aminopeptidase activity at the beginning of incubation was four times greater than alanine-aminopeptidase
Fig. 1. (A) Evolution of the bacterial growth during the incubation of labelled globulin peptides with mixed rumen bacteria in vitro. OD, Optical density; Viable bacteria, Viable bacteria counts per ml. (B) Evolution of fermentation parameters during the incubation of labelled globulin peptides with mixed rumen bacteria in vitro. N, Total nitrogen in supernatants expressed in mg/ml (mean of three incubations); NH3, Ammoniac in supernatants expressed in mM (mean of three incubations).
activity. However, it only increased by 60% in 5 h whereas alanine-aminopeptidase activity increased 2.8-fold.
3.3. Changes in the peptide profile
We calculated the amounts of total nitrogen in each compartment by multiplying the quantity of dry matter recovered for each peptide fraction or the bacteria by the concentration of total nitrogen in dry matter estimated by mass spectrometry in the corresponding compartment. The described concentrations are averages across treat-ments. The amounts of nitrogen in the five peptide fractions changed differently (Table 1). The nitrogen in fractionaremained roughly constant and even seemed to be increasing after 2 h. This fraction did not appear to be broken down by the bacteria. The nitrogen in fractionbdecreased by 111 mg/ml in 5 h (42.3% of its initial amount, SE 8.9%) and three quarters of this loss occurred in the first 30 min. Fewer peptides were supplied from fractiona to fractionb than peptides lost from fractionb.
Fractionsganddshowed the same type of evolution with 45% (SE 10.2%) of fractiong and 66% (SE 9.1%) of fraction dbeing lost in 5 h. Here again, about 75% of this loss occurred during the first 30 min. Fractionsb,g andd were more rapidly broken down than they were produced, and, the rate of disappearance decreased (b<g<d) as the size of the peptides increased (b>g>d). However, the drop in fraction e, which occurred mainly during the first 2 h, was less marked. Only 34% (SE 10.1%) of the initial nitrogen had disappeared from this fraction after 5 h of incubation.
3.4. Distribution of the15N enrichment in the various compartments
The total amount of 15N recovered at each incubation time should equal the initial amount of 15N. This was partly made up of the excess 15N provided by the labelled fraction initially added and partly of the15N naturally present in each compartment. We considered that the initial labelled fraction was the only fraction to present an enrichment
Table 1
Evolution of nitrogen amounts in the different peptide fractions during the incubation of globulin peptides with mixed rumen bacteria in vitro
g 510.8 8E-06 323 24.7 390.6 42.5 408.2 67.2 308 31.8
d 353.5 0 138.1 36.2 165.7 23.8 97.59 12.5 107.7 10.5
e 300.1 0 212.8 42.1 198.2 38.1 183.5 25.4 205.5 17.9
aa, peptides weighing >10 000 Da;b, peptides weighing between 5000 and 10 000 Da;g, peptides weighing
between 2000 and 5000 Da;d, peptides weighing between 1000 and 2000 Da;e, peptides weighing <1000 Da.
bMean: results are expressed in mg nitrogen per ml (mean of three incubations). cStandard error.
in15N at the beginning of the incubation. Therefore, the initial labelling was measured only for this fraction and zero values were arbitrarily attributed to the enrichments of the other fractions and of the bacteria. The natural proportion of 15N present in each compartment was assumed to be 0.3663%.
The labelling of the released ammonia increased differently depending on the initially labelled fraction (Fig. 3). It seemed that less15N accumulated in ammonia at the end of the incubation as the size of the initial labelled peptides increased. When the excess15N was added as fraction a, no trace of its accumulation in ammonia was observed. Incubation with labelled fractionbgave an enrichment of ammonia of 0.04%. Incubation with labelled fraction ggave 0.11% and incubation with labelled fraction d Ð 0.16%. Although the enrichment in15N in ammonia increased, the proportion of total excess15N associated with this fraction remained very low, because of the small amounts of ammonia released.
Bacteria were increasingly labelled throughout the incubation (Fig. 4), but this increase did not seem to be correlated with the size of labelled peptides added. Bacterial enrichment, like those of the different peptide fractions and of ammonia, depended greatly on the level of the initial labelling, and thus on the total quantity of excess15N in the medium. That is why we considered this quantity in order to study the change in the distribution of the total recovered excess15N among the different compartments.
The results for fractions g and d were similar (Figs. 5 and 6). The labelling of the fraction containing peptides of molecular weight lower than those of the initially labelled fraction increased because of the gradual replacement of hydrolysed unlabelled
Fig. 3. Evolution of the 15N-labelling of ammonia in relation to the initially labelled fraction during the
Fig. 4. Evolution of the15N-labelling of bacteria in relation with the initially labelled fraction during the
incubation of globulin peptides with mixed rumen bacteria in vitro (mean of three incubations).a, Peptides weighing >10 000 Da;b, peptides weighing between 5000 and 10 000 Da;g, peptides weighing between 2000 and 5000 Da;d, peptides weighing between 1000 and 2000 Da.
Fig. 5. Evolution of the15N-labelling of the different compartments during the incubation of labelled fractiond
with mixed rumen bacteria in vitro. (mean on three incubations).a, Peptides weighing between 1000 and 2000 Da;e: peptides weighing <1000 Da.
peptides with labelled peptides from fractions g or d. The enrichment remained unchanged after 30 min.
If labelled peptides were provided by fractiond, fractioneaccumulated half the excess 15
N in 30 min (Table 2). Its distribution between these two fractions remained rather constant thereafter.
About 35% (SE 14.5%) of the excess15N was found in fractioneafter only half an hour when fractiongwas the labelled fraction added (Table 2). A small part of this 15N was recovered in fractiond(about 15%, SE 8.18%). The shift of excess15N from fraction g to fraction e occurred at least partially via fraction d. The proportion of excess 15N associated with this latter fraction remained low but constant throughout the incubation.
Enrichment of labelled fraction b remained roughly stable (about 0.05%) (Fig. 7). Fractions g andd showed increasing labelling in the first hour of incubation (0.046% for fraction g and 0.045 for fraction d). Then, their enrichment decreased a little to 0.024% (fractiong) and 0.025% (fraction d) after 5 h. The inputs of excess15N and the outputs by hydrolysis of these fractions in smaller peptides became balanced. The labelling of fractioneincreased less, and much more slowly (0.02% after 30 min, 0.037% after 5 h).
The change in the distribution of the excess15N during the incubation of fraction b enabled us to more accurately define the flows of peptides from one fraction to another or to the bacteria. The proportions of excess15N associated with the different compartments were maximum at different incubation times (Table 2). Fractiongconcentrated the largest part of this nitrogen in half an hour, while the maximum accumulation in fraction d
Fig. 6. Evolution of the15N-labelling of the different compartments during the incubation of labelled fractiong
Evolution of15N in excess amounts recovered in the different compartments during the incubations of globulin peptides with mixed rumen bacteria in vitro (mean of
a a 100 18.56a,A 1.88 13.37a,B 1.80 23.83a,C 4.35 31.84a,D 5.77
b 0 19.01b,A 4.65 20.17b,A 4.25 19.94b,A 2.71 15.05b,A 2.14
g 0 25.01c,A 2.75 27.84c,A 0.52 27.34c,A 7.59 26.79c,A 4.89
d 0 13.81d,A 4.22 16.56d,A 2.94 10.66d,A 5.04 12.01d,A 1.58
e 0 23.05e,A 3.76 20.91e,A 5.54 17.11e,A 2.67 19.08e,A 2.34 bacteria 0 0.57f,A 0.12 1.15f,B 0.39 1.11f,C 0.47 1.59f,D 0.40
b b 100 27.45a,A 13.48 27.83a,A 3.16 37.14a,A 2.89 35.99a,A 14.27
g 0 45.76b,A 5.79 36.65b,A 10.16 34.19b,A 8.79 25.40b,A 7.11
d 0 13.97c,A 4.17 31.31c,A 6.11 8.85c,A 2.17 11.64c,A 3.25
e 0 12.21d,A 3.43 18.01d,A 3.32 17.28d,A 8.59 20.85d,A 4.98 bacteria 0 0.61e,A 0.08 1.49e,B 0.73 2.54e,C 0.25 6.13e,D 4.00
g g 100 52.20a,A 3.32 53.62a,A 2.04 68.10a,A 4.33 57.67a,A 4.36
d 0 12.17b,A 8.18 29.49b,A 8.35 13.50b,A 6.35 15.18b,A 3.08
e 0 35.23c,A 14.56 33.83c,A 1.60 17.21c,A 2.20 23.27c,A 7.96 Bbcteria 0 0.39d,A 0.05 0.93d,B 1.09 1.18d,C 0.48 3.88d,D 2.28
d d 100 36.99a,A 9.78 48.97a,A 1.23 67.64a,A 11.08 65.40a,A 14.29
e 0 62.19b,A 9.77 65.69b,A 7.12 45.53b,A 5.78 65.91b,A 0.00 bacteria 0 0.83c,A 0.18 1.66c,B 1.16 2.01c,C 1.53 3.28c,D 0.00
aData with different upper-case letters on the same row are different (5%); data with different lower-case letters in the same column are different (1%).
occurred at 1 h. The proportion of excess 15N associated with fraction e was still increasing after 5 h of incubation, and the bacteria showed a substantial accumulation of 15
N only after 1 h of incubation.
All the other fractions, containing smaller peptides, were in turn enriched with 15N after incubation with fractionawhile the labelling of this fraction decreased from 0.05% at the beginning to 0.02% after 5 h (Fig. 8). The excess15N, initially brought by fraction a, was rather equally distributed among all the peptide fractions at 30 min, and this distribution remained stable until the end of the incubation (Table 2).
4. Discussion
The results for the total nitrogen in fractionaseem to indicate that few, if any, peptides in this fraction were broken down (Table 1). However, there was a small15N-enrichment in smaller fractions as early as 30 min after the beginning of the incubation with labelled fractiona, certainly consecutively to a hydrolysis of this fraction.
As, originally, no protein or peptide longer than those contained in fractiona were placed in the medium, no other added peptides fraction could regenerate it. Nevertheless, the enrichment of residual fraction a decreased throughout incubation. This evolution could only be the consequence of the hydrolysis of unlabelled bacterial cells and the release of unlabelled proteins into the culture medium. These could then have diluted the excess 15N in the different peptide fractions. This is supported by the probable
Fig. 7. Evolution of the15N-labelling of the different compartments during the incubation of labelled fractionb
with mixed rumen bacteria in vitro.(mean on three incubations).b: Peptides weighing between 5000 and 10 000 Da;g, peptides weighing between 2000 and 5000 Da;d, peptides weighing between 1000 and 2000 Da;
disappearance of some bacterial cells, that could be responsible for the decrease in optical density.
The longer the peptides in the initial labelled fraction, the smaller the part of the total excess15N recovered in this fraction at the end of the incubation. Hence, long peptides are more rapidly hydrolysed than shorter ones (Figs. 5±8; Table 2). This was not so for the evolution of total nitrogen in fractions a and b, respectively, corresponding to peptides of molecular weights > 10 000 Da and 5000±10 000 Da (Table 1). Wallace et al. (1990) used synthetic peptides of low molecular weight to show that the rates of hydrolysis of these peptides depended on their size. However, this was not borne out by more recent studies from Wallace (1992) and Armstead and Ling (1993) who worked with mixtures of peptides. Depardon et al. (1995, 1996) are the only ones who have shown that peptides of over 2000 Da disappear more rapidly than the others.
Whatever the15N-labelled fraction initially added, all the fractions containing smaller peptides were enriched rapidly. However, the rate of enrichment differed from fraction to fraction.
The delay before bacteria accumulate excess15N might represent the time needed for a sufficient amount of this nitrogen to supply the peptide fraction which can be taken up by bacteria. The upper size limiting the bacterial absorption of peptides is still not well defined. Peptides weighing <1000 Da seem to be directly assimilated by bacteria without any previous extracellular hydrolysis (Alves et al., 1985; Westlake and Mackie, 1990). The small peptides (fractione) were effectively released into the medium and rapidly used by bacteria.
Fig. 8. Evolution of the15N-labelling of the different compartments during the incubation of labelled fractiona
with mixed rumen bacteria in vitro (mean on three incubations).a, Peptides weighing >10 000 Da;b, peptides weighing between 5000 and 10 000 Da;g, peptides weighing between 2000 and 5000 Da;d, peptides weighing between 1000 and 2000 Da;e, peptides weighing <1000 Da.
Several studies have suggested that the presence of such an intermediate pool of peptides in the rumen causes delays in the utilisation of the nitrogen from proteins by bacteria. This apparent delay was estimated by Sauvant and van Milgen (1995) at about 8 h, including the lag time for peptide production during protein hydrolysis.
Many studies have examined the different enzymatic activities of the rumen content (Brock et al., 1982; Kopecny and Wallace, 1982; Wallace and Brammal, 1985). Thus, DAP-1 activity is the most important peptidase activity in the rumen, quantitatively speaking (Wallace et al., 1990). It is produced by only few bacterial species, such asP. ruminicola (McKain et al., 1992). Leucine aminopeptidase is not widely distributed among the bacteria in the rumen:S. bovisis one of the few bacterial strains to produce it (Wallace and McKain, 1991). On the contrary, alanine-aminopeptidase activity is produced by many strains.
These studies have scarcely examined the evolution of these activities throughout an incubation. We found that the DAP-1, Ala- and Leu-aminopeptidase activities were initially present in the culture medium, although weak, which may explain the rapid appearance of an enrichment in small peptides. Though these activities released low molecular weight peptides or free amino acids from long peptides, they did not result in a rapid absorption of these compounds. Thus, there is no early increase of15N enrichment in bacteria. The lag time in the accumulation of15N in the bacteria was all the more long as the excess15N was initially provided by larger peptides. The ratio of the total amount of excess 15N recovered in large peptides to the total quantity of excess 15N rapidly decreased during the same time. The release by endopeptidases of intermediate peptides was one of the major features of the early incubation. These medium-sized peptides themselves generated absorbable nitrogenous compounds and their availability increased during incubation. The exopeptidase activities gradually released sufficient amounts of labelled small peptides or free amino acids for the bacteria to accumulate the excess15N. Depardon et al. (1995, 1996) reported that aminopeptidase activities of a mixed bacterial inoculum appeared only after 1 h of incubation and were maximal after 3 h. We found that the proteolytic activities per viable cell decreased, but this was counter-balanced by an increase in the cell count. This difference was probably due to different evolution of the composition of the cultured microflora.
5. Conclusion
Two techniques often used separately when studying the effect of peptide size on their utilisation by bacteria were here associated: peptide labelling and HPLC gel filtration. This allowed us to obtain information on the release and the extracellular degradation of globulins peptides by bacteria that could not have been obtained otherwise by the sole measurements of total nitrogen.
Peptides seemed to be hydrolysed all the more rapidly as they were long. Even though all the different size of peptides seemed to be produced from these long ones, a large part of them have to undergo successive hydrolysis before they reach a proper size to be assimilated by bacteria.
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