Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol82.Issue1-2.Nov1999:
Animal Feed Science and Technology
82 (1999) 75±89
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
excess 15N in each of the initially labelled fractions gradually decreased and labelled compounds
were found in smaller peptides. Bacteria were labelled with 15N only after at least 30 min. This
delay increased with the length of the incubated labelled peptide. Large peptides (a and b) were
hydrolysed most rapidly and extensively. About 80% (SE 1.9) of the excess 15N coming from
fraction a was 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. Dipeptidylaminopeptidase type 1 and aminopeptidase activities combined with endopeptidase activities to
produce nitrogenous compounds that could be absorbed by bacteria. The monitoring of 15N 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
*
Corresponding author. Tel.: 33-383-595-889; fax: 33-383-595-804.
0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 0 9 7 - 8
76
A. Lambert et al. / Animal Feed Science and Technology 82 (1999) 75±89
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.
Peptides were obtained by hydrolysing the pea globulins for 10 h with pronase E
(protease XVI from Streptomyces griseus, Sigma, L'isle d'Abeau Chenes, France) at
398C in 0.1 M phosphate buffer, pH 6.8 (1 mg pronase per 100 mg globulins in 5 ml
buffer). Proteins were precipitated at the end of the hydrolysis by adding perchloric acid
(5% final concentration) and removed by centrifugation (28 000 g, 20 min, 48C).
Thereafter, 2 M K2CO3 was added to the supernatant according to the protocol of Chen
et al. (1987), as modified by Wallace et al. (1990). The KClO4 insoluble salts were
removed by a second centrifugation. Solutions of unlabelled peptides were immediately
A. Lambert et al. / Animal Feed Science and Technology 82 (1999) 75±89
77
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 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 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 the
15
N 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 the 15N naturally present in each compartment. We
considered that the initial labelled fraction was the only fraction to present an enrichment
82
A. Lambert et al. / Animal Feed Science and Technology 82 (1999) 75±89
Fig. 3. Evolution of the 15N-labelling of ammonia in relation to 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.
in 15N 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 less 15N accumulated in ammonia at the end of
the incubation as the size of the initial labelled peptides increased. When the excess 15N
was added as fraction a, no trace of its accumulation in ammonia was observed.
Incubation with labelled fraction b gave an enrichment of ammonia of 0.04%. Incubation
with labelled fraction g gave 0.11% and incubation with labelled fraction d Ð 0.16%.
Although the enrichment in 15N in ammonia increased, the proportion of total excess 15N
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 excess 15N in
the medium. That is why we considered this quantity in order to study the change in the
distribution of the total recovered excess 15N 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
A. Lambert et al. / Animal Feed Science and Technology 82 (1999) 75±89
83
Fig. 4. Evolution of the 15N-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 the 15N-labelling of the different compartments during the incubation of labelled fraction d
with mixed rumen bacteria in vitro. (mean on three incubations). a, Peptides weighing between 1000 and
2000 Da; e: peptides weighing
82 (1999) 75±89
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
excess 15N in each of the initially labelled fractions gradually decreased and labelled compounds
were found in smaller peptides. Bacteria were labelled with 15N only after at least 30 min. This
delay increased with the length of the incubated labelled peptide. Large peptides (a and b) were
hydrolysed most rapidly and extensively. About 80% (SE 1.9) of the excess 15N coming from
fraction a was 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. Dipeptidylaminopeptidase type 1 and aminopeptidase activities combined with endopeptidase activities to
produce nitrogenous compounds that could be absorbed by bacteria. The monitoring of 15N 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
*
Corresponding author. Tel.: 33-383-595-889; fax: 33-383-595-804.
0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 0 9 7 - 8
76
A. Lambert et al. / Animal Feed Science and Technology 82 (1999) 75±89
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.
Peptides were obtained by hydrolysing the pea globulins for 10 h with pronase E
(protease XVI from Streptomyces griseus, Sigma, L'isle d'Abeau Chenes, France) at
398C in 0.1 M phosphate buffer, pH 6.8 (1 mg pronase per 100 mg globulins in 5 ml
buffer). Proteins were precipitated at the end of the hydrolysis by adding perchloric acid
(5% final concentration) and removed by centrifugation (28 000 g, 20 min, 48C).
Thereafter, 2 M K2CO3 was added to the supernatant according to the protocol of Chen
et al. (1987), as modified by Wallace et al. (1990). The KClO4 insoluble salts were
removed by a second centrifugation. Solutions of unlabelled peptides were immediately
A. Lambert et al. / Animal Feed Science and Technology 82 (1999) 75±89
77
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 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 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 the
15
N 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 the 15N naturally present in each compartment. We
considered that the initial labelled fraction was the only fraction to present an enrichment
82
A. Lambert et al. / Animal Feed Science and Technology 82 (1999) 75±89
Fig. 3. Evolution of the 15N-labelling of ammonia in relation to 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.
in 15N 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 less 15N accumulated in ammonia at the end of
the incubation as the size of the initial labelled peptides increased. When the excess 15N
was added as fraction a, no trace of its accumulation in ammonia was observed.
Incubation with labelled fraction b gave an enrichment of ammonia of 0.04%. Incubation
with labelled fraction g gave 0.11% and incubation with labelled fraction d Ð 0.16%.
Although the enrichment in 15N in ammonia increased, the proportion of total excess 15N
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 excess 15N in
the medium. That is why we considered this quantity in order to study the change in the
distribution of the total recovered excess 15N 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
A. Lambert et al. / Animal Feed Science and Technology 82 (1999) 75±89
83
Fig. 4. Evolution of the 15N-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 the 15N-labelling of the different compartments during the incubation of labelled fraction d
with mixed rumen bacteria in vitro. (mean on three incubations). a, Peptides weighing between 1000 and
2000 Da; e: peptides weighing