Resistance of Proline-Containing Peptides to Ruminal Degradation In Vitro

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1992, p /92/ $2./ Copyright C) 1992, American Society for Microbiology Vol. 58, No. 12 Resistance of Proline-Containing Peptides to Ruminal Degradation In Vitro C.-M. J. YANG' AND J. B. RUSSELL2.3* Department ofanimal Science' and Section of Microbiology, 2* Cornell University, and Agricultural Research Service, U.S. Department ofagriculture,3 Ithaca, New York Received 15 June 1992/Accepted 1 October 1992 Mixed ruminal bacteria utilized an enzymatic digest of casein at a rate faster than that for an enzymatic digest of gelatin, but neither amino acid source was completely utilized even when the incubation period was as long as 96 h. Since the reaction of ninhydrin with the residual nonammonia, nonprotein nitrogen was more than twofold stronger when the samples were hydrolyzed with 6 N HCI, it appeared that much of the residual nitrogen was from peptides. Approximately 66% of the nonammonia, nonprotein, ninhydrin-reactive material could not be recovered as amino acids, but there was a significant decrease in total amino acid nitrogen when the samples were pretreated with a C1, Sep-Pak column to remove peptides. The resistant peptides had an abundance of proline, and subsequent incubations showed that synthetic dipeptides which contained proline were hydrolyzed slowly. Lysine appears to be the amino acid which is most apt to limit ruminant production. Dipeptides containing proline and lysine were hydrolyzed at least fivefold slower than lysine-alanine. Methionine, another potentially limiting amino acid, was also degraded at a slower (2.5-fold) rate when it was present as part of a proline dipeptide. Much of the protein which is ingested by ruminants is deaminated by ruminal microorganisms (1). Some of this ammonia can be used as a nitrogen source for microbial growth, but the production rate of ammonia often exceeds the utilization rate (21). "5N studies indicated that as much as 25% of the protein can be lost as ammonia, which is subsequently converted to urinary urea (15). Many insoluble proteins are not completely degraded in the rumen (11), but these escape or bypass supplements (fish meal, brewer's grains, etc.) are more expensive than forage proteins and soybean meal (lla). In the early 198s, Brock et al. (2) examined the effects of various proteinase inhibitors on ruminal protein degradation. Virtually all of the inhibitors tested had some effect, but only 5 mm Merthiolate, a general antimicrobial agent, produced more than 5% inhibition. On the basis of these results, it appeared that ruminal microorganisms produced a variety of different proteinases and that it would be difficult to manipulate protein degradation at this step. Because amino acids are generally present at low concentrations in ruminal fluid (26), it had been assumed that proteolysis was the rate-limiting step in ruminal protein degradation and the role of peptidases was largely ignored. When Mangan (14) added casein to the rumen, there was an increase in nonammonia, nonprotein nitrogen, and this observation indicated that peptides could accumulate. Some workers have suggested that peptide accumulation might be an artifact of casein (3), a rapidly degraded, nonfeedstuff protein, but significant amounts of nonammonia, nonprotein, ninhydrin-reactive material were also noted in the rumens of cattle fed soybean protein (8). Results presented here showed that mixed ruminal bacteria were unable to degrade all of the peptides from enzymatic digests of casein and gelatin, even when the incubation period was as long as 96 h. These small peptides (approxi- * Corresponding author mately 2 amino acids) had large amounts of proline. On the basis of this earlier work, it appeared that proline-containing peptides might be degraded at a slower rate than other peptides. Because lysine and methionine are the amino acids which are most apt to limit animal performance (2), we examined the degradation of dipeptides which contained proline and either lysine or methionine. Lysine-proline, proline-lysine, methionine-proline, and proline-methionine were degraded at a much slower rate than lysine-alanine and methionine-alanine. MATERIALS AND METHODS Peptide utilization. Ruminal contents were obtained from a ruminally fistulated, nonlactating holstein cow (65 kg). The cow was fed 2.5 kg of timothy hay (9% crude protein) and 2.5 kg of a commercial concentrate mix (16% crude protein) twice daily. At 1.5 h after feeding, ruminal contents were squeezed through four layers of cheesecloth. The ruminal fluid was allowed to stand for 1 h (39 C, anaerobic). After gas production had buoyed small feed particles to the top and protozoa had sedimented to the bottom, bacteria were collected from the middle section of the flask. The bacteria were harvested by centrifugation (1,3 x g, 15 min, 15 C, CO2 atmosphere) and transferred anaerobically to a basal medium containing (per liter) 292 mg of K2HPO4.3H2, 24 mg of KH2PO4, 48 mg of Na2SO4, 48 mg of NaCl, 1 mg of MgSO4. 7H2, 64 mg of CaCl2. H2, 4, mg of Na2CO3, 6 mg of cysteine, and either casein (Trypticase;.5 g of N per liter; BBL Laboratories, Cockeysville, Md.) or gelatin hydrolysate (.5 g of N per liter; U.S. Biochemical, Cleveland, Ohio). The final cell concentration was one optical density unit (Gilford 26 spectrophotometer; 6 nm, 1-mm light path) or 312 mg of protein per liter. Triplicate bottles (8 ml) were sealed with butyl rubber stoppers and aluminum seals and incubated at 39 C. Samples (3 ml) were centrifuged (1, x g, 5 C, 1 min), and the cells were Downloaded from on October 18, 218 by guest

2 VOL. 58, 1992 PROLINE-CONTAINING PEPTIDES RESIST RUMINAL DEGRADATION 3955 washed once with.9% NaCI. Cell-free supematants and cells were frozen (-2 C). Peptide and amino acid utilization was estimated by a ninhydrin procedure (18). Cell-free supernatant samples (2,ul) were dried in an oven (11 C) to remove ammonia and hydrolyzed with HCI (6 N, 11 C, 24 h under N2 gas). The samples were adjusted to ph 5.4 with NaOH and assayed by ninhydrin, with glycine as a standard. Peptide size was estimated from the ninhydrin reaction before and after HCI hydrolysis. The amino acid composition of the residual peptides and free amino acids (96 h) was estimated by high-pressure liquid chromatography (HPLC). Cell-free samples (25,ul) were hydrolyzed with HCl and derivatized with phenylisothiocyanate prior to being added to the HPLC column (1). This procedure gave the total amino acid composition of the samples. Peptides were then estimated by determining the difference from samples which had been run through a C18 Sep-Pak column to remove peptides (Millipore Corporation, Milford, Mass.). Dipeptide hydrolysis. Mixed ruminal bacteria were obtained (see above) from a cow which was fed.56 kg (dry-matter basis) of chopped timothy hay (9% crude protein, 41% acid detergent fiber, 65% neutral detergent fiber, and 17% nonstructural carbohydrates) every 2 h with a carousel feeder. Particle-free ruminal fluid prepared as previously described was dispensed anaerobically (1 ml) into tubes (Bellco Glass, Inc., Vineland, N.J.) that contained dipeptides (2 mm final concentration). The tubes were incubated at 39 C for 6 h. Samples (1 ml) were centrifuged (13, x g, 22 C, 5 min) to remove the bacteria. The cell-free supernatants were frozen at -2C. Peptides and amino acids resulting from peptide hydrolysis were assayed by an HPLC procedure which employed dansyl chloride derivatization. Cell-free samples (5,ul) were treated with 95,ul of Na2CO3 (4 mm, ph 9.5) and 5,ul of dansyl chloride (1.5 mg/ml in acetonitrile) and incubated at 39 C for 1 h in the dark. The reaction with dansyl chloride was terminated by adding an excess of methylamine (2,u1; 2%, wtlvol). Dansyl derivatives were analyzed by HPLC (254 nm; Beckman model 16 UV detector) using an acetonitrile gradient. Samples (2 each) were injected into sodium phosphate buffer (3 mm, ph 6.5) which was passing through a C18 reversed-phase column at 1. ml/min (28C). Acetonitrile was increased linearly to 55% during the next 3 min. Other analyses. Ammonia was measured by the colorimetric method of Chaney and Marbach (5). Cell samples were boiled in.2 N NaOH for 15 min, and protein was assayed by the method of Lowry et al. (13). Reagents. Lysine-proline, proline-lysine, methionine-proline, and proline-methionine were synthesized by Vega Biotechnologies, Inc. (Tucson, Ariz.). Other dipeptides were purchased from the Sigma Chemical Company (St. Louis, Mo.). All peptides contained L-amino acids. All other chemicals were reagent grade. Experimental design. All experiments were performed in duplicate, and the coefficient of variation was less than 1%. RESULTS Utilization of casein hydrolysate. Mixed ruminal bacteria which were suspended in anaerobic buffer without enzymatic casein (Trypticase) or gelatin hydrolysate produced little ammonia or material which would react with ninhydrin (data not shown). When casein hydrolysate (equivalent to j~ o Trypticase Gelatin Hydrolysate Time (h) FIG. 1. Utilization of Trypticase and gelatin hydrolysate (.5 g of N per liter each) by mixed ruminal bacteria (312 mg of protein per liter) in vitro. Bars, standard deviations. 2.5 g of glycine) was added, there was a rapid decrease in total amino acids during the first 24 h of incubation (Fig. 1). Thereafter, there was little further decrease in amino acid nitrogen. On the basis of the ninhydrin reaction, it appeared that only 6% of the Trypticase was utilized. Since the ratio of ninhydrin reactions after and before HCI hydrolysis was 2.2, it appeared that much of this residual amino acid nitrogen was from short peptides. The amino acid composition of Trypticase was well balanced (Fig. 2a), and approximately 8% of the amino acids was found in peptides (Fig. 3a). When amino acid disappearance was estimated by the HPLC procedure, it appeared that approximately 1% of the amino acids in Trypticase resisted 96 h of incubation with mixed ruminal bacteria (Fig. 2b). Approximately 3% of these residual amino acids was found in the peptide fraction (Fig. 3b). Branched-chain amino acids accounted for 7% of the residual amino acid nitrogen (Fig. 2b), and the peptide fraction had large amounts of proline and glutamine-glutamate as well as branched-chain amino acids (Fig. 3b). 8 7 c o 6 E 5-4 F2 3 2 Kt 1 8 Z 7 o 6 E 5 4 ` 3 o 2 t 1 4 i 96 h Trypticase b.48 g N/liter..l,.E,1., x x L- :% vb L. f O' L. _ '4, u, u < o Cl o I < 1- < IL 1- >: L) _5,.L, FIG. 2. The amino acid composition of Trypticase which resisted degradation by ruminal bacteria in vitro. (a) Composition at h; (b) amounts remaining at 96 h. Downloaded from on October 18, 218 by guest

3 3956 YANG AND RUSSELL X8-7- c 6- E 5 X 4. Cx E 5 'I 4.I-- a 3-2- ~1. be 1 h Trypticase a.394 g N/liter mm. m - 96 h Trypticase.15 9 N/liter *i' m- u...iuiue. x4 x L =Ft L q* L _ 4, ua < o o I <: 1 < CL 1- u = Q-X O. J FIG. 3. The amino acid composition of Trypticase peptides which resisted degradation by ruminal bacteria in vitro. (a) Composition at h; (b) amounts remaining at 96 h. Utilization of gelatin hydrolysate. Gelatin hydrolysate was degraded at a slower rate than Trypticase, and less than 25% of the ninhydrin-reactive material was utilized by mixed bacteria (Fig. 1). Gelatin hydrolysate contained longer peptides than casein hydrolysate (5.5 versus 2.8 amino acids), but the ninhydrin ratio was only 2 at 96 h. The ruminal bacteria left 6% of the ninhydrin-reactive material, but only half of this ninhydrin-reactive material could be accounted for in terms of amino acids (Fig. 4b). Approximately 25% of the residual amino acids was found in the peptide fraction C E X 4- I- 3- B 2-1 X8 7-6 E 5- X 4- l be 1 h Gelatin hydrolysate a.5 g N/liter Nn,,. 96 h Gelatin hydrolysate b.94 g N/liter x x L M L L b L. _ w =P M < o () x - a. 1 LX > u X a. 5 - FIG. 4. The amino acid composition of gelatin hydrolysate which resisted degradation by ruminal bacteria in vitro. (a) Composition at h; (b) amounts remaining at 96 h. b h Gelatin hydrolysate c 6 - E 5 - * 4 - *3 - X g N/liter *o 7-96 h Gelatin hydrolysate b.24 g N/liter o 6- E X 2 1 -xx L' Rs L a L. =r* m 4ps <~~~ <) 1 I o - > 3XLO FIG. 5. The amino acid composition of gelatin hydrolysate peptides which resisted degradation by ruminal bacteria in vitro. (a) Composition at h; (b) amounts remaining at 96 h. (Fig. Sb). Gelatin hydrolysate had large amounts of glycine, proline, and alanine (Fig. 4a), and these amino acids were utilized poorly by mixed ruminal bacteria (Fig. 4b). Glycine, proline, and alanine accounted for 65% of the residual peptide (Fig. Sb). Hydrolysis of proline-containing dipeptides. Mixed ruminal bacteria degraded the dipeptide methionine-alanine at a rapid rate (initial rate,.143 h-1), and nearly 7% was hydrolyzed by 6 h (Fig. 6a). When proline was substituted E a. a- a- cl Ly s- Ala a Lys-Pro 13 Pro-Ly s APPL. ENVIRON. MICROBIOL Time (h) FIG. 6. Degradation of Met-Ala, Met-Pro, and Pro-Met (a) and Lys-Ala, Lys-Pro, and Pro-Lys (b) by mixed ruminal bacteria (1,6 mg of protein per liter) in vitro. b Downloaded from on October 18, 218 by guest

4 VOL. 58, 1992 PROLINE-CONTAINING PEPTIDES RESIST RUMINAL DEGRADATION 3957 TABLE 1. Dipeptide Initial rate of dipeptide hydrolysis by mixed ruminal bacteria in vitroa ~~~~~~~~~~~(h-1) Initial rate Methionine-alanine Methionine-glycine...1 Methionine-glutamate Methionine-proline...55 Proline-methionine...69 Lysine-alanine...19 Lysine-glycine Lysine-proline...25 Proline-lysine...44 Leucine-alanine...2 Leucine-leucine Serine-leucine Leucine-proline...16 Proline-leucine...33 Alanine-alanine Alanine-glycine Alanine-isoleucine Proline-alanine...17 Valine-valine Valine-glycine Valine-proline...5 Proline-valine Glycine-glycine...94 Glycine-alanine Glycine-threonine...2 Glycine-isoleucine Glycine-proline...56 Proline-glycine...77 a Particle-free ruminal fluid (1,6 mg of bacterial protein per liter). The incubation period was to 6 h, the temperature was 39 C, and the initial dipeptide concentration was 2 mm. for alanine, there was a marked decrease (more than 2.5- fold) in the initial rate of peptide hydrolysis (Table 1), and less than 6% of the peptide was degraded (Fig. 6a). The rate of proline-methionine hydrolysis was only slightly greater than the rate observed with methionine-proline (Table 1). Lysine-alanine was degraded very rapidly (Table 1), and by 6 h more than 8% of this peptide had been hydrolyzed (Fig. 6b). When lysine was provided as lysine-proline or proline-lysine, the initial rates of dipeptide hydrolysis were more than fourfold slower (Table 1) and less than 25% of the lysine-proline or proline-lysine was hydrolyzed during the 6-h incubation period (Fig. 6b). Leucine dipeptides which contained alanine, serine, or leucine in addition to leucine were hydrolyzed rapidly, but leucine dipeptides containing proline were degraded at a much slower rate (Table 1). Glycine-glycine was hydrolyzed at half the rate of leucineleucine. Alanine, valine, and glycine dipeptides were also hydrolyzed at a slower rate when they were present as proline dipeptides. The only exceptions were proline-alanine and proline-valine. DISCUSSION For many years, it was generally accepted that "free amino acids are intermediate products in the breakdown of proteins by ruminal microorganisms" (4). Because the degradation rates of free amino acids in the rumen are generally rapid (.25 to.6 h-' [4]), it appeared that proteolysis was the rate-limiting step in ruminal protein degradation. Recent work, however, showed that significant amounts (as much as 6% of the crude protein) of nonammonia, nonprotein, ninhydrin-reactive nitrogen could pass out of the rumen (7, 8). Because the ratio of ninhydrin reactions after and before hydrolysis was greater than 2, it appeared that significant amounts of peptide nitrogen could flow out of the rumen to the lower gut. Wallace and McKain (22) also noted some increase in the amount of nonammonia, nonprotein, ninhydrin-reactive material in the period soon after feeding, but they indicated that only a small portion of this material was amino acids. When mixed ruminal bacteria were incubated in vitro with casein or gelatin hydrolysate, much of the nonammonia, nonprotein, ninhydrin-reactive nitrogen was never utilized (Fig. 1). Because the ninhydrin ratio was approximately 2 at the end of the incubation, it appeared that some peptides were resistant to ruminal hydrolysis. At time zero, the ninhydrin reaction and amino acid analyses gave similar estimates of amino acid nitrogen (.5 versus.45 g of N per liter), but by 96 h the ninhydrin reaction was considerably greater (3- and 2.7-fold for Trypticase and gelatin hydrolysate, respectively) than the amount which could be accounted for by the amino acid analyses (Fig. 1, 2, and 4). Wallace and McKain (22) indicated that amino acids accounted for less than 1% of the nonammonia, nonprotein, ninhydrin-reactive nitrogen in ruminal fluid. The authors suggested that ninhydrin was reacting with amino sugars (e.g., N-acetylhexosamines), but there has been no support for this hypothesis. Because our initial incubations (Fig. 1 to 5) had only trace amounts of ruminal fluid and there was little increase in nonammonia, nonprotein, ninhydrin-reactive nitrogen in incubations lacking protein hydrolysate, it is unlikely that amino sugars can explain the difference between ninhydrin and amino acids at 96 h. When Chen et al. (9) fractionated Trypticase with 9% isopropyl alcohol, mixed ruminal bacteria utilized an alcohol-soluble portion at a much slower rate (twofold) than the alcohol-insoluble material and the alcohol-soluble portion had a very high proline content. The peptides from Trypticase and gelatin hydrolysate which resisted a 96-h incubation had an abundance of proline (Fig. 3b and Sb), and this observation supported the idea that proline-containing peptides might be hydrolyzed slowly by mixed ruminal bacteria. The study of peptide degradation is confounded by the number of amino acid combinations (n'2) and the commercial availability of synthetic peptides, but Wallace et al. (24) noted that proline-glycine-glycine, glycine-proline-glycineglycine, glycine-proline, and proline-leucine-glycine-glycine were degraded at slower rates than a variety of other peptides. Broderick et al. (3) reported that mixed ruminal microorganisms took up glycine-alanine twice as fast as glycine-proline. When Schwab et al. (2) infused casein or amino acids into the abomasums of lactating dairy cattle, a combination of lysine and methionine produced 43% of the total increase in milk protein. On the basis of these results, it appeared that lysine and methionine were the most limiting amino acids. There have been attempts to protect lysine and methionine by capsules which are ruminally inert but soluble in the acidic abomasum (17). While this approach may have merit, problems can arise. If the capsule does not have some resistance to a low ph, it can decompose in silage acid. If the capsule is too acid resistant, the lysine or methionine may be Downloaded from on October 18, 218 by guest

5 3958 YANG AND RUSSELL poorly absorbed from the intestines (16). Our experiments indicated that lysine and methionine dipeptides which contained proline were degraded slowly by bacteria in ruminal fluid (Fig. 6). Because the initial rates of proline dipeptide degradation (Table 1) were lower than the ruminal fluid dilution rate (12), it is likely that these peptides could escape ruminal degradation. The only exceptions were prolinealanine and proline-valine. Based on the latter comparison, proline may provide better protection when it is present at the C-terminal end of the dipeptide. Further work is needed to examine the intestinal absorption of proline-containing peptides. The idea that peptide nitrogen might accumulate in the rumen is not particularly new. In 1964, Winter et al. (25) noted that there are large amounts of nitrogen that could be precipitated by tungstic acid but not trichloroacetic acid, and Mangan (14) noted a large increase in nonammonia, nonprotein nitrogen when casein was infused into the rumen. Since that time, there has been considerable controversy regarding the magnitude and potential importance of peptide flow from the rumen (3, 6-8, 19, 22-24). Because peptides are intermediates in protein degradation as well as a nitrogen source for ruminal microbial protein synthesis and deamination, peptide flow could be influenced by (i) the quantity and type of food consumed each day, (ii) the solubility and degradability of the protein fraction, (iii) the availability of carbohydrate to drive the synthesis of microbial protein from peptides and amino acids, (iv) the passage rates of solids and liquids from the rumen, and (v) the method of measuring peptides and amino acids. Although peptide flow from the rumen may represent only a minor portion of amino acids entering the intestines, it appears that certain peptide bonds may be highly resistant to ruminal degradation. The latter observation indicates that the availability of a particular amino acid may be affected by the primary structure (amino acid sequence) of a protein as well as its secondary and tertiary structures (folding, solubility, and disulfide bridges). ACKNOWLEDGMENTS This research was supported by the U.S. Dairy Forage Research Center, Madison, Wis. We thank Ted Thannhauser, Cornell Biotechnology Center, Ithaca, N.Y., for performing the amino acid analyses. REFERENCES 1. Annison, E. F Nitrogen metabolism in the sheep. Biochem. J. 64: Brock, F. M., C. W. Forsberg, and J. G. Buchanan-Smith Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors. Appl. Environ. Microbiol. 44: Broderick, G. A., R. J. Wallace, and N. J. 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McKain Some observations on the susceptibility of peptides to degradation by rumen microorganisms. Asian-Australas. J. Anim. Sci. 2: Wallace, R. J., and N. McKain A survey of peptidase activity in rumen bacteria. J. Gen. Microbiol. 137: Wallace, R. J., C. J. Newbold, and N. McKain Patterns of peptide metabolism by rumen microorganisms, p In S. Hoshino, R. Onodera, H. Minato, and H. Itabashi (ed.), The rumen ecosystem: the microbial metabolism and its regulation. Japan Scientific Press/Springer-Verlag, Tokyo. 25. Winter, K. A., R. R. Johnson, and B. A. Dehority Metabolism of urea nitrogen by mixed cultures of rumen bacteria grown on cellulose. J. Dairy Sci. 47: Wright, D. E., and R. E. Hungate Amino acid concentrations in rumen fluid. Appl. Microbiol. 15: Downloaded from on October 18, 218 by guest