Power spectrum analysis of EEG in a translational nonhuman primate model after chronic exposure to low levels of the common marine neurotoxin, domoic acid.

Domoic acid (DA), the focus of this research, is a marine algal neurotoxin and epileptogen produced by species in the genus Pseudo-nitzschia. DA is found in finfish and shellfish across the globe. The current regulatory limit for DA consumption (20 ppm in shellfish) was set to protect humans from acute toxic effects, but there is a growing body of evidence suggesting that regular consumption of DA contaminated seafood at or below the regulatory limit may lead to subtle neurological effects in adults. The present research uses a translational nonhuman primate model to assess neurophysiological changes after chronic exposure to DA near the regulatory limit. Sedated electroencephalography (EEG) was used in 20 healthy adult female Macaca fascicularis, orally administered 0.075 and 0.15 mg DA/kg/day for at least 10 months. Paired video and EEG recordings were cleaned and a Fast Fourier Transformation was applied to EEG recordings to assess power differences in frequency bands from 1-20 Hz. When DA exposed animals were compared to controls, power was significantly decreased in the delta band (1-4 Hz, p < 0.005) and significantly increased in the alpha band (5-8 Hz, p < 0.005), theta band (9-12 Hz, p < 0.01), and beta band (13-20 Hz, p < 0.05). The power differences were not dose dependent or related to the duration of DA exposure, or subtle clinical symptoms of DA exposure (intentional tremors). Alterations of power in these bands have been associated with a host of clinical symptoms, such as deficits in memory and neurodegenerative diseases, and ultimately provide new insight into the subclinical toxicity of chronic, low-dose DA exposure on the adult primate brain.

Ricinoleic acid inhibits methanogenesis and fatty acid biohydrogenation in ruminal digesta from sheep and in bacterial cultures.

Ricinoleic acid (RA; 12-hydroxy-cis-9-18:1) is the main fatty acid component of castor oil. Although a precursor for CLA synthesis in lactic acid bacteria, RA was found previously not to form CLA in ruminal digesta but to have some inhibitory properties. The present study was undertaken to evaluate the potential of RA to modulate ruminal biohydrogenation and methanogenesis. Ruminal digesta from 4 sheep receiving a mixed hay-concentrate diet was incubated in vitro with 0.167 g/L of linoleic acid (LA; cis-9,cis-12-18:2) or with a combination of LA and RA or LA and castor oil (LA, RA, and castor oil added to a final concentration of 0.167 g/L) in the presence and absence of lipase. The CLA rumenic acid (cis-9,trans-11-18:2) accumulated when either RA or castor oil and lipase was present. Vaccenic acid (VA; trans-11-18:1) also accumulated, and a decrease of the rate of production of stearic acid (SA; 18:0) was observed. When LA was incubated with castor oil in the absence of lipase, no effects on biohydrogenation were observed. Ricinoleic acid at 0.02 g/L did not affect growth of Butyrivibrio fibrisolvens but it inhibited growth of Butyrivibrio proteoclasticus. Butyrivibrio proteoclasticus but not B. fibrisolvens metabolized RA to 12-hydroxystearate. Linoleic acid metabolism by B. proteoclasticus appeared to be unaffected by RA addition whereas rumenic acid accumulation increased (P = 0.015 at 12 h) when RA was added. A 28% decrease (P = 0.004) in methane was obtained in 24 h in vitro incubations of diluted buffered ruminal fluid with added 0.2 g RA/L. There was no effect on the total concentration of VFA after 24 h as a result of RA addition, but the molar proportions of acetate and butyrate were decreased (P = 0.041 and P < 0.001, respectively) whereas that of propionate increased (P < 0.001). It was concluded that, at least in vitro, RA or the combination of castor oil and lipase inhibit biohydrogenation, causing the accumulation of rumenic acid and VA, with potential health benefits for ruminant products. The effect appeared to be mediated via an inhibitory effect on the biohydrogenating activity of B. proteoclasticus. An added environmental benefit could be a concomitant decrease in methane emissions. In vivo studies are now required to confirm the potential of these additives.

Quantification of ruminal Clostridium proteoclasticum by real-time PCR using a molecular beacon approach.

AIMS: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18:2) via conjugated 18:2 metabolites (mainly cis-9,trans-11-18:2, conjugated linoleic acid) to vaccenic acid (trans-11-18:1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18:0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. METHODS AND RESULTS: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2-9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. CONCLUSIONS: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. SIGNIFICANCE AND IMPACT OF THE STUDY: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.

Metabolic properties of Eubacterium pyruvativorans, a ruminal 'hyper-ammonia-producing' anaerobe with metabolic properties analogous to those of Clostridium kluyveri.

Eubacterium pyruvativorans I-6(T) is a non-saccharolytic, amino-acid-fermenting anaerobe from the rumen, isolated by its ability to grow on pancreatic casein hydrolysate (PCH) as sole C source. This study investigated its metabolic properties and its likely ecological niche. Additional growth was supported by pyruvate, vinyl acetate, and, to a lesser extent, lactate and crotonate, and also by a mixture of amino acids (alanine, glycine, serine and threonine) predicted to be catabolized to pyruvate. No single amino acid supported growth, and peptides were required for growth on amino acids. Alanine, followed by leucine, serine and proline, were used most extensively during growth, but only alanine and asparate were extensively modified before incorporation. Growth on PCH, but not on pyruvate, was increased by the addition of acetate, propionate and butyrate. l-Lactate was fermented incompletely, mainly to acetate, but no lactate-C was incorporated. Propionate and butyrate were utilized during growth, forming valerate and caproate, respectively. Labelling experiments suggested a metabolic pattern where two C atoms of butyrate, valerate and caproate were derived from amino acids, with the others being formed from acetate, propionate and butyrate. The metabolic strategy of E. pyruvativorans therefore resembles that of Clostridium kluyveri, which ferments ethanol only when the reaction is coupled to acetate, propionate or butyrate utilization. The fermentative niche of E. pyruvativorans appears to be to scavenge amino acids, lactate and possibly other metabolites in order to generate ATP via acetate formation, using volatile fatty acid elongation with C(2) units derived from other substrates to dispose of reducing equivalents.

Eubacterium pyruvativorans sp. nov., a novel non-saccharolytic anaerobe from the rumen that ferments pyruvate and amino acids, forms caproate and utilizes acetate and propionate.

Two similar gram-positive rods were isolated from 10(-6) dilutions of ruminal fluid from a sheep receiving a mixed grass hay/concentrate diet, using a medium containing pancreatic casein hydrolysate as sole source of carbon and energy. The isolates did not ferment sugars, but grew on pyruvate or trypticase, forming caproate as the main fermentation product and valerate to a lesser extent. Acetate and propionate were utilized. One of these strains, I-6T, was selected for further study. Strain I-6T was a non-motile coccal rod, 1.2 x 0.4 microm, with a gram-positive cell wall ultrastructure and a G + C content of 56.8 mol%. No spores were visible, and strain I-6T did not survive heating at 80 degrees C for 10 min. Its rate of NH3 production was 375 nmol (mg protein)(-1) min(-1), placing it in the 'ammonia-hyperproducing' (or HAP) group of ruminal bacteria. 16S rDNA sequence analysis (1296 bases) indicated that it represents a novel species within the 'low-G + C' gram-positive group, for which the name Eubacterium pyruvativorans sp. nov. is proposed. Among cultivated bacteria, strain I-6T was most closely related (89% identity) to other asaccharolytic Eubacterium isolates from the mouth and the rumen. It was 98% identical to uncultured bacterial sequences amplified by others from ruminal digesta.

Ammonia production by ruminal microorganisms and enumeration, isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen.

Excessive NH(3) production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH(3) production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH(3) production. Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH(3) production from Trypticase varied from 1.8 to 19.7 nmol mg of protein(-1) min(-1) depending on the substrate, its concentration, and the method used. Monensin (5 micro M) inhibited NH(3) production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml (P = 0.011). Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 micro M monensin. Many fewer bacteria (0.002% of the total) grew on amino acids alone. Nineteen isolates capable of growth on Trypticase were obtained from four sheep. 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups. All were sensitive to monensin, and all except one group (group III, similar to Atopobium minutum), produced NH(3) at >250 nmol min(-1) mg of protein(-1), depending on the medium, as determined by a batch culture method. All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity. Groups I, II, and IV were most closely related to asaccharolytic ruminal and oral Clostridium and Eubacterium spp. Group V comprised one isolate, similar to Desulfomonas piger (formerly Desulfovibrio pigra). Group VI was 95% similar to Acidaminococcus fermentans. Growth of the Atopobium- and Desulfomonas-like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars. This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH(3) production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.

Influence of supplementary fibrolytic enzymes on the fermentation of corn and grass silages by mixed ruminal microorganisms in vitro.

This study was done to determine the effectiveness of supplementary enzymes at increasing the fiber digestion by ruminal microorganisms and to assess whether enzyme activity limits the rate of fiber digestion in ruminal digesta. In vitro comparisons of enzyme activities in two feed enzyme preparations (A and B) with enzyme activities extracted from ruminal fluid indicated that the addition of fibrolytic enzymes at the application rates recommended by the manufacturers would not be expected to increase significantly glycanase and polysaccharidase activities in ruminal fluid. Preparations A and B both increased (P < 0.001) the rate of gas production from freeze-dried corn and grass silages in in vitro incubations with ruminal fluid, but only at concentrations much higher than recommended application rates. Autoclaved controls had little or no effect. Ultrafiltration of enzyme B indicated that most stimulation was due to components >100 kDa, which is consistent with the cause of the stimulation being enzyme activity. Fibrolytic enzymes from other sources were also able to stimulate gas production: increased rates of gas production were observed in seven out of eight combinations of "cellulase" and corn or grass silage (P < 0.05). The comparison of glycanase and polysaccharidase activities with gas-stimulatory activity in the different enzyme preparations indicated that the highest correlation was between increased gas production and enzyme activity against microgranular cellulose (P < 0.05). In a wider range of fibrolytic enzyme preparations, those with endo-(beta-1,4)- or exo-(beta-1,4)-xylanase activity equal to that of preparation A did not produce similar increased rates of fermentation of corn silage when glucanase activity was low (P > 0.05). In contrast, preparations with glucanase activity similar to enzyme A gave at least as great (P < 0.05) an improvement in gas production than enzyme A, irrespective of xylanase activity. It was concluded that enzyme activity, probably a type of endo-(beta-1,4)-glucanase activity, limits the rate of fermentation of corn and grass silage in the rumen. Enzyme supplements of the type used in these experiments are unlikely to possess sufficient activity to overcome this limitation by direct application to ruminal digesta, implying that treatment of the ration prefeeding will be key to harnessing the potential of exogenous fibrolytic enzymes in ruminant nutrition.

Proteolytic activity of ruminal digesta during the feeding cycle in sheep receiving grass hay/concentrate or maize silage/concentrate diets.

Proteolytic activity was measured by the digestion of 14C-labelled casein in digesta removed from the rumen of four sheep receiving a grass hay/concentrate diet and four sheep receiving a maize silage/concentrate diet. Samples were removed immediately before feeding and at 2-h intervals after feeding up to 12 h. Animals on both diets produced similar proteolytic activities (1.83 (S.D. 0.41) and 2.14 (S.D. 0.61) mg 14C-casein hydrolysed (ml ruminal fluid)-1 h-1 with the maize silage- and grass hay-based diets, respectively). Time after feeding had no effect on proteolytic activity, but between-animal variation was consistent and highly significant, with the highest-activity animals having activities 64 and 74% higher than the lowest-activity animals on the two diets, respectively.

Influence of 1-[(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl]pyrrolidine-2-carboxylic acid and diphenyliodonium chloride on ruminal protein metabolism and ruminal microorganisms.

The effects of 1-[(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl]pyrrolidine-2-carboxy lic acid (LY29) and diphenyliodonium chloride (DIC) on the degradation of protein to ammonia were determined in a mixed rumen microbial population taken from sheep on a grass hay-concentrate diet. Both compounds decreased NH3 production by inhibiting deamination of amino acids. LY29, but not DIC, inhibited growth of the high-activity ammonia-producing species, Clostridium aminophilum and Clostridium sticklandii.

Peptidase activity of human colonic bacteria.

Aminopeptidase activities of mixed faecal suspensions from four human donors and 12 of the most numerous species of human colonic bacteria were measured using alanine oligopeptides and various dipeptidyl- and amino acyl-arylamidase substrates. The pattern of hydrolysis of Ala(4)and Ala(5)in faecal suspensions, whereby Ala(2)was the first breakdown product, suggested that the main mechanism of peptide hydrolysis was dipeptidyl peptidase. Dipeptidyl p-nitroanilides and 4-methoxynaphthylamides were broken down more rapidly than amino acyl derivatives in three out of four individuals tested, consistent with this conclusion. The predominant Bacteroides spp. of the intestine also had greater dipeptidyl peptidase activity than amino acyl aminopeptidase activity, while Bifidobacterium, Clostridium, Enterococcus and Propionibacterium spp. had a more variable pattern of peptidase activities. Thus, peptide hydrolysis in the human intestine, as in the rumen, appears to be mainly a two-stage process which is initiated by dipeptidyl peptidases present in the most numerous Bacteriodes spp.

Peptidases of the rumen bacterium, Prevotella ruminicola.

Prevotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested. Streptococcus bovis hydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobacter amylophilus, Ruminococcus spp. and Veillonella parvula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P. ruminicola. Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Ala4and Ala5; another was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro-4-methoxynapthylamide and was similar to dipeptidyl peptidase type IV XDPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. All of the enzymes, and particularly those active against Ala2-p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures.

Influence of 1,10-phenanthroline and its analogues, other chelators and transition metal ions on dipeptidase activity of the rumen bacterium, Prevotella ruminicola.

Prevotella ruminicola plays a prominent role in the breakdown of peptides in the rumen, a process which contributes to excessive ammonia production and inefficient nitrogen retention in ruminants. Various metal ions and chelators were examined to assess how the metal ion-dependent dipeptidase activity of P. ruminicola M384 might be inhibited. Using sonicated extracts, Cu2+, Cr2+ and Hg2+ were most inhibitory, decreasing Ala2 breakdown to 15, 15 and 5% of control activity, whereas Co2+, Mn2+ and Zn2+ stimulated activity by 189, 30 and 26%, respectively. The chelators, EDTA, EGTA, TPEN and 1,10-phenanthroline, were inhibitory, as were several phenanthroline analogues. Among the stereoisomers of 1,10-phenanthroline tested, derivatives methylated on C-2 and C-9 were less effective than the parent molecule, but 3,4,7,8-tetramethyl-1,10-phenanthroline (TMP) was more inhibitory. Titration of the most effective inhibitors showed that EDTA, TPEN and TMP had similar potency and were effective at 0.1 mmol l-1 and above. Thus some metal ions and chelators are potent inhibitors of P. ruminicola dipeptidase, although they are unlikely to be sufficiently specific to peptide metabolism to be useful in vivo.

Inhibition by 1,10-phenanthroline of the breakdown of peptides by rumen bacteria and protozoa.

The rate of peptide breakdown in the rumen frequently exceeds the rate at which the amino acids released can be used for microbial growth. The final step in this often wasteful process involves the cleavage of dipeptides. The main rumen bacterial species with high dipeptidase activity, Prevotella ruminicola, Fibrobacter succinogenes, Lachnospira multipara and Megasphaera elsdenii, had activities which were inhibited > 95% by 1,10-phenanthroline, a chelator of divalent metal ions and metalloprotease inhibitor. Dipeptidase activity in digesta taken from the rumen of sheep decreased by 33% in the presence of 1,10-phenanthroline, while mixed bacteria from the same samples were inhibited by 80% and the activity of mixed protozoa decreased by only 15%. Thus a substantial amount of dipeptide breakdown appears to be due to ciliate protozoa in the mixed population. Extensive washing of the protozoa increased the sensitivity of protozoal dipeptidase activity to 1,10-phenanthroline, suggesting that protozoa too have a metallo-dipeptidase activity but that it is normally protected from inhibition by 1,10-phenanthroline. Breakdown of the pentapeptide, Ala5, was also inhibited 27% by 1,10-phenanthroline in the mixed population, and when Trypticase, a pancreatic casein hydrolysate containing a mixture of oligopeptides, dipeptides and amino acids, was incubated with rumen fluid, the production of ammonia and free amino groups was inhibited 71% by 1,10-phenanthroline. It was concluded that metal ion chelation inhibits oligopeptidase and dipeptidase activities of rumen micro-organisms and may be a means of controlling ammonia production from peptides in the rumen.

Cleavage of di- and tripeptides by Prevotella ruminicola.

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala2 and Ala3 hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala2 and Ala3 by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala2 hydrolysis by EDTA-treated cells was inhibited by the Ca2+/H+ ionophore, tetronasin. Ala3 hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B(1)4 had activities which ran at the same R(f); strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R(f) for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M(r) of the dipeptidases from strains M384 and B(1)4 were 115,000 and 114,500 respectively, and 112,500 and 121,500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.

Breakdown of different peptides by Prevotella (Bacteroides) ruminicola and mixed microorganisms from the sheep rumen.

Several di-, tri-, and oligopeptides were incubated individually in vitro with rumen fluid from two sheep receiving a mixed grass hay/concentrate diet and with washed cells of Prevotella (formerly Bacteroides) ruminicola M384 and P. ruminicola B(1)4. The rates of breakdown of most peptides were similar in the rumen fluid from the two sheep. Acidic and proline-containing peptides tended to be more slowly degraded than neutral or basic peptides. The dipeptide at the N-terminus of higher peptides was observed as an early product of hydrolysis, confirming that a dipeptidyl aminopeptidase type of activity was present. The relative rates of breakdown of dipeptides by P. ruminicola were different from that of rumen fluid, but the hydrolysis of higher peptides followed a similar pattern, and dipeptides from the N-terminus were detected as early products.

Selective isolation of bacteria with dipeptidyl aminopeptidase type I activity from the sheep rumen.

Five-hundred-and-six fresh isolates of rumen bacteria were tested for their ability to hydrolyse the synthetic substrate for dipeptidyl aminopeptidase type I, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA), using a gel overlay technique. Twelve positive isolates were small Gram-negative rods which resembled Bacteroides ruminicola in their biochemical and morphological properties. SDS-PAGE of whole cell extracts indicated that two were similar to B. ruminicola strain B14, six resembled B. ruminicola strain M384, and four were similar to B. ruminicola GA33. All hydrolysed GlyArg-MNA, Ala2 and Ala5, and showed no activity against Leu-MNA. Ala3 and Ala2, but no Ala4, was produced from Ala5. The different groups had different, distinctive activity profiles. The two remaining positive isolates were Lactobacillus spp. with an exceptionally high Leu-MNA activity. It was concluded that, although different strains may only be distantly related, B. ruminicola forms the most important group of bacteria in the rumen to possess a dipeptidyl aminopeptidase type I activity.

A survey of peptidase activity in rumen bacteria.

Twenty-nine strains of 14 species of rumen bacteria were screened for their ability to hydrolyse Ala2, Ala5, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA) and Leu-MNA. Several species, notably Megasphaera elsdenii, were active against Ala2, and a smaller number, including Bacteroides ruminicola, Butyrivibrio fibrisolvens, Ruminococcus flavefaciens, Lachnospira multipara and Ruminobacter amylophilus, broke down Ala5. Streptococcus bovis had an exceptionally high leucine arylamidase activity. However, only Ba. ruminicola hydrolysed GlyArg-MNA. Further investigation revealed that only Ba. ruminicola and Bu. fibrisolvens hydrolysed Ala5 to Ala3 and Ala2, with little ALa4 being produced, in a manner similar to rumen fluid. The activity of Ba. ruminicola against synthetic peptidase substrates, including GlyArg-MNA, LysAla-MNA, ArgArg-MNA, GlyPro-MNA, LeuVal-MNA, and Ala3-p-nitroanilide, was similar to that of rumen fluid, whereas the activity of Bu. fibrisolvens was quite different. Since the main mechanism by which peptides are broken down in the rumen is similar to dipeptidyl aminopeptidase type I, for which GlyArg-MNA is a diagnostic substrate, it was concluded that Ba. ruminicola was the most important single species in peptide breakdown in the rumen.

Effects of the ionophore tetronasin on nitrogen metabolism by ruminal microorganisms in vitro.

The effects of tetronasin on ruminal protein metabolism were investigated in vitro using ruminal fluid from cattle receiving tetronasin in the diet, ovine ruminal fluid from animals not receiving tetronasin and pure cultures of proteolytic ruminal bacteria. Ruminal fluid from cattle receiving tetronasin in a predominantly barley diet had lower proteolytic (76% of control, P less than .10) and deaminative (58% of control, P less than .05) activities than controls after 42 d. The effect of deamination disappeared after 84 d, although the proteolytic activity remained lower (P less than .10) than that of controls. When tetronasin was added in vitro to ruminal fluid from sheep not receiving the ionophore, proteolytic activity (14C-labeled casein hydrolysis) was unaffected, but the rate of ammonia production from amino acids was decreased by 87% (P less than .01). Oligopeptide breakdown was inhibited to a lesser extent (21%, P less than .05). Dipeptidase activity (dialanine hydrolysis) was not affected. The addition of tetronasin to cultures of the ruminal bacteria Ruminobacter amylophilus and Bacteroides ruminicola had no influence on their protease, deaminase or dipeptidase activities. However, when the bacteria were adapted to grow in the presence of tetronasin, deamination of amino acids was severely inhibited (87 to 100%, P less than .01), even when tetronasin was absent from the incubation mixture. Tetronasin had no effect on the proteolytic activity of adapted cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of peptide metabolism by ruminal microorganisms.

Methods were developed for the determination of oligoalanine and other short-chain peptides and peptide analogs in ruminal fluid by using reverse-phase high-pressure liquid chromatography. Chromatographic analysis of the breakdown of (Ala)3 and (Ala)4 in ruminal fluid in vitro revealed that the predominant mechanism of hydrolysis was a dipeptidyl peptidase-like activity. Hydrolysis proceeded from the N terminal of the peptide chain; N-acetyl-(Ala)3 was broken down at 11% of the rate of breakdown of (Ala)3 or (Ala)3-p-nitroanilide. (Ala)2-p-nitroanilide was hydrolyzed most rapidly of the arylamide substrates tested, but fluorogenic 4-methoxy-2-naphthylamide (MNA) compounds were more convenient and potentially more versatile substrates than p-nitroanilides. Gly-Arg-MNA was the most rapidly hydrolyzed dipeptidyl peptidase substrate, suggesting that ruminal peptidase activity was predominantly of a type I specificity.