In vitro metabolism of phenylalanine by ruminal bacteria,protozoa, and their mixture

An in vitro study was conducted to examine the metabolism of phenylalanine (Phe) by mixed rumen bacteria (B), mixed rumen protozoa (P), and a combination of the two (BP). Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrated mixture twice a day. Microbial suspensions were anaerobically incubated at 39 degrees C for 12 h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analysed by HPLC. The net degradation rate (&mgr;mol/g microbial nitrogen) of Phe in B was about 1.5-fold higher than that in P. Phe was converted mainly into phenylacetic acid (PAA) and unknown compound(s) that presumably involved tyrosine in B, P, and BP during the 12 h incubation period. Small amounts of benzoic acid (BZA), and traces of phenylpropionic acid (PPR) and phenyllactic acid (PLA) were also produced from Phe. PAA production in B was found to be higher than that in P, whereas it was significantly higher in BP. Although BZA production was less than one-tenth that of PAA production, it was higher in P than in B and BP. PPR was detected in both B and BP, but not in P. PLA was detected only in B. The production of unknown compound(s) was higher in B than in P and BP.     

Degradation of tryptophan and related indolic compounds by ruminal bacteria,protozoa and their mixture in vitro

Summary.  In vitro experiments were conducted to examine the degradation of d- and l-isomers of tryptophan (Trp) and 10 related indolic compounds by mixed rumen bacteria (B), protozoa (P) and a combination of the two (BP). The analyses were carried out by HPLC. d-Trp (1.0 mM) was not degraded by rumen microorganisms during the 24-h incubation period. The net degradation of 1 mM l-Trp was 46.5%, 8.7% and 80.0% by B, P and BP suspensions, respectively. Trp was degraded into indoleacetic acid, indolelactic acid and indole by rumen bacteria and protozoa, and into skatole, p-cresol and indolepropionic acid by rumen bacteria only. Of them, indoleacetic acid was the major product of Trp found in B (15.4%) and P (3.1%), and skatole in BP (43.2%). This is the first report of the production of indolelactic acid and p-cresol from Trp by rumen microbes. Starch, d-glucose, salinomycin and monensin inhibited the production of skatole and indole from Trp, and skatole from indoleacetic acid by rumen bacteria. Received August 2, 2001 Accepted June 21, 2002 Published online November 14, 2002 Acknowledgements The authors are extremely grateful to Dr. H. Ogawa, Professor, the University of Tokyo and Dr. T. Hasegawa, Associate Professor, Miyazaki University, for inserting a permanent fistula in goats. The present study was financially supported by research grants from Kyowa Hakko Kogyo Co. Ltd. and Daiichi Seiyaku Co., Japan. Nazimuddin Mohammed thanks the Ministry of Education, Science, Sports and Culture of Japan (Monbusho) for the award of a research studentship from 1996. Authors' address: Dr. Nazimuddin Mohammed, Laboratory of Agricultural Production Technology, Faculty of Agriculture, Field Science Center, Tokyo University of Agriculture and Technology, Saiwai-cho 3-5-8, Fuchu-shi, Tokyo 183-8509, Japan     

Synthesis of phenylalanine and production of other related compounds from phenylpyruvic acid and phenylacetic acid by ruminal bacteria,protozoa, and their mixture in vitro

Phenylalanine (Phe) synthesis and the production of other related compounds by mixed ruminal bacteria (B), protozoa (P), and a combination of the two mixture (BP) in an in vitro system were quantitatively investigated using phenylpyruvic acid (PPY) and phenylacetic acid (PAA) as substrates. Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrated mixture twice a day. Microbial suspensions were anaerobically incubated at 39 degrees C for 12 h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analysed by HPLC. A large quantity of Phe was produced from both PPY and PAA not only in B but also in P. In B suspensions, free Phe also accumulated in the medium only when PPY was used as a substrate. The ability of B to synthesize Phe from both PPY and PAA (expressed as unit 'per microbial nitrogen') was 5.1 and 24.8% higher than P, respectively. Phe production from PPY in B and P was 43.5 and 55.2% higher than that from PAA. Large amounts of PAA (17-27%) were produced from PPY in all microbial suspension and production amounts were similar in B and P. Small amounts of benzoic acid (BZA) were produced from PPY and PAA in B, P, and BP, and higher BZA production was observed in P as compared to B. Phenylpropionic acid (PPR) was produced in B from both PPY and PAA, but not in P or BP. A trace amount of phenyllactic acid (PLA) was detected only from PPY in B. Higher concentrations of an unknown compound from PPY and PAA were found to be accumulated in the body protein of B and also in the medium of P, and production of the compound from both PPY and PAA was also higher in B than P.     

Effects of salinomycin and vitamin B(6) on the in vitro synthesis of lysine from the stereoisomers of 2,6-diaminopimelic acid by mixed rumen protozoa,bacteria, and their mixture

An in vitro study was conducted to examine the effects of salinomycin (SL) and vitamin B(6) (pyridoxine hydrochloride) (B(6)) on the production of lysine from the three stereoisomers of 2,6-diaminopimelic acid (DAP-SI) by mixed rumen protozoa (P), mixed rumen bacteria (B), and their mixture (PB). P, B, and PB were isolated from the rumen of goats given a concentrate mixture and lucerne cubes, separately incubated for 12 h with and without DAP-SI (5 mM) as a substrate and SL (5 &mgr;g/ml) and/or B(6) (10 &mgr;g/ml) as additives. In P suspensions, SL and B(6) reduced the amount of DAP-SI by 2.1 times (p<0.001, where p is probability) and 19.9% (p<0.05), respectively, and also increased the production of lysine by 2.4 times (p<0.001) and 26.8% (p <0.05), respectively, during 12 h incubation. In B suspensions, the reductions of DAP-SI with a single addition of SL or B(6) were 8.5% (p<0.001) and 2.7%, respectively, and lysine production increased by 54.3 and 32.9% (p<0.001), respectively, during 12 h incubation. In PB suspensions, the reductions of DAP-SI were 21.9 and 11.7% (p<0.001) with a single addition of SL or B(6), respectively, and the production of lysine increased by 81.4 and 39.4% (p<0.001), respectively, during 12 h incubation. When SL and B(6) were added together to the P, B, and PB suspensions, lysine production further increased by 12.3, 21.3, and 12.4% more than the cases of adding SL only during 12 h incubation, respectively. SL and B(6) were demonstrated to enhance the production of lysine from DAP-SI by mixed rumen protozoa, mixed rumen bacteria and their mixture in this study.     

Tryptophan biosynthesis and production of other related compounds from indolepyruvic acid by mixed ruminal bacteria,protozoa, and their mixture in vitro

Tryptophan (Trp) biosynthesis and the production of other related compounds by mixed ruminal bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated by using 1 mM of indole-3-pyruvic acid (IPA) as substrate. Ruminal microorganisms were anaerobically incubated at 39 degrees C for 12 h. Trp and other related compounds in both the supernatants and the microbial hydrolyzates of the incubation were analyzed by HPLC. As a whole, about 334, 440, and 436 &mgr;M of Trp were produced from IPA in 12 h by B, P, and BP suspensions, respectively. In the B suspension, a greater portion of synthesized Trp (242 &mgr;M) from IPA was accumulated as free form in the medium, whereas a large amount of Trp (92 &mgr;M) was incorporated into cell protein in a 12-h incubation. On the other hand, in the P suspension, a large amount of Trp (475 &mgr;M) from IPA was also found as free form in the supernatant in a 12-h incubation. Protozoa did not incorporate Trp into cell protein, but they liberated endogenous Trp (34 &mgr;M) into the medium. The net productions of Trp from IPA were 344.3 and 447.7 &mgr;mol/g of microbial nitrogen in 12 h by B and P suspensions, respectively. The ability of P to synthesize Trp from IPA was about 30% higher than that of B in 12 h. Trp produced from IPA by B, P, and BP suspensions were simultaneously degraded into its related compounds, and among them, indoleacetic acid (IAA) was a major product found in all microbial suspensions. Productions of IAA were 124, 25, and 99 &mgr;M from IPA in 12 h by B, P, and BP suspensions, respectively. The formation of indolelactic acid (ILA) from IPA was observed for the first time in all microbial suspensions, and it was about 84, 24, and 54 &mgr;M in 12 h by B, P, and BP, respectively. Higher IAA and ILA productions were observed in B when compared with P. A small amount of skatole (SKT) (26 &mgr;M) was produced from IPA in B, whereas a sizable amount of SKT (38 &mgr;M) was found in BP after a 12-h incubation. p-Cresol (CRL) was also produced from IPA by both B (43 &mgr;M) and BP (65 &mgr;M) suspensions in 12 h, and this is also the first discovery to show the formation of CRL from IPA by B and BP suspensions. BP suspension was more active to produce both SKT and CRL from IPA, though P suspension has no ability to produce either SKT or CRL from IPA during a 12-h incubation.     

Comparative studies on the metabolism of linoleic acid by rumen bacteria,protozoa, and their mixture in vitro

Linoleic acid was differentially catabolized by the various rumen microbial fractions, such as rumen bacteria (B), protozoa (P), and their mixture (BP). The predominant isomer of conjugated linoleic acids (CLA) synthesized by B, P, and BP from linoleic acid was 9c11t-CLA. The formation of 9c11t-CLA was higher (P < 0.05) in P suspension (53.6 μg/mg microbial nitrogen) compared with B (38.3 μg/mg microbial nitrogen) and BP (28.8 μg/mg microbial nitrogen) suspensions by 12 h of incubation. The second most abundant CLA isomer was 10t12c. The accumulation of 10t12c-CLA in BP suspension was 2.3 times lower (P < 0.05) than that in B suspension (84.8 μg/mg microbial nitrogen) by 12 h of incubation. The accumulation of 10t-18:1 in BP suspension during 6- and 12-h incubation periods were not different (P > 0.05) than that in B suspension (6.8 and 14.0 μg/mg microbial nitrogen, respectively). However, the accumulation of 11t-18:1 in BP suspension at 6- and 12-h incubations were 2.7 and 3.3 times higher (P < 0.05), respectively, than that in B suspension. There were no significant accumulations of 11t-18:1, 10t-18:1, and 18:0 in P suspension throughout the incubation period. It was concluded that B, P, and BP metabolized linoleic acid to different isomers of CLA, whereas B, including BP, was only capable of biohydrogenating the CLA isomers to 18:0 by the reduction of 18:1 isomers. P was incapable of biohydrogenating LA, but its association with B in the BP suspension altered the biohydrogenation of LA significantly compared with B alone.     

Aromatic amino acid biosynthesis and production of related compounds from p-hydroxyphenylpyruvic acid by rumen bacteria,protozoa and their mixture

Summary. Aromatic amino acid biosynthesis and production of related compounds from p-hydroxyphenylpyruvic acid (HPY) by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated. Microbial suspensions prepared from mature, fistulated goats fed Lucerne (Medicago sativa) cubes and a concentrate mixture were anaerobically incubated at 39°C for 12 h. Tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp) and other related compounds in both supernatants and hydrolyzates of all incubations were analyzed by HPLC. Large amounts of Tyr (27.0, 47.0 and 50.8% of disappeared HPY in B, P and BP, respectively) were produced from 1 mM HPY during a 12-h incubation period. The formation of Tyr in P was 1.8 and 1.6 times higher than those in B and BP, respectively. Appreciable amounts of Phe (3–12% of the disappeared HPY) and Trp (2–10% of the disappeared HPY) were also produced from HPY in B, P, and BP. Phe synthesis in B and P was almost similar but Trp synthesis in B was 1.8 times higher than that in P. The biosynthesis of both Phe and Trp from HPY in BP was higher than those in B plus P. A large amount of p-hydroxyphenylacetic acid (about 45% of the disappeared HPY) was produced from HPY in B which was 1.9 times higher than that in P. p-Hydroxybenzoic acid produced from HPY in P was 1.6 times higher than that in B. Considerable amounts of phenylpropionic acid, phenyllactic acid, and phenylpyruvic acid (2–6% of the disappeared HPY) were produced only in B. Received March 21, 2001 Accepted July 4, 2001     

Catabolism of methionine and threonine in vitro by mixed ruminal bacteria and protozoa

Summary. In vitro studies were conducted to examine the metabolism of methionine (Met) and threonine (Thr) using mixed ruminal bacteria (B), mixed ruminal protozoa (P), and a combination of these two (BP). Rumen microorganisms were collected from fistulated goats fed with lucerne cubes (Medicago sativa) and a concentrate mixture twice a day. Microbial suspensions were anaerobically incubated with or without 1 mM each of the substrates at 39°C for 12 h. Met, Thr and their related amino compounds in both the supernatants and microbial hydrolyzates of the incubation were analyzed by HPLC. Met was degraded by 58.7, 22.1, and 67.3% as a whole in B, P, and BP suspensions, respectively, during 12 h incubation. In the case of Thr, these values were 67.3, 33.4, and 76.2% in B, P, and BP, respectively. Met was catabolized by all of the three microbial suspensions to methionine sulfoxide and 2-aminobutyric acid. Catabolism of Thr by B and BP resulted in the production of glycine and 2-aminobutyric acid, while P produced only 2-aminobutyric acid. From these results, the existence of diverse catabolic routes of Met and Thr in rumen microorganisms was indicated. Received August 2, 2000 Accepted February 27, 2001     

T-2 toxin metabolism by ruminal bacteria and its effect on their growth

The effect of T-2 toxin on the growth rates of different bacteria was used as a measure of its toxicity. Toxin levels of 10 micrograms/ml did not decrease the growth rate of Selenomonas ruminantium and Anaerovibrio lipolytica, whereas the growth rate of Butyrivibrio fibrisolvens was uninhibited at toxin levels as high as 1 mg/ml. There was, however, a noticeable increase in the growth rate of B. fibrisolvens CE46 and CE51 and S. ruminantium in the presence of low concentrations (10 micrograms/ml) of T-2 toxin, which may indicate the assimilation of the toxin as an energy source by these bacteria. Three tributyrin-hydrolyzing bacterial isolates did not grow at all in the presence of T-2 toxin (10 micrograms/ml). The growth rate of a fourth tributyrin-hydrolyzing bacterial isolate was unaffected. B. fibrisolvens CE51 degraded T-2 toxin to HT-2 toxin (22%), T-2 triol (3%), and neosolaniol (10%), whereas A. lipolytica and S. ruminantium degraded the toxin to HT-2 toxin (22 and 18%, respectively) and T-2 triol (7 and 10%, respectively) only. These results have been explained in terms of the presence of two different toxin-hydrolyzing enzyme systems. Studies with B. fibrisolvens showed the presence of a T-2 toxin-degrading enzyme fraction in a bacterial membrane preparation. This fraction had an approximate molecular weight of 65,000 and showed esterase activity (395.6 mumol of p-nitrophenol formed per min per mg of protein with p-nitrophenylacetate as the substrate.     

Studies on the possibility of histidine biosynthesis from histidinol, imidazolepyruvic acid, imidazoleacetic acid, and imidazolelactic acid by mixed ruminal bacteria, protozoa, and their mixture in vitro

The possibility of histidine (His) synthesis using a main biosynthetic pathway involving histidinol (HDL) and also the recycling capability of imidazolic compounds such as imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) to produce His were investigated using mixed ruminal bacteria (B), protozoa (P), and a mixture of both (BP) in an in vitro system. Rumen microorganisms were anaerobically incubated at 39 degrees C for 18 h with or without each substrate (2 mM) mentioned. His and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by high-performance liquid chromatography. B, P, and BP suspensions failed to show His synthesizing ability when incubated with HDL. His was synthesized from ImPA by B, P, and BP. Expressed in units "per gram of microbial nitrogen (MN)", ImPA disappearance was greatest in B (72.7 micromol/g MN per hour), followed by BP (33.13 micromol/g MN per hour) and then P (18.6 micromol/g MN per hour) for the 18-h incubation period. The production of His from ImPA in B (240.0, 275.9, and 261.2 micromol/g MN in 6, 12, and 18 h incubation, respectively) was about 3.5 times higher than that in P (67.3, 83.8, and 72.7 micromol/g MN in 6, 12, and 18 h incubation, respectively). Other metabolites produced from ImPA were ImLA, ImAA, histamine (HTM), and urocanic acid (URA), found in all microbial suspensions. ImLA as a substrate remained without diminution in all microbial suspensions. Although ImAA was found to be degraded to a small extent (3.4-6.3%) only after 18 h incubation, neither His nor other metabolites were detected on the chromatograms. These results have been demonstrated for the first time in rumen microorganisms and suggest that His may be an essential amino acid for rumen microorganisms.     

In vitro metabolism of 2,2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal protozoa and bacteria

Bacillus megaterium GW1 and Escherichia coli W7-M5 were specifically radiolabeled with 2,2'-diamino[G-3H]pimelic acid [( 3H]DAP) as models of gram-positive and gram-negative bacteria, respectively. These radiolabeled bacterial mutants were incubated alone (control) and with mixed ruminal bacteria or protozoa, and the metabolic processes, rates, and patterns of radiolabeled products released from them were studied. Control incubations revealed an inherent difference between the two substrates; gram-positive supernatants consistently contained 5% radioactivity, whereas even at 0 h, those from the gram-negative mutant released 22%. Incubations with ruminal microorganisms showed that the two mutants were metabolized differently and that protozoa were the major effectors of their metabolism. Protozoa exhibited differential rates of engulfment (150 B. megaterium GW1 and 4,290 E. coli W7-M5 organisms per protozoan per h), and they extensively degraded [3H]DAP-labeled B. megaterium GW1 at rates up to nine times greater than those of ruminal bacteria. By contrast, [3H]DAP-labeled E. coli W7-M5 degradation by either ruminal bacteria or ruminal protozoa was more limited. These fundamental differences in the metabolism of the two mutants, especially by ruminal protozoa, were reflected in the patterns and rates of radiolabeled metabolites produced; many were rapidly released from [3H]DAP-labeled B. megaterium GW1, whereas few were slowly released from [3H]DAP-labeled E. coli W7-M5. Most radiolabeled products derived from [3H]DAP-labeled B. megaterium GW1 were peptides of bacterial peptidoglycan origin. The ruminal metabolism of DAP-containing gram-positive and gram-negative bacteria, even with the same peptidoglycan chemotype, is thus likely to be profoundly different.(ABSTRACT TRUNCATED AT 250 WORDS)     

T-2 toxin metabolism by ruminal bacteria and its effect on their growth.

The effect of T-2 toxin on the growth rates of different bacteria was used as a measure of its toxicity. Toxin levels of 10 micrograms/ml did not decrease the growth rate of Selenomonas ruminantium and Anaerovibrio lipolytica, whereas the growth rate of Butyrivibrio fibrisolvens was uninhibited at toxin levels as high as 1 mg/ml. There was, however, a noticeable increase in the growth rate of B. fibrisolvens CE46 and CE51 and S. ruminantium in the presence of low concentrations (10 micrograms/ml) of T-2 toxin, which may indicate the assimilation of the toxin as an energy source by these bacteria. Three tributyrin-hydrolyzing bacterial isolates did not grow at all in the presence of T-2 toxin (10 micrograms/ml). The growth rate of a fourth tributyrin-hydrolyzing bacterial isolate was unaffected. B. fibrisolvens CE51 degraded T-2 toxin to HT-2 toxin (22%), T-2 triol (3%), and neosolaniol (10%), whereas A. lipolytica and S. ruminantium degraded the toxin to HT-2 toxin (22 and 18%, respectively) and T-2 triol (7 and 10%, respectively) only. These results have been explained in terms of the presence of two different toxin-hydrolyzing enzyme systems. Studies with B. fibrisolvens showed the presence of a T-2 toxin-degrading enzyme fraction in a bacterial membrane preparation. This fraction had an approximate molecular weight of 65,000 and showed esterase activity (395.6 mumol of p-nitrophenol formed per min per mg of protein with p-nitrophenylacetate as the substrate.     

In vitro metabolism of 2,2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal protozoa and bacteria.

Bacillus megaterium GW1 and Escherichia coli W7-M5 were specifically radiolabeled with 2,2'-diamino[G-3H]pimelic acid [( 3H]DAP) as models of gram-positive and gram-negative bacteria, respectively. These radiolabeled bacterial mutants were incubated alone (control) and with mixed ruminal bacteria or protozoa, and the metabolic processes, rates, and patterns of radiolabeled products released from them were studied. Control incubations revealed an inherent difference between the two substrates; gram-positive supernatants consistently contained 5% radioactivity, whereas even at 0 h, those from the gram-negative mutant released 22%. Incubations with ruminal microorganisms showed that the two mutants were metabolized differently and that protozoa were the major effectors of their metabolism. Protozoa exhibited differential rates of engulfment (150 B. megaterium GW1 and 4,290 E. coli W7-M5 organisms per protozoan per h), and they extensively degraded [3H]DAP-labeled B. megaterium GW1 at rates up to nine times greater than those of ruminal bacteria. By contrast, [3H]DAP-labeled E. coli W7-M5 degradation by either ruminal bacteria or ruminal protozoa was more limited. These fundamental differences in the metabolism of the two mutants, especially by ruminal protozoa, were reflected in the patterns and rates of radiolabeled metabolites produced; many were rapidly released from [3H]DAP-labeled B. megaterium GW1, whereas few were slowly released from [3H]DAP-labeled E. coli W7-M5. Most radiolabeled products derived from [3H]DAP-labeled B. megaterium GW1 were peptides of bacterial peptidoglycan origin. The ruminal metabolism of DAP-containing gram-positive and gram-negative bacteria, even with the same peptidoglycan chemotype, is thus likely to be profoundly different.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of plant secondary compounds on in vitro methane,ammonia production and ruminal protozoa population

Aims

The objective of this study was to evaluate the potential of secondary plant metabolites from 38 sources to serve as antimethanogenic additives in ruminant diets. The effect of leaf tannins from these different plant sources on rumen fermentation, protozoal populations and methanogenesis was also studied.

Methods and Results

Samples (200 mg dry matter, DM) were incubated without and with polyethylene glycol (PEG)‐6000 (400 mg DM) as a tannin binder during 24‐h incubation in the in vitro Hohenheim gas system. In the leaf samples, total phenol (g kg^?1 DM) was maximum in Pimenta officinalis (312) followed by Oenothera lamarckiana (185) and Lawsonia inermis (105). Of the 38 samples, condensed tannins exceeded 4·0 g kg^?1 in only Alpinia galanga (7·50), Cinnamomum verum (4·58), Pelargonium graveolens (18·7) and Pimenta officinalis (23·2) and were not detected in seven samples. When the bioactivity of the leaf samples was assessed using the tannin bioassay, the percentage increase in the amount of gas produced during incubation of samples with the tannin‐binding agent PEG‐6000 over the amount produced during incubation without the tannin binder ranged from nil (zero) to 367%, with the highest being recorded with A. galanga leaves. The ratio of methane reduction per ml of total gas reduction was maximum with Rauvolfia serpentina (131·8) leaves, followed by Indigofera tinctoria (16·8) and Withania somnifera (10·2) leaves. Total and differential protozoal counts increased with added PEG in twenty‐two samples, maximum being in Pimenta officinalis. Increased accumulation of total volatile fatty acids during incubation with added PEG‐6000 was recorded, and the values ranged from zero to 61%. However, the increase was significant in only 11 of the 38 tannin sources tested indicating noninterference of tannin on in vitro fermentation of carbohydrates by the majority of samples tested. Conversely, in 26 of 38 plant sources, the leaf tannins reduced N‐digestibility as evidenced by increased accumulation of NH₃‐N with added PEG.

Conclusions

Our study unequivocally demonstrated that plants containing secondary metabolites such as Rauvolfia serpentine, Indigofera tinctoria and Withania somnifera have great potential to suppress methanogenesis with minimal adverse effect of feedstuff fermentation.

Significance and Impact of the Study

It was established that methanogenesis was not essentially related to the density of protozoa population in vitro. The tannins contained in these plants could be of interest in the development of new additives in ruminant nutrition.     

Transport and metabolism of vitamin B6 in lactic acid bacteria.

Streptococcus faecalis 8043 concentrates extracellular [3H]pyridoxal or [3H]pyridoxamine primarily as the corresponding 5'-phosphates. Accumulation of pyridoxamine requires an exogenous energy source and is inhibited by glycolysis inhibitors. A membrane potential is not required for transport of pyridoxamine, and an artificially generated potential does not drive uptake in this organism. Based on this and other evidence, it is concluded that S. faecalis accumulates pyridoxamine by facilitated diffusion in conjunction with trapping by pyridoxal kinase. Pyridoxamine-P is not concentrated, but equilibrates with that provided externally. Lactobacillus casei 7469 concentrates radioactivity only from pyridoxal, which appears internally as pyridoxal-P, suggesting that it too absorbs the vitamin by facilitated diffusion plus trapping. The specificity of the growth requirement of S. faecalis and L. casei for vitamin B6 parallels the specificity of the transport systems for this vitamin in these organisms. Lactobacillus delbrueckii 7469, however, which specifically requires pyridoxamine-P or pyridoxal-P for growth, accumulates both these compounds and pyridoxine-P from the medium, apparently by active transport, but not pyridoxine, pyridoxamine, or pyridoxal. While pyridoxal-P and pyridoxamine-P are interconvertible in this organism, pyridoxine-P is not further metabolized, thus accounting for the specificity of the growth requirement. These and previous results show (a) that different organisms may employ quite different transport machinery in utilization of a given external nutrient, and (b) that the specificity of the growth requirement for a given form of a vitamin frequently arises from the specificity of transport, but that internal metabolism of the compounds also plays a significant role in some organisms.     

Fermentation of plant cell walls by ruminal bacteria, protozoa and fungi and their interaction with fibre particle size

The objective of the experiment was to evaluate the contribution of various ruminal microbial groups to the fermentation of cell walls of corn stover with different particle sizes based on ruminal gas production in vitro. Physical, chemical, and antibiotical methods were used to differentiate groups of bacteria, protozoa and fungi in rumen fluid, offering following rumen microbial groups: whole rumen fluid (WRF), bacterial (B), protozoal (P), fungal (F), bacterial plus protozoal (B + P), bacterial plus fungal (B + F), protozoal plus fungal (P + F), and negative control (CON). Cell walls from corn stover were ground and ball milled to produce two different particle sizes. The results showed that digestion of the cell walls was undertaken by the interaction among ruminal bacteria, protozoa and fungi, and such co-actions seemed to fail alternation by one of three microbial groups or any combinations. However, B + P group showed a significant contribution to the degradation of milled cell walls, and B + F group revealed a great synergy effect on the ground cell walls degradation. Particle size of cell walls also had a considerable influence on their fermentation extent instead of the fermentative patterns by various rumen microbial groups.     

Fermentation of plant cell walls by ruminal bacteria,protozoa and fungi and their interaction with fibre particle size

Abstract

The objective of the experiment was to evaluate the contribution of various ruminal microbial groups to the fermentation of cell walls of corn stover with different particle sizes based on ruminal gas production in vitro. Physical, chemical, and antibiotical methods were used to differentiate groups of bacteria, protozoa and fungi in rumen fluid, offering following rumen microbial groups: whole rumen fluid (WRF), bacterial (B), protozoal (P), fungal (F), bacterial plus protozoal (B + P), bacterial plus fungal (B + F), protozoal plus fungal (P + F), and negative control (CON). Cell walls from corn stover were ground and ball milled to produce two different particle sizes. The results showed that digestion of the cell walls was undertaken by the interaction among ruminal bacteria, protozoa and fungi, and such co-actions seemed to fail alternation by one of three microbial groups or any combinations. However, B + P group showed a significant contribution to the degradation of milled cell walls, and B + F group revealed a great synergy effect on the ground cell walls degradation. Particle size of cell walls also had a considerable influence on their fermentation extent instead of the fermentative patterns by various rumen microbial groups.     

家蚕体内维生素B6的存在形态和转换代谢

采用不含桑叶粉末、以去维生素牛乳酪蛋白为蛋白源的准合成饲料饲育家蚕Bombyx mori幼虫,探讨了家蚕体内维生素B（VB₆）化合物的存在形态和转换代谢途经。随饲料中盐酸吡哆醇（PN-HCl）添加量的增加,幼虫体内吡哆醇(PN)含量相应变化,其次是吡哆醛(PL);而辅酶型磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)含量存在稳定性。饲料中的吡哆醇以单纯扩散的形式进入体液;体液中的吡哆醇被各种组织吸收后,在各自的吡哆醛激酶和PNP/磷酸吡哆胺氧化酶的作用下,转变成辅酶型磷酸吡哆醛。家蚕不同于哺乳动物,没有特定的辅酶型磷酸吡哆醛形成组织和辅酶型磷酸吡哆醛的转送机制。同时家蚕体内缺乏具储存VB₆功能的辅酶型磷酸吡哆醛结合蛋白,推测这是用缺乏VB₆的饲料饲育各龄起蚕,幼虫当龄死亡的主要原因。     

Evolution of vitamin B6 (pyridoxine) metabolism by gain and loss of genes

Vitamin B(6) (VB6) functions as a cofactor of many diverse enzymes in amino acid metabolism. Three metabolic pathways for pyridoxal 5'-phosphate (PLP; the active form of VB6) are known: the de novo pathway, the salvage pathway, and the fungal type pathway. Most unicellular organisms and plants biosynthesize VB6 using one or two of these three biosynthetic pathways. However, animals such as insects and mammals do not possess any of the pathways and, thus, need to intake VB6 in their diet to survive. It is conceivable that breakdowns of these pathways occurred in the evolutionary lineages of insects and mammals, and one of the major reasons for this would be the loss of pertinent genes. We studied the evolution of VB6 biosynthesis from the view of the gain and loss of 10 pertinent genes in 122 species whose genome sequences were completely determined. The results revealed that each gene in the pathways was lost more than once in the entire evolutionary lineages of the 122 species. We also found the following three points regarding the evolution of PLP biosynthesis: (1) the breakdown of the PLP biosynthetic pathways occurred independently at least three times in animal lineages, (2) the de novo pathway was formed by the generation of pdxB in gamma-proteobacteria, and (3) the order of the gene loss in VB6 metabolism was conserved among different evolutionary lineages. These results suggest that the evolution of VB6 metabolism was subject to gains and frequent losses of related genes in the 122 species examined. This dynamic nature of the evolutionary changes must have been responsible for the breakdowns of the pathways, resulting in profound differentiation of heterotrophy among the species.     

Effect of monensin, fish oil or their combination on in vitro fermentation and conjugated linoleic acid (CLA) production by ruminal bacteria

An in vitro study was conducted to examine the effect of adding monensin, fish oil, or their combination on rumen fermentation and conjugated linoleic acid (CLA) production by mixed ruminal bacteria when incubated with safflower oil. Concentrate (1 g/100 ml) with safflower oil (0.2 g/100 ml) was added to a mixed solution (600 ml) of strained rumen fluid and buffer (control). Monensin (10 ppm), fish oil (0.02 g/100 ml), or monensin plus fish oil was also added into control mixture. All the culture solutions prepared were incubated anaerobically at 39 °C for 12 h. A higher pH and ammonia concentration were observed from the culture solution containing monensin at 12 h of incubation than those from the control or the culture containing fish oil. Monensin increased (P < 0.007) the C₃ content over all the collection times of culture solution while reducing the C₄ content at 6 h (P < 0.018) and 12 h (P < 0.001) of incubations. Supplementation of monensin, fish oil or their combination changed the content of C₁₈-fatty acids of ruminal culture. Monensin alone reduced (P < 0.021) the content of cis-9, trans-11 CLA compared to fish oil at all sampling times, but increased (P < 0.041) the trans-10, cis-12 CLA production compared to fish oil addition and the control which were similar at incubation for 12 h. The combination of monensin and fish oil increased the content of cis-9, trans-11 CLA (P < 0.023) and transvaccenic acid (TVA, P < 0.018) significantly compared to the control or monensin alone at incubation for 12 h.