Mixed Ruminal Bacteria Research Articles

Enzymatic Study of Linoleic and Alpha-Linolenic Acids Biohydrogenation by Chloramphenicol-Treated Mixed Rumen Bacterial Species.

In the rumen, dietary polyunsaturated fatty acids (PUFA) are reduced by a multistage reaction called biohydrogenation (BH). BH leads to a high proportion of saturated fat in ruminant products, but also products some potential bioactive intermediates like conjugated linoleic and linolenic acids. BH is composed of two kinds of reactions: first an isomerization of PUFA followed by reductions (two for linoleic acid, C18:2n-6; three for α-linolenic acid, C18:3n-3). There is little knowledge about BH enzymes as BH bacterial species are the subject of a lot of studies. Nevertheless, both aspects must be explored to control BH and enhance the fatty acids profile of ruminant products. In the present study, an alternative approach was developed to study the enzymes produced in vivo by mixed ruminal bacteria, using inactivation of bacteria by chloramphenicol, an inhibitor of protein synthesis in prokaryotes, before in vitro incubation. To study C18:2n-6 and C18:3n-3 BH several experiments were used: (1) with different incubation durations (0 to 3) to estimate average rates and efficiencies of all BH reactions, and intermediates production; and (2) with different initial quantities of PUFA (0.25 to 2 mg) to estimate Michaelis–Menten enzymatic parameters, Km and Vmax. A last experiment explored the effect of pH buffer and donor cow diet on C18:2n-6 isomerization pathways. Concerning C18:2n-6 BH, this study confirmed the high saturability of its isomerization, the inhibition of both trans11 and trans10 pathways by a low pH, and the last reduction to stearic acid as the limiting-step. Concerning C18:3n-3, its BH was faster than C18:2n-6, in particular its isomerization (Vmax = 3.4 vs. 0.6 mM/h, respectively), and the limiting-step was the second reduction to t11-C18:1. Besides, our mixed isomerases had a higher affinity for C18:2n-6 than for C18:3n-3 (Km = 2.0 × 10-3 vs. 4.3 × 10-3 M, respectively), but due to their high saturability by C18:2n-6, they had a lower efficiency to isomerize C18:2n-6 than C18:3n-3. Chloramphenicol-treated ruminal fluid would be a meaningful method to study the BH enzymes activities.

Redox mediators modify end product distribution in biomass fermentations by mixed ruminal microbes in vitro.

The fermentation system of mixed ruminal bacteria is capable of generating large amounts of short-chain volatile fatty acids (VFA) via the carboxylate platform in vitro. These VFAs are subject to elongation to larger, more energy-dense products through reverse β-oxidation, and the resulting products are useful as precursors for liquid fuels production. This study examined the effect of several redox mediators (neutral red, methyl viologen, safranin O, tannic acid) as alternative electron carriers for mixed ruminal bacteria during the fermentation of biomass (ground switchgrass not subjected to other pretreatments) and their potential to enhance elongation of end-products to medium-chain VFAs with no additional run-time. Neutral red (1 mM) in particular facilitated chain elongation, increasing average VFA chain length from 2.42 to 2.97 carbon atoms per molecule, while simultaneously inhibiting methane accumulation by over half yet maintaining total C in end products. The ability of redox dyes to act as alternative electron carriers suggests that ruminal fermentation is inherently manipulable toward retaining a higher fraction of substrate energy in the form of VFA.

Addition of potassium carbonate to continuous cultures of mixed ruminal bacteria shifts volatile fatty acids and daily production of biohydrogenation intermediates

A recent study reported a 0.4 percentage unit increase in milk fat of lactating dairy cattle when dietary K was increased from 1.2 to 2% with potassium carbonate. Because milk fat yield has been associated with ruminal production of certain conjugated linoleic acid (CLA) isomers, 2 studies were conducted to determine if increasing potassium carbonate in the rumen would alter patterns of fermentation and biohydrogenation. In experiment 1, 5 dual-flow continuous fermenters were injected just before each feeding with a 10% (wt/wt) stock potassium carbonate solution to provide the equivalent of 1.1 (K1), 2.2 (K2), and 3.3 (K3) % of diet dry matter (DM) as added K. One of the remaining fermenters received no K (K0) and the last fermenter (NaOH) was injected with adequate NaOH stock solution (10%, wt/wt) to match the pH observed for the K3 treatment. For experiment 2, 6 dual-flow continuous fermenters were used to evaluate 6 treatments arranged in a 2×3 factorial to examine 2 levels of soybean oil (0 and 3.64% of diet DM) and added K at 0, 1.6, and 3.3% of diet DM. In both experiments, fermenters were fed 55 to 57g of DM/d of a typical dairy diet consisting of 1:1 forage (10% alfalfa hay and 90% corn silage) to concentrate mix in 2 equal portions at 0800 and 1630h, and fed the respective diets for 10-d periods. Potassium carbonate addition increased pH in both experiments. Acetate:propionate ratio and pH in experiment 1 increased linearly for K0 to K3. Acetate:propionate ratio was lower for NaOH compared with K3 but the pH was the same. The trans-11 18:1 and cis-9,trans-11 CLA production rates (mg/d) increased linearly from K0 to K3, but K3 and NaOH did not differ. Production of trans-10 18:1 decreased and that of trans-10,cis-12 tended to decrease from K0 to K3, but production of trans-10,cis-12 CLA remained high for NaOH. Addition of K to the cultures in experiment 2 decreased propionate and increased acetate and acetate:propionate ratio for the 0% fat diet but not for the 3.64% fat diet. Addition of K increased stearic acid and cis-9,trans-11 CLA but decreased daily production of trans-10 C18:1 and trans-10,cis-12 CLA. The results indicate that increasing potassium carbonate in the diet shifts both fermentation and biohydrogenation pathways toward higher milk fat percentage in dairy cows, but the effects are only explained in part by elevation of pH.

In vitro degradation of lysine by ruminal fluid-based fermentations and by Fusobacterium necrophorum

The objective of these studies was to characterize some factors affecting lysine degradation by mixed ruminal bacteria and by ruminal Fusobacterium necrophorum. Mixed ruminal bacteria degraded lysine, and addition of pure cultures of F. necrophorum did not increase lysine degradation. Addition of acetic or propionic acid strikingly reduced NH3 production from lysine by mixed ruminal bacteria at pH 6, but not at pH 7. Although typical ruminal environments with acidic pH and normal concentrations of volatile fatty acids might inhibit lysine degradation by F. necrophorum, ruminal fluid contained enough bacteria with a lysine-degrading capacity to ferment 50mM lysine in vitro. Of 7 strains of ruminal F. necrophorum tested, all grew on both lactate and lysine as the primary energy source. Both subspecies of ruminal F. necrophorum (necrophorum and funduliforme) used lysine as a primary C and energy source. Lysine and glutamic acid were effectively fermented by F. necrophorum, but alanine and tryptophan were not, and histidine and methionine were fermented only to a minor extent. The end products of lactate fermentation by F. necrophorum were propionate and acetate, and those of lysine degradation were butyrate and acetate. Fermentation of glutamic acid by F. necrophorum yielded acetate and butyrate in a ratio near to 2:1. The minimum inhibitory concentration of tylosin for F. necrophorum was not dependent on whether bacteria were grown with lactate or lysine, but F. necrophorum was more susceptible to monensin when grown on lysine than on lactate. Although F. necrophorum is generally resistant to monensin, the ionophore may reduce lysine degradation by F. necrophorum in the rumen. The essential oil components limonene, at 20 or 100μg/mL, and thymol, at 100μg/mL, inhibited F. necrophorum growth, whereas eugenol, guaiacol, and vanillin had no effect. Our findings may lead to ways to minimize ruminal lysine degradation and thus increase its availability to the animal.

Bovicin HC5 inhibits wasteful amino acid degradation by mixed ruminal bacteriain vitro

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5) that inhibits pure cultures of hyper ammonia-producing bacteria (HAB). Experiments were preformed to see if: (1) S. bovis HC5 cells could inhibit the deamination of amino acids by mixed ruminal bacteria taken directly from a cow, (2) semi-purified bovicin was as effective as S. bovis HC5 cells, and 3) semi-purified and the feed additive monensin were affecting the same types of ammonia-producing ruminal bacteria. Because purified and semi-purified bovicin HC5 was as effective as S. bovis HC5 cells, it appeared that bovicin HC5 was penetrating the cell membranes of HAB before it could be degraded by peptidases and proteinases. Mixed ruminal bacteria that were successively transferred and enriched nine times with trypticase did not become significantly more resistant to either bovicin HC5 (50 AU mL(-1)) or monensin (5 microM), and amplified rDNA restriction analysis indicated that bovicin HC5 and monensin appeared to be selecting against the same types of bacteria.

Effects of essential oils on proteolytic, deaminative and methanogenic activities of mixed ruminal bacteria

The objective of this study was to evaluate in vitro the effects of three essential oils (EO) [cinnamon leaf (250 mg L-1), garlic oil (100 and 250 mg L-1), and juniper berry oil (20 mg L-1)] and two EO compounds (EOC) [anethol (20 mg L-1) and p-cymene (20 mg L-1)] on proteolytic, deaminative and methanogenic activities of mixed ruminal bacteria. Concentrations of total VFA were similar (P > 0.05) among treatments. With the exception of cinnamon and garlic oils, which reduced (P < 0.05) the proportion of propionate, the other EO and EOC had no effect on the proportions of individual VFA, compared with the control. Proteolytic activity of ruminal bacteria was unaffected (P > 0.05) by treatments; however, bacterial deaminative activity and NH3 concentration were increased (P < 0.05) by the addition of EO (except for cinnamon leaf oil and garlic oil at 250 mg L-1) and EOC. Except for anethol, methanogenic activity of ruminal bacteria was reduced (P < 0.05) by EO and EOC, which was reflected by a marked decrease in methane concentration. This study shows that at the concentrations evaluated, anethol, garlic oil (100 mg L-1), juniper berry oil, and p-cymene may not be beneficial to improve efficiency of N utilization in ruminants because they enhance deaminative activity, while cinnamon and garlic oil (250 mg L-1) could be good alternatives to antibiotics because they reduce methanogenic activity of ruminal bacteria. Key words: Essential oil, proteolysis, deamination, methanogenesis, in vitro

Sequence determination of 16s ribosomal RNA for ruminal ammoniabacters of Holstein cows

Protein degradation in the rumen often proceeds at a rate which exceeds the ability of the microbial population to utilize the resulting breakdown products. Bacteria are the most active proteolytic organisms in the rumen, and many species of rumen bacteria are known to be proteolytic (Wallaceet al., 1985). The commonly isolated proteolytic bacteria are also able to break down peptides and amino acids, and it was assumed that they were responsible for the ruminal degradation of intact protein through to ammonia. However, studies comparing the specific activities of ammonia production between mixed ruminal bacteria and the well-known proteolytic bacteria noted that no individual bacterium had an activity which could explain the activity of the mixed ruminal culture (Russellet al., 1988). Subsequently, three gram-positive, monensin-sensitive, ammonia hyper producing (HAP) bacteria were isolated from the rumen. The present study reports the sequence of 16sr RNA for ruminal ammoniabacters of Iranian Holstein cows.

Effects of tannic acid and quebracho tannins on in vitro ruminal fermentation of wheat and corn grain

AbstractCurrent understanding of the effect of moderate amounts of tannins on the utilization of protein contained in forages indicates beneficial effects when fed to ruminants. Nevertheless, there is little knowledge about the effects of tannins on the ruminal hydrolysis of starch‐rich concentrates. Modulation of the hydrolysis of starch may reduce the occurrence of metabolic disorders, such as acidosis, in ruminants consuming high‐grain diets. The effects of commercial tannic acid (hydrolysable tannins) and quebracho tannins (condensed tannins) (50 g kg−1 DM) on the in vitro fermentation of ground wheat and corn grains by mixed ruminal bacteria was examined. The architecture and chemical composition of wheat and corn endosperms might explain differences in fermentation rates, as well as in response to similar concentrations of tannins. Regardless of the source of tannin, microbial fermentation was inhibited in both grains, as demonstrated by a decline in gas production, DM disappearance, volatile fatty acids and ammonia production. However, these effects were more pronounced for wheat than corn grain, mostly during the initial stages of the incubation. Scanning electron microscopy revealed that both sources of tannins inhibited the microbial hydrolysis of the endosperm protein matrix. Tannins did not prevent bacterial attachment to starch granules, but starch hydrolysis was slowed indirectly as a result of a tannin‐mediated reduction in the degradation of the surrounding protein matrix. Tannins are likely to be more effective at modulating the rate of starch digestion in grains that possess a readily degradable protein matrix. Copyright © 2006 Society of Chemical Industry

Factors affecting lysine degradation by ruminal fusobacteria

Fusobacterium necrophorum can readily be enriched from the rumen with lysine, and its deamination rate is very rapid. The addition of F. necrophorum JB2 to mixed ruminal bacteria significantly increased lysine degradation, but only if the ratio of ruminal fluid to basal medium was less than 25%. If more ruminal fluid (pH 6.1) was added, ammonia production decreased by as much as 80%. Clarified, autoclaved ruminal fluid was also inhibitory. When F. necrophorum JB2 was grown in a lysine-limited continuous culture (0.1 h(-1) dilution rate) and pH was decreased using HCl, optical density decreased linearly, and the culture washed out at pH 5.6. Batch cultures of F. necrophorum JB2 deaminated as much lysine at pH 6.1 as at pH 6.6, but only if fermentation acids were not present. Sodium acetate (100 mM) had little effect at pH 6.6, but the same concentration inhibited ammonia production by 80% at pH 6.1. The idea that fermentation acids could prevent the enrichment of fusobacteria in vivo was supported by the observation that dietary lysine supplementation did not enhance the lysine deamination rate of the mixed ruminal bacteria.

Short Communication: Amino Acids Antagonistic to the Amino Acids Inhibitory for Growth Rate of Mixed Ruminal Bacteria

Antagonism of some amino acids (AA) to the inhibitory effects of other AA (Ile, Phe, and Thr) on the growth rate of mixed ruminal bacteria was investigated. In vitro growth rate of the mixed ruminal bacteria was inhibited when the 3 inhibitory AA (1mM each) were each added to individual control treatments in which an ammonium salt was included as a sole N source. The inhibitory effect caused by Ile was relieved by addition of Leu or Val (equimolar to Ile), and no significant inhibition was shown when both Leu and Val were added together with Ile. The growth inhibition caused by Phe was also alleviated by supplementing with Trp, and was completely negated by adding Tyr. The inhibitory effect of Thr, on the other hand, was not affected by addition of Lys or Met (which are synthesized using a common pathway with Thr), but was mitigated by supplementation with Glu, Ser, Val, Ala, or Gln. Among the antagonistic AA, Leu, Val, Trp, Tyr, and Glu were indispensable for the maximum growth rate of the ruminal bacteria under the experimental condition of supplementation of amino-N, the removal of which from a mixture of 20 protein AA caused the growth rate to decline. Removals of Ile along with Leu or Val or both, of Phe along with Trp or Tyr, and of Thr along with Glu recovered the promotion of bacterial growth rate. It was concluded that inhibitions of the bacterial growth rate caused by Ile, Phe, or Thr could be antagonized by some other AA (Leu, Val, Tyr, Trp, or Glu), and the role of these latter AA as relievers of the inhibitory effects could explain why they are indispensable for maximum growth rate of ruminal bacteria.

Effect of Monensin and Fish Oil Supplementation on Biohydrogenation and CLA Production by Rumen Bacteria In vitro When Incubated with Safflower Oil

An in vitro study was conducted to examine the effect of monensin or fish oil addition on bio-hydrogenation of C18- unsaturated fatty acids and CLA production by mixed ruminal bacteria when incubated with safflower oil. Commercially manufactured concentrate (1%, w/v) with safflower oil (0.2%, w/v) were added to mixed solution (600 ml) of strained rumen fluid and McDougalls artificial saliva (control). Monensin (Rumensin  , 10 ppm, w/v, MO), mixed fish oil (0.02%, w/v, absorbed to 0.2 g alfalfa hay, FO) or similar amounts of monensin and fish oil (MO+FO) to MO and FO was also added into the control solution. All the culture solutions prepared were incubated in the culture jar anaerobically at 39°C up to 12 h. Higher pH (p<0.047) and ammonia concentration (p<0.042) were observed from the culture solution containing MO at 12 h incubation than those from the culture solutions of control or FO. The MO supplementation increased (p<0.0001-0.007) propionate proportion of culture solution but reduced butyrate proportion at 6 h (p<0.018) and 12 h (p<0.001) of incubations. Supplementation of MO or MO+FO increased (p<0.001) the proportions of C18:2 .T he MO alone reduced (p<0.022-0.025) the proportion of c9,t11-CLA compared to FO in all incubation times. The FO supplementation increased the proportion of c9,t11-CLA. An additive effect of MO to FO in the production of c9,t11-CLA was observed at 6 h incubation. In vitro supplementation of monensin reduced hydrogenation of C18-UFAs while fish oil supplementation increased the production of CLA. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 2 : 221-225)

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 C 3 content over all the collection times of culture solution while reducing the C 4 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 C 18-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.

Enrichment of fusobacteria from the rumen that can utilize lysine as an energy source for growth

Ruminal lysine degradation is a wasteful process that deprives the animal of an essential amino acid. Mixed ruminal bacteria did not deaminate lysine (50 mM) at a rapid rate, but lysine degrading bacteria could be enriched if Trypticase (5 mg/mL) was also added. Lysine degrading isolates produced acetate, butyrate and ammonia, were non-motile, stained Gram-negative and could also utilize lactate, glucose, maltose or galactose as an energy source for growth. Lactate was converted to acetate and propionate, and 16S rDNA indicated that their closest relatives were Fusobacterium necrophorum. Growing cultures produced ammonia at rates as high as 2400 nmol/mg protein/mL/min. Washed cell suspensions took up 14C lysine (3 μM) at an initial rate of 6 nmol/mg protein/min, and glucose addition did not affect the transport. Cells washed aerobically had the same transport rate as those handled anaerobically, but only if the transport buffer contained sodium. The affinity constant for sodium was 8 mM, and sodium could not be replaced by lithium. Cells treated with the sodium/proton antiporter, monensin (5 μM), did not take up lysine, but a protonophore that inhibited growth (tetrachlorosalicylanilide, 10 μM) had no effect. An artificial membrane potential created by potassium diffusion did not increase the rate of lysine transport, and an Eadie-Hofstee plot indicated the transport rate was directly proportional to the lysine concentration. Decreasing the pH from 6.7 to 5.5 caused an 85% decrease in the rate of lysine transport. The addition of F. necrophorum JB2 (130 μg protein/mL) to mixed ruminal bacteria increased lysine degradation 10-fold, but only if the pH was 6.7 and monensin was not present. Further work will be needed to see if dietary lysine enriches fusobacteria in vivo.

Effects of ionophores, vitamin B 6 and distiller's grains on in vitro tryptophan biosynthesis from indolepyruvic acid, and production of other related compounds by ruminal bacteria and protozoa

Tryptophan (Trp) is an essential amino acid (AA) and it may be limiting to ruminants when feed rations are based on corn (maize). Distiller's grains (DGs) from barley are the dried residue remaining after the starch fraction of corn is fermented using selected yeasts and enzymes to produce alcohol. DG is a rich source of nutrients, because it contains polyphenol, peptides, yeast extracts (nucleic acids and amino acids), citric acid, fermented barley extract and fiber. The ionophore-type feed additives (monensin and salinomycin) modify the movement of monovalent (sodium and potassium) and divalent (calcium) ions across biologic membranes, modify the rumen microflora pattern, decrease acetate and methane production, increase propionate, may improve nitrogen utilization and can increase dry matter digestibility in ruminants. Vitamin B 6 (B 6) acts as a coenzyme in many enzymatic reactions that are involved in the metabolism of AA. An in vitro study was conducted to examine the effects of salinomycin (SL), monensin (MN), B 6 and DG on the production of tryptophan (Trp) and other related compounds from indolepyruvic acid (IPA) by mixed ruminal bacteria (B), protozoa (P) and their mixture (BP). Ruminal microbial suspensions were anaerobically incubated at 39 °C for 12 h, and analyses of Trp and its related metabolites were carried out by HPLC. When 1 mM of IPA was used as a substrate, it was mainly converted to Trp and indoleacetic acid (IAA), resulting in concentrations of 300 and 131, 410 and 28, and 435 and 112 μM in B, P and BP suspensions, respectively. A considerable amount of skatole (SKT) (in B and BP) and indolelactic acid (ILA) (in B, P and BP), and small amount of p-cresol (CRL) (in B and BP) were also produced from IPA. The production of Trp from IPA with SL, MN, B 6 and DG were increased ( P < 0.05) by 18.9–55.3, 9.0–50.4 and 5.1–16.1% in B, P and BP, respectively. The increased Trp production with these additives occurred by the simultaneous inhibition of IAA from IPA. On the other hand, addition of B 6 into B, P and BP enhanced ( P < 0.05) IAA from IPA by 18.9, 26.5 and 5.1%, respectively. Trp produced from IPA with SL was 13.8, 8.8 and 5.0% higher than that with MN in B, P and BP, respectively. Salinomycin and MN completely inhibited the SKT and CRL (in B and BP), and partially inhibited ILA (in B, P and BP) from IPA. On the other hand, B 6 stimulated the production of SKT and CRL in B and BP, and ILA in B, P and BP from IPA. No effects were observed on the production of SKT, CRL and ILA by DG. We conclude that addition of these additives to feeds may increase duodenal Trp supply in ruminants. In addition, SL and MN may also inhibit the production of clinically important Trp metabolites (SKT, CRL and ILA).

A quantitative study on arginine catabolism by mixed ruminal bacteria, protozoa and their mixture in vitro

ABSTRACTThe catabolism of arginine (Arg) by mixed rumen bacteria (B), mixed rumen protozoa (P), and their mixture (BP) was quantitatively investigated in an in vitro system in order to confirm the metabolic pathway of Arg and provide basic information for enzymatic and molecular studies as well as an understanding of the quantitative distribution of metabolites. Rumen microbial suspensions (B, P, and BP) collected from fistulated goats were anaerobically incubated with or without 1 mmol/L Arg at 39°C for 12 h. Arg and other related compounds such as citrulline (Cit), ornithine (Orn), proline (Pro) and 5‐aminovaleric acid (5AV) in both supernatant and hydrolyzates of B, P, and BP suspensions were analyzed by HPLC. The metabolic pathways of Arg in mixed rumen bacteria and mixed rumen protozoa were considered to be as follows: rumen bacteria, Arg → Cit → Orn → Pro → 5AV → VFAs + NH3; rumen protozoa, Arg → Cit → Orn → Pro → 5AV. The disappearance of Arg (1 mmol/L) was approximately 52.9 and 88.2% in B, 33.9 and 55.6% in P, and 52.8 and 85.2% in BP during 6 and 12 h incubations, respectively. When expressed in units of ‘per gram (g) of microbial nitrogen (MN)’, the net degradation rate of Arg in BP (50.3 µmol/g MN/h) was approximately 46% lower than that of B during a 12 h incubation period. The presence of protozoa tended to inhibit the production of Orn from Cit and the production of 5AV from Pro which were thought to be rate‐limiting steps of Arg metabolism in rumen microorganisms. As a result, protozoa appeared to have a saving effect on Arg metabolism, that is, protozoa protected Arg from wasteful exhaustion in the rumen.

PH Affects the In vitro Formation of cis-9, trans-11 CLA and trans-11 Octadecenoic Acid by Ruminal Bacteria When Incubated with Oilseeds

The effect of pH on the fermentation characteristics and the formation of cis-9, trans-11 conjugated linoleic acid (CLA) and trans-11 octadecenoic acid by mixed ruminal bacteria was examined in vitro when incubated with linseed or rapeseed. Concentrate (1%, w/v) with ground linseed (0.6%, w/v) or rapeseed (0.5%, w/v) was added to 600 ml mixed solution of strained rumen fluid with artificial saliva (1:1, v/v), and was incubated anaerobically for 12 h at 39°C. The pH of culture solution was maintained at level close to 4.5, 5.3, 6.1 and 6.9 with 30% H 2 SO 4 or 30% NaOH solution. pH increment resulted in increases of ammonia and total volatile fatty acid (VFA) concentration in culture solutions containing both oilseeds. Fermentation did not proceeded at pH 4.5. Molar proportion of acetate decreased but that ofpropionate increased as pH increased when incubated with oilseeds. While the hydrogenating process was very slow at the pH range of 4.5 to 5.3, rapid hydrogenation was found from the culture solutions of pH 6.1 and 6.9 when incubated with linseed or rapeseed. As pH in culture solution of linseed or rapeseed increases proportions of oleic acid (cis-9 C 1 8 : 1 ) and trans-11 octadecenoic acid increased but those of linoleic acid and linolenic acid decreased. The CLA proportion increased with pH in culture solution containing rapeseed but CLA was mostly not detected from the incubation of linseed.

Factors affecting conjugated linoleic acid and trans-C18:1 fatty acid production by mixed ruminal bacteria.

The objective of this study was to identify environmental factors that influence conjugated linoleic acid (CLA) and trans-C18:1 fatty acid production by mixed ruminal bacteria. Ruminal contents were collected from a 600-kg ruminally fistulated Hereford steer maintained on pasture. Mixed ruminal bacteria were obtained by differential centrifugation under anaerobic conditions and added to a basal medium that contained a commercial emulsified preparation of soybean oil and a mixture of soluble carbohydrates (cellobiose, glucose, maltose, and xylose). Culture samples were collected from batch culture incubations at 0, 2, 4, 6, 8, 12, 24, 26, 28, 30, 32, and 48 h. Continuous culture incubations were conducted at dilution rates of 0.05 and 0.10 h(-1) with extracellular pH values of 5.5 and 6.5, and 0.5 and 1.0 g/L of mixed soluble carbohydrates. Culture samples were obtained from the culture vessel once steady-state conditions had been achieved. In batch culture, trans-C18:1 concentrations increased over time and reached a maximum at 48 h. Little CLA was produced during the first 8 h, but cis-9, trans-11 CLA concentrations remained high between 24 and 30 h. When mixed ruminal bacteria were maintained in continuous culture on 0.5 g/L of mixed soluble carbohydrates, concentrations of trans-C18:1 and cis-9, trans-11 CLA were reduced (P < 0.05) at a dilution rate of 0.05 h(-1) and an extracellular pH of 5.5. Similar effects were also observed when 1.0 g/L of mixed soluble carbohydrates was used. When extracellular pH was lowered to 5.0, neither trans-C18:1 or CLA isomers were detected. In conclusion, our results suggest that culture pH appears to have the most influence on the production of trans-C18:1 and CLA isomers by mixed ruminal bacteria.

The effect of bovicin HC5, a bacteriocin from Streptococcus bovis HC5, on ruminal methane production in vitro.

Methane represents a loss of feed energy to ruminant animals, and nutritionists have sought methods of inhibiting ruminal methane production. When mixed ruminal bacteria (approximately 400 mg protein ml(-1)) from a cow fed timothy hay were incubated in vitro with carbon dioxide and hydrogen (0.5 atm) for less than 8 h, the first-order rate of methane production was 17 micromol ml(-1). Semi-purified bacteriocin from Streptococcus bovis HC5 (bovicin HC5) inhibited methane production, by as much as 50%, and even a low concentration of bovicin HC5 (128 activity units (AU) ml(-1)) caused a significant decrease. Mixed ruminal bacteria that were transferred successively retained their ability to produce methane from carbon dioxide and hydrogen, and the first-order rate of methane production did not decrease. Cultures that were treated with bovicin HC5 (128 AU ml(-1)) gradually lost their ability to produce methane, and methane was not detected after four transfers. These latter results indicated that ruminal methanogens could not adapt and become resistant to bovicin HC5. When the chromosomal DNA was amplified with 16S rDNA primers specific to archaea, digested with restriction enzymes (HhaI and HaeIII) and separated on agarose gels, approximately 12 fragments were observed. DNA from control and treated cultures (third transfer) had the same fragment pattern indicating bovicin HC5 was not selective. Given the perception that the routine use of antibiotics in animal feeds should be avoided, bacteriocins may provide an alternative strategy for decreasing ruminal methane production.

The effect of bovicin HC5, a bacteriocin from Streptococcus bovis HC5, on ruminal methane production in vitro

Methane represents a loss of feed energy to ruminant animals, and nutritionists have sought methods of inhibiting ruminal methane production. When mixed ruminal bacteria (approximately 400 mg protein ml −1) from a cow fed timothy hay were incubated in vitro with carbon dioxide and hydrogen (0.5 atm) for less than 8 h, the first-order rate of methane production was 17 μmol ml −1. Semi-purified bacteriocin from Streptococcus bovis HC5 (bovicin HC5) inhibited methane production, by as much as 50%, and even a low concentration of bovicin HC5 (128 activity units (AU) ml −1) caused a significant decrease. Mixed ruminal bacteria that were transferred successively retained their ability to produce methane from carbon dioxide and hydrogen, and the first-order rate of methane production did not decrease. Cultures that were treated with bovicin HC5 (128 AU ml −1) gradually lost their ability to produce methane, and methane was not detected after four transfers. These latter results indicated that ruminal methanogens could not adapt and become resistant to bovicin HC5. When the chromosomal DNA was amplified with 16S rDNA primers specific to archaea, digested with restriction enzymes ( HhaI and HaeIII) and separated on agarose gels, approximately 12 fragments were observed. DNA from control and treated cultures (third transfer) had the same fragment pattern indicating bovicin HC5 was not selective. Given the perception that the routine use of antibiotics in animal feeds should be avoided, bacteriocins may provide an alternative strategy for decreasing ruminal methane production.

Biosynthesis of arginine from citrulline and related compounds by mixed ruminal bacteria, protozoa and their mixture in vitro

Biosynthesis of arginine from citrulline and related compounds by mixed ruminal bacteria, protozoa and their mixture in vitro