Ruminal microbe of biohydrogenation of trans-vaccenic acid to stearic acid in vitro.
ABSTRACT: Optimization of the unsaturated fatty acid composition of ruminant milk and meat is desirable. Alteration of the milk and fatty acid profile was previously attempted by the management of ruminal microbial biohydrogenation. The aim of this study was to identify the group of ruminal trans-vaccenic acid (trans-11 C18:1, t-VA) hydrogenating bacteria by combining enrichment studies in vitro.The enrichment culture growing on t-VA was obtained by successive transfers in medium containing t-VA. Fatty acids were detected by gas chromatograph and changes in the microbial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands of the enrichment cultures were identified by 16S rRNA gene sequencing.The growth of ruminal t-VA hydrogenating bacteria was monitored through the process of culture transfer according to the accumulation of stearic acid (C18:0, SA) and ratio of the substrate (t-VA) transformed to the product (SA). A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Bacteria corresponding to predominant DGGE bands in t-VA enrichment cultures clustered with t-VA biohydrogenated bacteria within Group B.This study provides more insight into the pathway of biohydrogenation. It also may be important to control the production of t-VA, which has metabolic and physiological benefits, through management of ruminal biohydrogenation bacterium.
Project description:This study aimed to investigate the effects of ruminal infusion of garlic oil (GO) on fermentation dynamics, fatty acid (FA) profile, and abundance of bacteria involved in biohydrogenation in the rumen. Six wethers fitted with ruminal fistula were assigned to two groups for cross-over design with a 14-d interval. Each 30-d experimental period consisted of a 27-d adaptation and a 3-d sample collection. Goats were fed a basal diet without (control) or with GO ruminal infusion (0.8 g/d). Ruminal contents collected before (0 h) and at 2, 4, 6, 8, and 10 h after morning feeding were used for fermentation analysis, and 0 h samples were further used for FA determination and DNA extraction. Garlic oil had no influence on dry matter intakes of concentrate and hay. During ruminal fermentation, GO had no effects on total VFA concentration and individual VFA molar proportions, whereas GO increased the concentrations of ammonia nitrogen and microbial crude protein (p<0.05). Compared with control, GO group took a longer time for total VFA concentration and propionate molar proportion to reach their respective maxima after morning feeding. The ratio of acetate to propionate in control reduced sharply after morning feeding, whereas it remained relatively stable in GO group. Fatty acid analysis showed that GO reduced saturated FA proportion (p<0.05), while increasing the proportions of C18, t11-18:1 (TVA), c9,t11-conjugated linoleic acid (c9,t11-CLA), t10,c12-CLA, and polyunsaturated FA (p<0.05). The values of TVA/(c9,t11-CLA+TVA) and C18:0/(TVA+ C18:0) were reduced by GO (p<0.05). Real-time PCR showed that GO tended to reduce Butyrivibrio proteoclasticus abundance (p = 0.058), whereas GO had no effect on total abundance of the Butyrivibrio group bacteria. A low correlation was found between B. proteoclasticus abundance and C18:0/(TVA+C18:0) (p = 0.910). The changes of fermentation over time suggested a role of GO in delaying the fermentation process and maintaining a relatively modest change of ruminal environment. The inhibitory effects of GO on the final step of biohydrogenation may be related to its antibacterial activity against B. proteoclasticus and other unknown bacteria involved.
Project description:BACKGROUND:Micro-algae could inhibit the complete rumen BH of dietary 18-carbon unsaturated fatty acid (UFAs). This study aimed to examine dose and time responses of algae supplementation on rumen fermentation, biohydrogenation and Butyrivibrio group bacteria in goats. METHODS:Six goats were used in a repeated 3?×?3 Latin square design, and offered a fixed diet. Algae were infused through rumen cannule with 0 (Control), 6.1 (L-Alg), or 18.3 g (H-Alg) per day. Rumen contents were sampled on d 0, 3, 7, 14 and 20. RESULTS:H-Alg reduced total volatile fatty acid concentration and acetate molar proportion (P?<?0.05), and increased propionate molar proportion (P?<?0.05), whereas L-Alg had no effect on rumen fermentation. Changes in proportions of acetate and propionate in H-Alg were obvious from d 7 onwards and reached the largest differences with the control on d 14. Algae induced a dose-dependent decrease in 18:0 and increased trans-18:1 in the ruminal content (P?<?0.05). H-Alg increased the concentrations of t9, t11-18:2 and t11, c15-18:2 (P?<?0.05). L-Alg only seemed to induce a transient change in 18-carbon isomers, while H-Alg induced a rapid elevation, already obvious on d 3, concentrations of these fatty acid rose in some cases again on d 20. Algae had no effect on the abundances of Butyrivibrio spp. and Butyrivibrio proteoclasticus (P?>?0.10), while H-Alg reduced the total bacteria abundance (P?<?0.05). However, this was induced by a significant difference between control and H-Alg on d 14 (-4.43 %). Afterwards, both treatments did not differ as increased variation in the H-Alg repetitions, with in some cases a return of the bacterial abundance to the basal level (d 0). CONCLUSIONS:Changes in rumen fermentation and 18-carbon UFAs metabolism in response to algae were related to the supplementation level, but there was no evidence of shift in ruminal biohydrogenation pathways towards t10-18:1. L-Alg mainly induced a transient effect on rumen biohydrogenation of 18-carbon UFAs, while H-Alg showed an acute inhibition and these effects were not associated with the known hydrogenating bacteria.
Project description: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 ?-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.
Project description:The role of marine lipids as modulators of ruminal biohydrogenation of dietary unsaturated fatty acids may be explained by the effects of their n-3 polyunsaturated fatty acids (PUFA) on the bacterial community. However, the impact of individual PUFA has barely been examined, and it is uncertain which bacteria are truly involved in biohydrogenation. In addition, despite interspecies differences in rumen bacterial composition, we are not aware of any direct comparison of bovine and ovine responses to dietary PUFA. Therefore, rumen fluid from cannulated cattle and sheep were used as inocula to examine in vitro the effect of 20:5n-3 (EPA), 22:5n-3 (DPA), and 22:6n-3 (DHA) on the bacterial community. Amplicon 16?S rRNA sequencing suggested that EPA and DHA had a greater contribution to the action of marine lipids than DPA both in cattle and sheep. Certain effects were exclusive to each ruminant species, which underlines the complexity of rumen microbial responses to dietary fatty acids. Based on changes in bacterial abundance, Barnesiella, Prevotella, Paraprevotella, Hallela, Anaerovorax, Succiniclasticum, Ruminococcus and Ruminobacter may be involved in the ruminal response in biohydrogenation to the addition of marine lipids, but further research is necessary to confirm their actual role in ruminal lipid metabolism.
Project description:Cold-pressed sunflower cake (CPSC), by-product of oil-manufacturing, has high crude fat and linoleic acid concentrations, being a promising supplement to modulate rumen fatty acid (FA) profile. This trial studied CPSC effects on ruminal fermentation, biohydrogenation and the bacterial community in dairy cows. Ten cows were used in a crossover design with two experimental diets and fed during two 63-day periods. The cows were group fed forage ad libitum and the concentrate individually. The concentrates, control and CPSC, were isoenergetic, isoproteic and isofat. The ruminal samples collected at the end of each experimental period were analyzed for short-chain fatty acid, FA and DNA sequencing. CPSC decreased butyrate molar proportion (4%, p = 0.005). CPSC decreased C16:0 (28%, p < 0.001) and increased C18:0 (14%, p < 0.001) and total monounsaturated FA, especially C18:1 trans-11 (13%, p = 0.023). The total purine derivative excretion tended to be greater (5%, p = 0.05) with CPSC, resulting in a 6% greater daily microbial N flow. CPSC did not affect the diversity indices but increased the relative abundances of Treponema and Coprococcus, and decreased Enterococcus, Ruminococcus and Succinivibrio. In conclusion, the changes in ruminal fermentation and the FA profile were not associated with changes in microbial diversity or abundance of dominant populations, however, they might be associated with less abundant genera.
Project description:This experiment aimed to determine the effects of camelina seed (CS) supplementation at different dietary fat levels on ruminal bacterial community composition and how it relates to changes in ruminal fermentation in a dual-flow continuous culture system. Diets were randomly assigned to 8 fermenters (1,200-1,250 mL) in a 2 × 2 factorial arrangement of treatments in a replicated 4 × 4 Latin square with four 10-day experimental periods that consisted of 7 days for diet adaptation and 3 days for sample collection. Treatments were: (1) no CS at 5% ether extract (EE, NCS5); (2) no CS at 8% EE (NCS8); (3) 7.7% CS at 5% EE (CS5); and (4) 17.7% CS at 8% EE (CS8). Megalac was used as a control to adjust EE levels. Diets contained 55% orchardgrass hay and 45% concentrate, and fermenters were equally fed a total of 72 g/day (DM basis) twice daily. The bacterial community was determined by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Sequencing data were analyzed using mothur and statistical analyses were performed in R and SAS. The most abundant phyla across treatments were the Bacteroidetes and Firmicutes, accounting for 49 and 39% of the total sequences, respectively. The bacterial community composition in both liquid and solid fractions of the effluent digesta changed with CS supplementation but not by dietary EE. Including CS in the diets decreased the relative abundances of Ruminococcus spp., Fibrobacter spp., and Butyrivibrio spp. The most abundant genus across treatments, Prevotella, was reduced by high dietary EE levels, while Megasphaera and Succinivibrio were increased by CS supplementation in the liquid fraction. Correlatively, the concentration of acetate was decreased while propionate increased; C18:0 was decreased and polyunsaturated fatty acids, especially C18:2 n-6 and C18:3 n-3, were increased by CS supplementation. Based on the correlation analysis between genera and fermentation end products, this study revealed that CS supplementation could be energetically beneficial to dairy cows by increasing propionate-producing bacteria and suppressing ruminal bacteria associated with biohydrogenation. However, attention should be given to avoid the effects of CS supplementation on suppressing cellulolytic bacteria.
Project description:Background:In ruminants, dietary C18:3n-3 can be lost through biohydrogenation in the rumen; and C18:3n-3 that by-passes the rumen still can be lost through oxidation in muscle, theoretically reducing the deposition of C18:3n-3, the substrate for synthesis of poly-unsaturated fatty acids (n-3 LCPUFA) in muscle. In vitro studies have shown that rumen hydrogenation of C18:3n-3 is reduced by supplementation with palm oil (rich in cis-9 C18:1). In addition, in hepatocytes, studies with neonatal rats have shown that cis-9 C18:1 inhibits the oxidation of C18:3n-3. It therefore seems likely that palm oil could reduce both rumen biohydrogenation of C18:3n-3 and muscle oxidation of C18:3n-3. The present experiment tested whether the addition of palm oil to a linseed oil supplement for goat kids would prevent the losses of C18:3n-3 and thus improve the FA composition in two muscles, Longissimus dorsi and Biceps femoris. To investigate the processes involved, we studied the rumen bacterial communities and measured the mRNA expression of genes related to lipid metabolism in Longissimus dorsi. Sixty 4-month-old castrated male Albas white cashmere kids were randomly allocated among three dietary treatments. All three diets contained the same ingredients in the same proportions, but differed in their fat additives: palm oil (PMO), linseed oil (LSO) or mixed oil (MIX; 2 parts linseed oil plus 1 part palm oil on a weight basis). Results:Compared with the LSO diet, the MIX diet decreased the relative abuandance of Pseudobutyrivibrio, a bacterial species that is positively related to the proportional loss rate of dietary C18:3n-3 and that has been reported to generate the ATP required for biohydrogenation (reflecting a decrease in the abundance of rumen bacteria that hydrogenate C18:3n-3 in MIX kids). In muscle, the MIX diet increased concentrations of C18:3n-3, C20:5n-3, C22:6n-3, and n-3 LCPUFA, and thus decreased the n-6/n-3 ratio; decreased the mRNA expression of CPT1? (a gene associated with fatty acid oxidation) and increased the mRNA expression of FADS1 and FADS2 (genes associated with n-3 LCPUFA synthesis), compared with the LSO diet. Interestingly, compared to Longissimus dorsi, Biceps femoris had greater concentrations of PUFA, greater ratios of unsaturated fatty acids/saturated fatty acids (U/S), and poly-unsaturated fatty acids/saturated fatty acids (P/S), but a lesser concentration of saturated fatty acids (SFA). Conclusions:In cashmere goat kids, a combination of linseed and palm oils in the diet increases the muscle concentration of n-3 LCPUFA, apparently by decreasing the relative abundance of rumen bacteria that are positively related to the proportional loss rate of dietary C18:3n-3, by inhibiting mRNA expression of genes related to C18:3n-3 oxidation in muscle, and by up-regulating mRNA expression of genes related to n-3 LCPUFA synthesis in muscle, especially in Longissimus dorsi.
Project description:Optimization of the fatty acid composition of ruminant milk and meat is desirable. Dietary supplementation of algae was previously shown to inhibit rumen biohydrogenation, resulting in an altered milk fatty acid profile. Bacteria involved in biohydrogenation belong to the Butyrivibrio group. This study was aimed at relating accumulation of biohydrogenation intermediates with shifts in Butyrivibrio spp. in the rumen of dairy cows. Therefore, an experiment was performed with three rumen-fistulated dairy cows receiving a concentrate containing algae (9.35 g/kg total dry matter [DM] intake) for 20 days. Supplementation of the diet with algae inhibited biohydrogenation of C(18:2) omega 6 (n-6) and C(18:3) n-3, resulting in increased concentrations of biohydrogenation intermediates, whereas C(18:0) decreased. Addition of algae increased ruminal C(18:1) trans fatty acid concentrations, mainly due to 6- and 20-fold increases in C(18:1) trans 11 (t11) and C(18:1) t10. The number of ciliates (5.37 log copies/g rumen digesta) and the composition of the ciliate community were unaffected by dietary algae. In contrast, supplementation of the diet with algae changed the composition of the bacterial community. Primers for the Butyrivibrio group, including the genera Butyrivibrio and Pseudobutyrivibrio, were specifically designed. Denaturing gradient gel electrophoresis showed community changes upon addition of algae without affecting the total amount of Butyrivibrio bacteria (7.06 log copies/g rumen DM). Clone libraries showed that algae affected noncultivated species, which cluster taxonomically between the genera Butyrivibrio and Pseudobutyrivibrio and might play a role in biohydrogenation. In addition, 20% of the clones from a randomly selected rumen sample were related to the C(18:0)-producing branch, although the associated C(18:0) concentration decreased through supplementation of the diet with algae.
Project description:The objective of this study was to evaluate the effect of condensed tannin inclusion (0, 10, 30 or 50 g/kg of dry matter (DM) total) from Acacia mearnsii extract on the fatty acid profile, physicochemical quality and sensorial analysis of salted and sun-dried meat from young Nellore bulls. The inclusion of condensed tannin extract in the young bulls' diets promoted a quadratic reduction in the lipid content. There was a linear increase in the water retention capacity, cooking weight loss and C18:3n-3 and a linear reduction in collagen, C16:0, C16:1cis-9, C18:1, MUFAs, and ?9-desaturaseC18 in the salted and sun-dried meat from young Nellore bulls supplemented with condensed tannin. The myristic fatty acid (C14:0) and the flavor sensory attribute presented a quadratic increase. The inclusion of condensed tannin extract in the young Nellore bulls' diets did not influence most of the physicochemical characteristics, fatty acids and nutraceutical compounds, including CLA, atherogenicity, thrombogenicity and the h:H index, the tenderness and the global appearance of the salted and sun-dried meat. Condensed tannins reduce ruminal biohydrogenation and improve the PUFA content of salted and sun-dried meat from young Nellore bulls.
Project description:Active plant metabolites (APM) are recognized as modifiers of ruminal microbial fermentation including methanogenesis and biohydrogenation of fatty acids (FA). Coleus amboinicus Lour. leaves (CAL) are rich in several APM, which could serve as ruminal fermentation modulators. A phytochemical analysis showed that CAL contain phenolic acids (10.4 mg·g-1 dry matter [DM]; high in rosmarinic acid), flavonoids (2.6 mg·g-1 DM), diterpenes (2 mg·g-1 DM), and linolenic acid (35.4 g (100 g)-1 FA). This study aimed to investigate the effect of CAL on ruminal methanogenesis and biohydrogenation as well as basic fermentation characteristics and microbial populations. The in vitro experiment was carried out using Hohenheim gas test system with 40 mL of buffered ruminal fluid incubated for 24 h at 39 °C in anaerobic conditions. Approximately 400 mg (DM basis) of total mixed ration (TMR) was used as a control substrate and the CAL substrate was used at doses of 10, 20, 40, and 80 mg DM replacing equal amounts of TMR. Addition of CAL decreased methane production up to 30% linearly as the amount of CAL increased (P < 0.05). In vitro dry matter digestibility and ammonia tended to increase with increasing doses of CAL. Concentration of total volatile fatty acids was not affected by the CAL although there appeared to be a minor positive linear trend; however, acetate, butyrate, and isobutyrate proportion increased quadratically (P < 0.001). CAL tended to linearly increase ?-linolenic acid and conjugated linoleic acid as well as increased stearic acid concentration in buffered ruminal fluid. CAL particularly increased total protozoa and bacterial populations during fermentation, but inhibited methanogens. It is concluded that the CAL may be promising to be used as a feed additive to decrease methanogenesis as well as biohydrogenation of FA in the rumen.