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Effects of saponins, quercetin, eugenol, and cinnamaldehyde on fatty acid biohydrogenation of forage polyunsaturated fatty acids in dual-flow continuous culture fermenters¹

M. Lourenço^*, P. W. Cardozo, S. Calsamiglia and V. Fievez^*,2

^* Laboratory for Animal Production and Animal Product Quality, Department of Animal Production, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium; and Animal Nutrition, Management, and Welfare Research Group, Department de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain

	Abstract

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Four different plant secondary metabolites were screened for their effect on rumen biohydrogenation of forage long-chain fatty acids, using dual-flow continuous culture fermenters. Treatments were as follows: control (no additive), positive control (12 mg/L of monensin), and plant extracts (500 and 1,000 mg/L of triterpene saponin; 250 and 500 mg/L of quercetin; 250 mg/L of eugenol; 500 mg/L of cinnamaldehyde). Monensin increased propionate, decreased acetate and butyrate proportions, and inhibited the complete biohydrogenation of fatty acids resulting in the accumulation of intermediates of the biohydrogenation process (C18:2 trans-11, cis-15 rather than C18:1 trans-11). Cinnamaldehyde decreased total VFA concentration and proportions of odd and branched-chain fatty acids in total fat effluent. Apparent biohydrogenation of C18:2n-6 and C18:3n-3 was also less, and a shift from the major known biohydrogenation pathway to a secondary pathway of C18:2n-6 was observed, as evidenced by an accumulation of C18:1 trans-10 and trans-10, cis-12 CLA. Quercetin (500 mg/L) increased total VFA concentration, but no shifts in the pathways or extent of biohydrogenation were observed. Eugenol resulted in the accumulation of C18:1 trans-15 and C18:1 cis-15, end products of an alternative biohydrogenation pathway of C18:3n-3. Triterpene saponins did not affect the fermentation pattern, the biohydrogenation pathways, or the extent of biohydrogenation. At the doses tested in this study, we could only show a direct relation between changes in the rumen fatty acid metabolism and the presence of cinnamaldehyde but not for eugenol, quercetin, or triterpene saponins.

Key Words: biohydrogenation • cinnamaldehyde • eugenol • flavonoid • in vitro • saponin

	INTRODUCTION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Forage feeding has been shown to increase n-3 PUFA, C18:1 trans-11, and cis-9, trans-11 CLA proportions in ruminant milk and meat, because forages are a major source of C18:3n-3 in ruminant diets (Dewhurst et al., 2006; Schmid et al., 2006; Scollan et al., 2006). In addition, some plant secondary metabolites are known to act positively on rumen feed digestion and ruminant production (Jouany and Morgavi, 2007). This might be related to their effect on rumen methanogenesis and fermentation (Wina et al., 2005; Calsamiglia et al., 2007; Jouany and Morgavi, 2007). Although some studies report milk fatty acid (FA) profiles when supplementing blends of secondary plant metabolites (Benchaar et al., 2006, 2007), to our knowledge, the direct effect of pure plant secondary metabolites on rumen long-chain PUFA metabolism has not yet been studied. Metabolites with rumen methane-inhibiting features might be of particular interest, because concomitant inhibition of rumen methanogenesis and rumen biohydrogenation of long-chain PUFA has been described for other compounds, such as ionophores (Fellner et al., 1997), medium-chain FA (Soliva et al., 2004; Fievez et al., 2006), and long-chain FA from fish oil (Fievez et al., 2003) and microalgae (Boeckaert et al., 2007; Fievez et al., 2007). The aim of this study was to screen the effects of 4 plant secondary metabolites with different chemical and functional groups on rumen fermentation and FA biohydrogenation using a dual-flow continuous culture fermentation system.

	MATERIALS AND METHODS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The research protocol was approved by the Campus Laboratory Animal Care Committee of the Universitat Autònoma of Barcelona (Spain).

Apparatus

Eight 1,320-mL dual-flow continuous culture fermenters (Hoover et al., 1976) were used in 4 replicated runs of 5 d (2 d for stabilization and 3 d for sample collection). On the first day of each run, rumen fluid diluted with artificial saliva (1:2, vol/vol) was inoculated in all fermenters. Rumen fluid was obtained from 2 rumen-fistulated Holstein dairy cows (650 kg of BW) fed a total mixed diet (35.7% alfalfa hay, 7.6% ryegrass hay, 15.5% ground corn, 11.6% barley grain, 11.9% corn gluten feed, 8.1% cottonseed, 4.6% molasses, 1.6% soybean meal, 1.3% calcium soaps of FA, and 2.1% mineral and vitamin mixture; DM basis). Artificial saliva was prepared according to Weller and Pilgrim (1974) and contained 0.4 g/L of urea to simulate recycled N. The saliva was continuously infused into the fermenters. Temperature (38.5°C), pH (6.4 ± 0.05), and liquid (10%/h) and solid (5%/h) dilution rates were maintained constant, and fermentation conditions were monitored with LabView Software (FieldPoint; National Instruments, Austin, TX). Solid and liquid fermenter effluents were collected separately, and collection vessels were maintained at 4°C to impede microbial action. Anaerobic conditions in the fermenters were maintained by infusion of 40 mL of N₂/min.

Diets and Treatments

Lolium perenne (perennial ryegrass) was cut, dried (70 to 80°C for 48 h), ground through a 1-mm mesh screen (Brabander, Duisburg, Germany), and stored at –20°C until used for fermenter feeding and FA analysis. Fermenters were fed 60 g of dried L. perenne in 2 equal daily portions (at 1000 and 2200 h) daily. Lolium perenne was chosen as a substrate to study the effect of selected plant secondary metabolites on rumen biohydrogenation of forage FA, because the selected plant secondary metabolites are present in low amounts or not present in L. perenne.

Plant secondary metabolites used in this study were chosen based on their effect on rumen fermentation and methanogenesis (Lourenço et al., 2008). Treatments were as follows: control (CON, no additive), positive control [monensin, MON (12 mg/L = 40 mg/d); Sigma-Aldrich, St. Louis, MO], and 4 plant secondary metabolites: 1) triterpene saponin from Quillaja bark [QB1000 (1,000 mg/L = 3.2 g/d) and QB500 (500 mg/L = 1.6 g/d), sapogenin content ~25%; Sigma-Aldrich]; 2) quercetin [Q500 (500 mg/L = 1.6 g/d) and Q250 (250 mg/L = 800 mg/d), C₁₅H₁₀O₇·2H₂O, 98% purity; Sigma-Aldrich]; 3) eugenol [E250 (250 mg/L = 800 mg/d), C₁₀H₁₂O₂, 98% purity; Pancosma SA, Bellegarde-sur-Valserine Cedex, France], and 4) cinnamaldehyde [C500 (500 mg/L = 1.6 g/d), C₉H₈O, 98% purity; Pancosma SA]. Average daily doses, for all treatments, are based on the daily input volume (3.2 L) in the fermenters. Doses of monensin, cinnamaldehyde, and eugenol were chosen based on previous in vitro research summarized by Calsamiglia et al. (2007). Doses of saponin and quercetin were chosen based on the literature (Hess et al., 2003, 2004; Wina et al., 2005; Pen et al., 2006 for saponin and Broudiscou and Lassalas, 2000 and Broudiscou et al., 2000 for quercetin). The daily average dose of the additives was divided into 2 equal fractions and added to the fermenters at the same time of feeding to achieve the expected average concentration.

Sampling

For sample collection, solid and liquid effluents were mixed and homogenized for 1 min with a homogenizer. A 250-mL sample was removed via aspiration, from which a subsample (100 mL) was collected for FA analysis. The rest of the sample was filtered through 1 layer of cheese cloth, and subsamples were collected for the determination of VFA concentration (4 mL). A solution (1 mL) composed of 0.2% (wt/wt) of mercuric chloride, 0.2% (wt/wt) 4-methylvaleric acid (used as internal standard), and 2% (vol/vol) orthophosphoric acid was added to samples for VFA analysis. All samples were frozen immediately after collection and kept at –20°C until analysis. Samples for FA analysis were freeze-dried without shelf heat (Dura-Dry MP, FTS Systems, New York, NY) and kept at –20°C until analysis.

Chemical Analyses

FA Analysis. Fatty acids of all fermenter effluents from d 3, 4, and 5 were extracted with chloroform/methanol (C/M; 2:1, vol/vol) as described by Lourenço et al. (2005) with some adaptations for smaller sample aliquots. Briefly, 1.25 g of freeze-dried sample was extracted overnight with 15 mL of C/M (2:1, vol/ vol), 10 mL of distilled water, and 10 mg of tridecanoic acid (C13:0; Sigma-Aldrich) as an internal standard. Samples were then centrifuged at 1,821 x g for 10 min, and the C/M layer was recovered. This procedure was repeated twice, adding 10 mL of C/M (2:1, vol/vol) in the second and third extraction steps. Finally, samples were washed with distilled water, and the C/M layer was recovered. Extracts were brought to a final volume of 50 mL with C/M (2:1, vol/vol). Fatty acids of dried perennial ryegrass samples of each experimental period were extracted with C/M (2:1, vol/vol) as described by Lourenço et al. (2005). Briefly, 2.5 g of dried sample was extracted overnight with 30 mL of C/M (2:1, vol/ vol), 20 mL of distilled water, and 10 mg of tridecanoic acid (C13:0; Sigma-Aldrich) as an internal standard. Samples were then centrifuged at 1,821 x g for 10 min, and the C/M layer was recovered. This procedure was repeated twice, adding 25 mL of C/M (2:1, vol/vol) in the second and 20 mL in the third extraction step. Finally, samples were washed with distilled water, and the C/M layer was recovered. Extracts were brought to a final volume of 100 mL with C/M (2:1, vol/vol). For preparation of FA methyl esters of effluent and dried perennial ryegrass samples, 10 mL of extract was used. Samples were methylated at 50°C with 0.5 M NaOH in methanol followed by HCl/methanol (1:1, vol/vol) according to Raes et al. (2001). The FA methyl esters were analyzed on a Hewlett-Packard 6890 gas chromatograph (Hewlett-Packard Co., Diegem, Belgium) with a CP-Sil88 column for FA methyl esters (100 m x 0.25 mm x 0.2 µm; Chrompack Inc., Middelburg, the Netherlands). More detailed information about the GLC conditions is given by Raes et al. (2004). The FA profile of dried perennial ryegrass is presented in Table 1.

Analysis of VFA. Samples for VFA analysis (from each of the 5 d of each experimental run) were prepared as described by Jouany (1982). Samples were centrifuged at 15,000 x g for 15 min at 7°C, and 1 mL of the supernatant was diluted with distilled water (1:1, vol/vol). The VFA were analyzed on a Hewlett-Packard 6890 gas chromatograph (Hewlett-Packard Co.) with a polyethylene glycol nitroterephtalic acid-treated capillary column for VFA (BP21; SGE Europe Ltd., Buckinghamshire, UK). More detailed information about the GLC conditions is given by Busquet et al. (2005b).

The VFA data from d 3, 4, and 5 were analyzed statistically. Data from d 1 and 2 were used to assess stability of fermenter conditions within 2 d after inoculating the fermenters.

Statistics

All statistical analyses were performed using SPSS (SPSS software for Windows, release 12.0, SPSS Inc., Chicago, IL). A general linear ANOVA model was used to evaluate the stability of fermenter conditions (based on total VFA and individual VFA proportions; only the means of CON are shown; Table 2) over the 5 d of the experimental period, according to Y_ij = µ + day_{i = 1,5} + R_{j = 1,4} + _ij, where µ = the overall mean; day_{i = 1,5} = the effect of sampling day (fixed effect); R_{j = 1,4} = the effect of experimental run (fixed effect); and _ij = the residual error.

	RESULTS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Total VFA and individual VFA concentrations remained constant from d 2 onward (Table 2), indicating that 2 d was sufficient for the stabilization of the rumen microbial population to the fermenter conditions. Nevertheless, day x treatment interactions indicate changes during the experimental days (d 3 to 5) for some of the treatments. For example, butyrate proportions in QB1000 (data not shown) were less on d 3 but similar to the CON on d 4 and 5. Described changes relate to the negative control (no additive, CON) unless stated otherwise.

Concentrations of VFA

Total VFA concentrations and proportions of individual VFA are presented in Table 3. The treatments CON and MON did not differ in terms of total VFA concentrations, but addition of MON resulted in greater proportions (P < 0.001) of propionate and in lesser proportions (P < 0.001) of acetate and butyrate. In contrast, Q500 increased (P = 0.001) total VFA concentrations, whereas C500 decreased (P < 0.001) total VFA concentrations by approximately one-third of CON. The treatment C500 further increased butyrate and decreased propionate proportions compared with both CON (P < 0.02 for butyrate and P < 0.001 for propionate) and MON (P = 0.05 for butyrate and P < 0.001 for propionate). For the other plant metabolites, only minor changes were observed compared with the CON: Q500 increased acetate proportions (P = 0.005) and decreased (P = 0.03) branched-chain VFA concentrations. The proportions of individual VFA for Q250 were intermediate between Q500 and the CON. Both total VFA concentrations and individual VFA proportions of QB1000, QB500, and E250 were similar to CON.

Total amount of FA and proportions of individual FA are presented in Tables 4, 5, 6, and 7. Compared with the CON, addition of C500 decreased (P = 0.002) the total amount of FA in the effluent (Table 4). The MON treatment increased (P = 0.009) total odd and branched-chain FA (OBCFA) proportions in the effluent fat (Table 4), whereas C500 decreased (P < 0.001) and E250 tended to decrease (P = 0.08) total OBCFA proportions compared with CON. For the other plant metabolites (QB1000, QB500, Q500, and Q250), no changes were observed in the proportions of total FA or total OBCFA.

	DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The aim of the current research was to screen the effect of plant secondary metabolites on rumen FA metabolism. However, as potential feed additives, these metabolites should partially inhibit rumen biohydrogenation of PUFA but without an overall reduction in microbial activity. Some variables such as total VFA concentrations in the effluent, proportions of OBCFA in the effluent fat, and fat proportions in the effluent provide an indication of rumen microbial population or activity. Indeed, flow of OBCFA has been suggested as a marker of microbial biomass (Vlaeminck et al., 2005). Further, the OBCFA pattern has also been shown to be associated with changes in the rumen microbial population (Vlaeminck et al., 2006). Moreover, rumen fermentation of dietary OM results in the partial conversion of nonfat OM into gaseous end products that escape from the fermenters. Hence, when degradation is decreased, greater amounts of OM are recovered in the effluent, therefore diluting the concentration of fat. The constant concentration of rumen undegradable dietary fat, combined with decreased de novo synthesis of OBCFA by rumen bacteria and the significantly greater recovery of the dietary OM, then results in a decreased effluent fat concentration.

Shifts in the fermentation pattern and a decreased apparent biohydrogenation of C18:2n-6 and C18:3n-3 were observed in the presence of MON, in agreement with what has been reported in literature (Fellner et al., 1997; Jenkins et al., 2003; Wang et al., 2005) for in vitro systems. However, in our study, MON resulted in an accumulation of C18:2 trans-11, cis-15 and C18:1 trans-10 rather than C18:1 trans-11 as observed in other studies (Wang et al., 2005). The former suggests the inhibition or retardation, or both, of biohydrogenation occurs at earlier steps of the biohydrogenation process, whereas C18:1 trans-10 accumulation suggests a shift from the major known (via C18:1 trans-11) to a secondary biohydrogenation pathway of C18:2n-6 (via C18:1 trans-10), as described by Jenkins et al. (2003).

The reduction in total VFA concentrations and the proportions of total OBCFA for C500 suggests that microbial activity was inhibited. This overall reduction in the rumen fermentation processes further decreased the total fat concentration of the effluent. Additionally, the day x treatment interactions for some individual OBCFA could suggest that some microbial populations seemed more sensitive to the presence of cinnamaldehyde than others. Indeed, the proportion of C13:0 iso on d 3 was greater than the CON, less than the CON on d 4, and similar to the CON on d 5; proportions of C14:0 iso and C16:0 iso on d 3 and 4 were less than for the CON and similar to the CON on d 5; and proportions of C15:0 iso and C17:0 anteiso on d 3 and 5 were similar to the CON and less than for the CON on d 4. On the other hand, proportions of C15:0 anteiso and C17:0 iso on d 3, 4, and 5 were always less than for the CON. This was further reflected in shifts in the proportions of individual OBCFA, shifts in the proportions of individual VFA, and in some shifts in the biohydrogenation pathways as suggested from the accumulation of C18:1 trans-10 and CLA trans-10, cis-12. Moreover, an inhibition of the biohydrogenation process is suggested from the accumulation of other intermediates (C18:2 trans-11, cis-15) and the decreased apparent biohydrogenation of C18:2n-6 and C18:3n-3. Accumulation of these FA decreased the precursor supply for later steps in the biohydrogenation process, explaining the lesser C18:1 trans-11, C18:1 trans-15, and C18:1 cis-15 proportions.

The dramatic overall reduction of the rumen microbial activity observed in this study has been reported in the literature with large doses of cinnamaldehyde (3,000 mg/L; Busquet et al., 2006), whereas moderate doses (31.2 and 312 mg/L; Busquet et al., 2005a), comparable to the currently applied average dose, were reported to cause some shifts in the proportions of individual VFA only. A possible reason for the different outcome of this study compared with that of Busquet et al. (2005a) might be the interaction between the basal diet used and the plant secondary metabolite. Busquet et al. (2005a) fed the fermenters a 50:50 alfalfa hay:concentrate diet, whereas in our study, a complete forage diet was used. Moreover, using a moderate dose (312 mg/L) of cinnamaldehyde, Busquet et al. (2005a) reported a different fermentation pattern, with greater butyrate, decreased acetate proportions, and no differences in propionate proportions, whereas in our study, in the presence of cinnamaldehyde, the proportions of acetate remained similar to the CON, the butyrate proportions increased, and the propionate proportions decreased.

Although total OBCFA proportions in the presence of eugenol (E250) were decreased compared with CON, suggesting a decreased total microbial biomass, total amounts of VFA and apparent biohydrogenation of C18:2n-6 and C18:3n-3 did not differ from CON. Moreover, no differences in the end products of rumen fermentation were observed, opposite to the results of Busquet et al. (2006; 300 mg/L, increased butyrate proportions) and Castillejos et al. (2006; Exp. 2, 500 mg/L, decreased acetate and increased propionate and butyrate proportions). Nevertheless, the biohydrogenation process seemed to be slightly altered based on the increased accumulation of C18:1 trans-15 and C18:1 cis-15, end products of an alternative biohydrogenation pathway of C18:3n-3, and on the lesser C18:0 proportions compared with CON. Further, the day x treatment interaction for some individual OBCFA also suggests a transient effect of eugenol in the rumen microbial population. Indeed, proportions of C15:0 iso, C15:0 anteiso, C17:0 iso, and C17:0 anteiso on d 3 and 5 were similar to the CON, and on d 4, the proportions of these OBCFA were less than for the CON.

The concentrations used might explain why some studies report different effects of eugenol on the fermentation pattern [Busquet et al., 2006 (3,000 mg/L) and Castillejos et al., 2006 (500 mg/L, Exp. 2)], whereas other studies [Busquet et al., 2005a (2.2 mg/L) and this study (250 mg/L)] reported no effects. With large doses of eugenol (3,000 mg/L), total VFA, butyrate, and branched-chain VFA proportions decreased and propionate proportions increased (Busquet et al., 2006), whereas with moderate doses (500 mg/L), total VFA, acetate, and branched-chain VFA proportions decreased, and propionate and butyrate proportions increased (Castillejos et al., 2006, Exp. 2).

The greater total amount of VFA in Q500 suggests a stimulation of the microbial activity. However, proportions of total OBCFA remained similar to CON, and no shifts in the extent or pathways of biohydrogenation were observed.

Triterpene saponins (QB1000 and QB500) did not affect rumen total microbial biomass or activity, as suggested from the similar total OBCFA proportions and total amounts of rumen VFA compared with CON. Additionally, neither the FA metabolism nor the fermentation pattern was affected by triterpene saponins. This divergence from previous studies [e.g., Wina et al. (2005) with increased or decreased propionate proportions or Pen et al. (2006) with increased propionate proportions] might be attributed to several factors: the basal diet used, the concentration of plant secondary metabolite applied, or the origin of the plant secondary metabolites, as suggested before by liwiski et al. (2002) and Pen et al. (2006). Indeed, triterpene saponins studied in literature were derived from a variety of plants including Quillaja saponaria (Pen et al., 2006), Sapindus saponaria (Wina et al., 2005), or tea extract (Wina et al., 2005). Additionally, the fast adaptation of the microbial population to saponins by its conversion to sapogenins (Teferedegne et al., 1999) could mask possible effects of the plant secondary metabolite on the rumen processes. Indeed, the day x treatment interaction found for butyrate proportions suggests an adaptation of the microbial population (in particular protozoa) to the presence of saponins from Quillaja bark (i.e., on d 3, the proportions of butyrate were less than for the CON, and on d 4 and 5, the butyrate proportions for QB1000 were similar to CON).

Conclusions

Monensin induced shifts in fermentation pattern and in biohydrogenation without affecting total VFA concentrations. Eugenol caused some minor inhibition of the biohydrogenation process, without affecting the fermentation pattern. Quercetin resulted in greater total VFA concentrations, suggesting a stimulation of the microbial activity. However, shifts in the pathways or extent of biohydrogenation did not occur for both quercetin and triterpene saponins. The use of cinnamaldehyde caused an overall inhibition of the microbial biomass and activity, based on the decreased VFA and OBCFA proportions, probably due to the large doses used in this study. Moreover, cinnamaldehyde caused a shift from the major biohydrogenation pathway to a secondary biohydrogenation pathway of C18:2n-6 as evidenced by the greater proportions of C18:1 trans-10 and CLA trans-10, cis-12 in the fermenter effluent. At the doses tested in this study, we could only show a direct relation between changes in the rumen FA metabolism and the presence of cinnamaldehyde, but not for eugenol, quercetin, or triterpene saponins. Other plant secondary metabolites and a wider range of doses merit further research as potential modifiers of rumen FA biohydrogenation.

	Footnotes

 
¹ M. Lourenço acknowledges receipt of a PhD grant from the Foundation for Science and Technology-Portugal and the financial support of this experiment by Commissie Wetenschappelijk Onderzoek, Belgium. Part of this research was also supported by the European Union community (project Food-CT-23006-36241 Prosafebeef).

² Corresponding author: Veerle.Fievez{at}UGent.be

Received for publication November 5, 2007. Accepted for publication June 11, 2008.

	LITERATURE CITED

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 


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