Bacterial detoxi fi cation of saponins in the crop of the avian foregut fermenter

The hoatzin is a folivorous bird with microbial fermentation in the crop that consumes plants that contain saponins. We investigated detoxifi cation activity of saponins by bacteria from the hoatzin crop, and how this activity was affected in the presence of methanogens. Strains and mixed cultures were grown in presence of 200 μg·ml-1 Quillaja saponins. Detoxifi cation was determined as loss of haemolytic activity. The methanogenesis was inhibited added bromoethanesulphonic acid (BES). Mixed cultures showed higher rate of saponin detoxifi cation than strains and it was reduced by BES. The hoatzin bacteria detoxify saponins, and methanogenic Archaea might act as a hydrogen sink.


INTRODUCTION
The toxic effects of saponins are due to their ability to form complexes with membrane sterols (Bangham and Horne, 1962).In ruminants, they inhibit rumen fermentation and may cause ruminant bloat (Sen et al., 1998), apparently through the inhibition of some rumen bacteria and protozoa (Wang et al., 2000;Wina et al., 2006).Microbial degradation of saponins produce the presumably less toxic GARCĺA-AMADO M.A. ET AL. sapogenins, with the consequent decrease of their cytolytic activity (Pillion et al., 1996).
The hoatzin diet comprises plants that contain saponins such as Acacia sp. and Pithecellobium ligustrinum (Domínguez-Bello et al., 1994).The aim of this study was to determine if bacterial cultures from the hoatzin crop could detoxify saponins and how this activity was affected in the presence of methanogens.

MATERIAL AND METHODS
To determine the effect of inoculum concentration on saponin detoxifi cation, three dilutions of crop contents (52, 13 and 0.65 mg•ml -1 ) were homogenized and grown anaerobically at 37ºC in broth M10 (Ogimoto and Imai, 1981) containing 200 µg.ml - Quillaja saponins.Detection of methanogenic bacteria was performed by PCR amplifi cation of a 0.76 kb region of the methyl coenzyme M reductase gene.The 16S rRNA gene sequence of one clone from crop contents and another from cultures were sequenced GenBank (AF506286 and AY089708).
Saponin detoxifi cation was determined by the loss of haemolytic activity of culture supernatants on rabbit red blood cells.Rabbit blood was diluted 1:2 in citrate buffer (%: dextrose 2, sodium citrate 0.8, citric acid 0.055 and NaCl 0.42), and centrifuged (2700 g per 5 min in a Sorvall T6000B).The pellet with the red blood cells was washed and resuspended in 5 volumes of 0.8% NaCl.Supernatants (50 µl) of cultures were added to a 96-well plate followed by addition of 50 µl of the red blood cell suspension.After 1 h, plates were centrifuged and supernatants were measured absorbance at 540 nm.
To investigate the importance of methanogens on saponin detoxifi cation, compared saponin detoxifi cation by crop contents of 52 mg•ml -1 grown with and without the methanogenesis inhibitor 2-bromoethanesulphonic acid (BES; 60 mM).

RESULTS
Mixed cultures of three crop dilutions (52, 13 and 0.65 mg•ml -1 ) were able to reduce haemolytic activity of Quillaja saponins by 80%, in few hours (Table 1).The rate of saponin detoxifi cation was a function of inoculum concentration, decreasing when the inoculum was diluted (P<0.05).Isolated strains of Streptococcus and a Bacillus species from saponin-containing cultures showed slower (days instead of hours) saponin detoxifi cation activity than mixed cultures.Inhibition of methanogens with BES caused a signifi cant decrease in the rate of saponin detoxifi cation (P<0.05;Table 2).

DISCUSSION
Crop cultures from the hoatzin were able to substantially reduce the haemolytic activity of Quillaja saponins, suggesting the crop capability to microbial detoxifi cation of dietary saponins, as has been shown in rumen bacteria that degrade Quillaja saponins to quillaic acid, a triterpenoid molecule which does not produce haemolysis (Pillion et al., 1996;Makkar and Becker, 1997).The reduction of saponin detoxifi cation by BES suggests a synergistic action of GARCĺA-AMADO M.A. ET AL. archaeal methanogens and eubacterial degraders.Consortia with methanogens has been described in the bacterial degradation of aromatic compounds in the cow rumen (Bisaillon et al., 1993), where methanogenics are a sink for H 2 .

Table 1 .
Effect of bacterial inoculum concentration (52 , 13 and 0.65 mg of crop content per ml of culture medium) on bacterial count, rate of saponin detoxifi cation and the time to reach detoxifi cation of 80% of saponin concentration by mixed crop cultures in two hoatzin crops counts and saponin detoxifi cation rates are expressed as mean ± SD values with different superscript are signifi cantly different (P<0.05);nd -non determined Methanogens were detected by PCR amplifi cation of the methyl coenzyme M reductase gene in both crop contents and in saponin-detoxifying cultures.The 16SrRNA sequence matched Methanobrevibacter sp.(Genbank AY196672) and Methanosphaera stadtmanae (AY196684).

Table 2 .
Effect of BES (60 mM) on the rate of saponin detoxifi cation by mixed crop cultures with an inoculum of 52 mg of crop content per ml of culture medium