Tjakradidjaja, Anita Sardiana. "Isolation and characterisation of tannin-resistant bacteria from the rumen of feral goats and camels." Thesis, 2012. http://hdl.handle.net/2440/79594.
Анотація:
Low availability and poor nutrient quality of tropical grasses result in low levels of animal production. Browse/shrub legumes such as mulga (Acacia sp.) and calliandra (Calliandra calothyrsus) can be used as supplements to improve animal production. However, their utilisation is limited by the presence of antinutritional compounds, such as tannins. Tannins are polyphenolic compounds that are capable of binding other nutrients to form stable complexes. Tannins comprise hydrolysable (HT) and condensed tannins (CT) with CTs being the major form found in the legumes. Their concentration in feeds determines their effect on animal production. Low levels of tannin (< 40 g.kg DM⁻¹) protect feed protein from degradation by rumen microbes, thereby increasing the amount of protein
passing through the rumen, and reducing the potential for bloat. However, high concentrations of tannin retard animal production through the inhibition of enzyme activities of some rumen bacteria, decreasing the availability of protein and fibre, and reducing feed intake. Tannins in high concentrations also bind nutrients such as protein and carbohydrates; tannin-protein or tannin-carbohydrate complexes are difficult to digest by rumen microbes or by enzymes secreted by ruminants in the gastrointestinal tract. This decreases the availability of protein and carbohydrate for the animals, reducing feed intake and decrease animal production. Several methods have been developed to overcome tannin problems in livestock feeds with a focus on the use of biological treatments. Such treatments emphasise the involvement of rumen microorganisms residing in the rumen of feral animals or of animals adapted to feeds with high tannin contents. This approach resulted in the isolation of the tannin-tolerant bacteria, Streptococcus gallollyticus and Selenomonas ruminantium K2, from the rumen of feral goats. However, these bacteria, alone or in combination, were unable to mimic the effect of whole rumen fluid on tannin detoxification. These bacteria may not, therefore, be the only ruminal species that are resistant to, or degrade tannins. Other tannin-resistant (TR) bacteria may also exist in the rumen. Although each of these TR-bacteria may have specific characteristics, they may interact synergistically to promote digestion of tannin-containing browse legumes. Therefore, the present project was aimed at isolating TR-bacteria from the rumen contents of feral goats and camels, and studying their ecology.
To achieve these objectives, the research project is divided into three sections as follows :
1. Optimising the method for extracting tannin from legume leaves and selecting the appropriate method to measure tannin content.
2. Isolating TR-bacteria in rumen fluid samples from feral goats and camels, and characterising them phenotypically and by molecular characterisation using restriction analysis of amplified 16S rDNA and 16S rDNA sequence analysis.
3. In vitro studies of dry matter (DM) disappearance of mulga and calliandra with populations of TR-bacteria grown in a monoculture or in a co-culture system.
Extracts of condensed tannins (CTs) were used as substrates for isolating TR-bacteria from feral goat and camel rumen fluids. CTs were extracted from legumes using 70% acetone, and used as a substrate in bacterial enrichment studies. The amount of soluble tannin extracted from the samples varied among plant species with mulga containing less free tannin than calliandra. Factors such as processing methods (fresh, freeze-drying and oven-drying), or physical and chemical treatments (autoclaving and phenol extraction) are tested for their effect on yield of extractable tannin. Freeze-drying was selected for processing mulga and calliandra leaves before extraction of CT by the method of Terrill et al. (1992). Measurement of tannins using different methods yielded different values of extractable tannin from the same legume. Since vanillin-HCl, butanol-HCl and H₂SO₄ methods are based on different principles, the standard used, the solvents, and other factors were tested for each method. The H₂SO₄ method was selected to measure the amount of free tannin extracted from the legumes. This selection was made on the basis that the results were comparable to those produced by the butanol-HCl assay procedure, but the H₂SO₄ method would have less interference from water in the microbiology studies. The amount of extractable tannin was expressed as equivalents to quebracho tannins when 70% acetone was used to dissolve the tannin extracts and the standard. Twenty TR-bacteria were successfully isolated from the rumen fluid of feral goats and camels using extractable CT of freeze-dried mulga and calliandra, as well as hydrolysable tannin (tannic acid). These isolates were divided morphologically into several groups : Gram-positive cocci (Group 1), Gram-positive cocci/rods (Group 2 and 4), Gramnegative cocci (Group 3), Gram-negative curved rods (Group 5) and Gram-negative slender rods (Group 6). These bacteria were identified by physiological and biochemical characterisations, and API testing as Streptococcus sp. (Group 1), Lactobacillus sp. (Group 2 and 4), Selenomonas ruminantium (Group 5) and Butyrivibrio sp. (Group 6). A further identification using restriction analysis of amplified 16S rDNA with four restriction endonucleases (AluI, HaeIII, MspI and TaqI) characterised bacteria that belong to the Streptococcus sp. (Group 1) and Selenomonas ruminantium (Group 5). Confirmation of genera of other TR-bacteria was made after integrating the RFLP analysis with amplified 16S rDNA sequence analysis. This identified the bacteria as : Lactobacillus sp. (Group 2 and 4), and Butyrivibrio sp. (Group 6). The genus of Gram-negative coccus (Group 3) was identified as Escherichia (E. coli) coli; however, a further clarification is necessary for the identification Group 3 isolate. These results provide an example of phenotypic identification and molecular characterisations using restriction analysis of amplified 16S rDNA and 16S rDNA sequence analysis for identifying TR-isolates from feral goat and camel rumen fluids. The TR-bacteria had different capabilities of degrading mulga and calliandra as
indicated by results of studies on in vitro DM degradability using monocultures of the TRbacteria. The highest DM degradabilities of both legumes were obtained in cultures
inoculated with Butyrivibrio spp. (G23A and G53C), and S. gallolyticus. These TR-bacteria are the main TR-bacteria degrading mulga and calliandra. The other TR-bacteria,
Lactobacillus spp. (G43C and G33A), E. coli (C43C) and Sel. ruminantium K2, used metabolic products from these main TR-bacteria. Co-culture between the main TR-bacteria (Butyrivibrio spp. (G23A and G53C) or S. gallolyticus) with the bacteria that utilize the metabolic products of tannin degradation (Lactobacillus spp. (G43C and G33A), E. coli (C43C) or Sel. ruminantium K2) could improve mulga and calliandra degradation.
Calliandra leaves were more degraded than mulga leaves because calliandra leaves have smoother physical characteristics, and lower concentrations of fibrous components and protein/fibre bound tannins than mulga leaves. Grinding the sample leaves and incubating cultures for 48 h improved degradation of legume leaves; incubation for 48 h was also suitable for TR-bacteria to achieve optimum growth and enzyme secretions. TR-bacterial characteristics and populations, and the nature of legume leaves are important factors affecting degradation of legumes containing tannins. Factors associated with TR-bacterial characteristics are the presence and activity of enzymes for degrading nutrients and antinutrients from the legumes, the presence of substances protecting TRbacteria from the antinutrients present in the legume, and the bacterial growth characteristics during the period of legume degradation. All these factors, consequently, affect TR-bacterial populations in cultures that contain legumes with tannins. Factors related to the nature of legume leaves are the physical characteristics, nutrient composition and concentration, especially the fibrous compounds and the types of tannins or other antinutrients. It is concluded that Streptococcus gallollyticus and Selenomonas ruminantium are not the only tannin-resistant bacteria. Other bacteria from the rumen of feral goats and camels also tolerate tannins extracted from freeze-dried mulga and calliandra leaves, including Butyrivibrio sp., Lactobacillus sp. and E. coli (Gram-negative coccus). These bacteria differ in their abilities to digest the legumes. However, improvement in the legume digestibility can result from the interactions between these tannin resistant bacteria.
Thesis (M.Sc.) -- University of Adelaide, School of Agriculture, Food and Wine, 2012