title: The influence of condensed tannin extracts on gut health in chickens
Creators: name: Dakheel, Mohammed M.
Publisher: name: University of Reading
Year: 2023

description: Tannins belong to the group of polyphenols, which occur naturally in many plants. They exist in the form of two different groups: hydrolysable tannins and condensed tannins (CTs), the latter are also called proanthocyanidins. This thesis investigated the antimicrobial activities in vitro of CTs and studied their impacts on the fermentation and metabolism in chicken caecum. An initial screening of a plant collection for CTs identified plant materials that contained different CT-types, e.g. high and low procyanidin/prodelphinidin or cis/trans, flavan-3-ol ratios and also of CTs with small or large mean degrees of polymerisation. After CT extraction and purification, the effects of these structural CT-features were studied and tested against two bacteria, avian pathogenic E. coli (APEC) as Gram negative bacteria and Staphylococcus epidermidis as Gram positive bacteria by the broth microdilution assays. Subsequent experiments investigated the effects of these different CT compositions on in vitro fermentation and metabolism, and determined the metabolic end products. These CTs were effective at minimal bactericidal concentrations (MBCs) of 5 – 10 mg/ml against APEC and at 5 – 1.25 mg/ml against S. epidermidis. At these concentrations they significantly (P≤0.05) inhibited bacterial growth. Scanning electron microscopy (SEM) revealed antimicrobial activity of these extracts on the bacterial cells of both bacteria, but CTs were more potent against the Gram positive compared to the Gram negative bacteria, which required higher MBC values of 5 - 10 mg/ml. Interestingly, this study found that low concentrations (0.6 mg/ml) of tilia flower extracts, which contain mostly procyanidins (PC), showed slightly enhancement of APEC growth. The anti-biofilm and anti-motility activity of the CTs were also evaluated. All CT extracts also affected significantly (P≤0.05) the biofilm formation of APEC depending on their concentrations and compositions. Significant (P≤0.05) anti-biofilm activity against APEC was observed for almost all CT extracts, especially at higher concentrations (10 – 2.5 mg/ml). More importantly, concentrations of 0.6 - 1.25 mg/ml, especially with extracts that contained low molecular weight of CTs, enhanced the bacterial cell attachment of APEC. This could be because surviving bacteria prefer to form the biofilm in stressful conditions, indicating further investigations due to CT’s high the anti-biofilm activity potential. In contrast, the CT extracts containing either high molar proportions of prodelphinidins (PD) or procyanidins (PC) interrupted and blocked swarming and swimming motility at 10h and 24h compared to the controls. These data support the theory that antimicrobial activity of the CTs, whether with high PC% and PD%, can elicit a positive relationship between anti-biofilm formation and anti-motility capacities. These findings are the first results that provide knowledge about the effect of different CTs on biofilm formation and motility of APEC. The most potent CTs chosen from the above experiments were then tested in vitro using a fermentation culture over a 24h period. A nuclear magnetic resonance (1H-NMR) experiment combined with multivariate statistics was used for the first time to investigate how CT compositions influenced either metabolic end products in a chicken contents or APEC metabolites in a nutrient medium. Both experiments showed interesting results of how CT compositions, in terms of PC% and PD%, impacted on metabolic compounds. Amino acids such as glutamate, leucine, lysine, pyroglutamate, phenylalanine, proline and sarcosine were significantly (P≤0.05/times) decreased. However, leucine and lysine were measured a highest significant difference (P≤0.05) by the interaction between treatments and times. Both CT compositions also influenced significantly (P≤0.05) on fermentation of some fatty acids such as acetate, butyrate and propionate were increased in the treatments. However, lactate was recorded statistically decreasing (P≤0.05) between the treatments. In addition, other metabolites such as some of carbohydrates (e.g. mannitol, glucose, fructose, lactose and galactose) were observed slightly fluctuations on their levels (but not significantly, P≥0.05) at different time-points. Conversely, APEC culture responded metabolically and statistically to these CT compositions. Both CT compositions showed different effect on some amino acids such as lysine, leucine, glutamate phenylalanine and pyroglutamate, which illustrated a significant value (P≤0.05) in CT-groups, especially high PD% treatment, whilst the values of these amino acids showed very close results between PC treated and controls. Similarly, the fatty acids, such as butyrate, iso-butyrate, valerate and iso-valerate were increased in high PD of CTs compared to the high PC and controls. These values did not record significant difference at P&gt;0.05 between the integration of times and treatments, but they showed a significant different (P≤0.05) at different time-points. However, CTs with high PD% led to a significant decreasing (P≤0.05) of lactate, which should normally be increased by E. coli mixed acid fermentation. This is new finding that shows the effect of CTs, particularly high PD% group can influence on the biological process of mixed acid fermentation. The most important results were that CTs with high PD% showed different effects to CTs compared to high PC % in APEC cultures, and that the PC results were close to the controls. Therefore, more research is recommended to investigate the effects of the different CT compositions and concentrations separately by using other NMR techniques e.g. 2D-NMR. In conclusion, this thesis identified the key structural features of CTs, which contain either high molar proportions of PD or PC that might be useful to improve the efficiency of feed utilisation and to reduce the incidence of pathogenic bacterial infections in chickens. This study was also the first investigation of the effects of different CT compositions on in vitro metabolism by 1H-NMR and provided new findings that could establish a powerful database for the future experiments by including the CT extracts in chicken diets.