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Title: The protective effects and related mechanisms of natural polysaccharides on probiotic bacteria against antibiotic damage
Authors: Mao, Yu-heng
Degree: Ph.D.
Issue Date: 2019
Abstract: Antibiotics are regarded as a class of the most successful drugs in history for their efficacy in the treatment of infectious diseases and having saved billions of lives. In recent decades, however, excessive use of antibiotics in poultry farming and aquafarming processes has imposed a health threat worldwide owing to the development of antimicrobial resistance as well as the many side effects of antibiotics on human health. The excess and unabsorbed antibiotics in the upper gut may enter the large intestine and disrupt the gut microbial balance by inhibiting the beneficial bacteria, increasing the colonization of resistant microbes and pathogenic organisms. The gut microbiota plays an important role in human health and an imbalanced gut microbiota is linked to a wide range of local and systemic disorders in the human body. This research project aimed to assess the protective effects and possible mechanisms of exopolysaccharides (EPS) produced by a medicinal fungus, Cs-HK1, against the inhibitive effects of antibiotics on pure cultures of several bifidobacterial species and on human fecal microflora in vitro fermentation. The antibiotics used in this study were selected from those that are widely used in therapy and animal husbandry, including enrofloxacin, penicillin, ampicillin, tetracycline and streptomycin. EPS fractions with different molecular weights (MW) were assessed together with some well-known prebiotic carbohydrates such as inulin, galactooligosaccharide (GOS) and konjac glucomannan (KGM), a high MW polysaccharide originated from the plant tuber of Amorphohallus konjac. In pure bifidobacterial cultures, the high-MW Cs-HK1 EPS (~2.252×10⁸ Da) and KGM (~8.8×10⁸ Da) showed the most significant protective effect on most of the bifidobacterial species against antibiotic inhibition, as shown by the drastic increase in the minimal inhibitory and bactericidal concentration (MIC and MBC) dramatically. In general, the protective effect was positively correlated to the MW of polysaccharide fractions and also varied with the bacterial species. Cs-HK1 EPS and KGM significantly increased the tolerance of bifidobacteria to the common antibiotics, suggesting their potential use as protection reagent for human gut bacteria, especially under the condition of intended or unexpected antibiotic exposure.
To understand the mechanisms of EPS and KGM's protective effect, we assessed the possible contribution by the nutritional effects and physical interactions. EPS, KGM and inulin were barely utilized as a carbon source for the bifidobacterial growth in the pure cultures. On the other hand, electron microscopic observation showed that the Cs-HK1 EPS formed a viscous layer around the bacterial cell, which could resist the access by antibiotics. Furthermore, the polysaccharide layer surrounding the bacteria cell also promoted the aggregation of bacterial cells to form biofilms, and EPS also enhanced the bifidobacterial adhesion to Caco-2 cell monolayer. Additionally, the EPS showed moderate ability to adsorb some of the antibiotics. These results suggested that EPS and other high MW polysaccharides protected bifidobacteria mainly through physical interactions. Human fecal fermentation was performed to evaluate the effects of EPS on the complex and diverse microbial community of intestinal microflora. EPS fractions with different MW ranges were tested in this system. While barely utilized by individual bifidobacterial species in pure cultures, all the EPS fractions were consumed by more than 75% during 24-48 h of the in vitro fecal fermentation. As a consequence, there was a significant increase in the production of short chain fatty acids (SCFAs) including acetic, propionic and butyric acid. The consumption rates and production levels of SCFAs varied slightly with the different EPS fractions. EPS also influenced the composition and diversity of fecal microflora, increasing the relative abundance of Firmicutes but suppressing that of Proteobacteria, which may be beneficial to human health. This result demonstrated the fermentability of EPS by gut microbiota and proposed the potential of Cs-HK1 EPS as prebiotic in the food industry. Similar to that in the pure bacterial cultures, Cs-HK1 EPS and KGM also protected the bacteria against antibiotic damage during in vitro fecal fermentation. Particularly, the Cs-HK1 EPS significantly increased the viability of major groups of gut bacteria compared with the well-known prebiotics, inulin and GOS. The presence of EPS in the fecal culture was able to maintain the operational taxonomic unit (OTU) of fecal microflora at the level of the control. The fermentative metabolism of EPS also increased the relative abundance of Clostridium. In contrast, inulin and GOS induced a significant decrease of the OTU number. The beneficial effects of EPS on the gut microbial diversity and microbial distribution in the presence of ampicillin further validated the protective effects of Cs-HK1 EPS on gut bacteria against antibiotic damage. In summary, the following are the major findings from this research project: (1) The high MW EPS produced by the medicinal fungus Cs-HK1 had protective effects against antibiotic inhibition of bifidobacteria in liquid cultures but could not be well utilized as a carbon source for bacterial growth. (2) The protective effect of EPS on bifidobacteria was mainly attributed to physical interactions, the formation of a viscous layer surrounding the bacterial cell that increased the resistance to antibiotics. (3) Cs-HK1 EPS also showed promising prebiotic function during in vitro human fecal fermentation, being well fermented by the fecal microflora to produce SCFAs and also beneficial for the stability and diversity of microbial populations. These findings lay a theoretical foundation for further development and application of Cs-HK1 EPS as new prebiotic nutraceutical and functional food products for improving gut health.
Subjects: Hong Kong Polytechnic University -- Dissertations
Polysaccharides -- Therapeutic use
Intestines -- Microbiology
Pages: xx, 152 pages : color illustrations
Appears in Collections:Thesis

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