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Yuen Hawii 0085O 10943.Pdf Effects of inulin, fructooligosaccharide, and breadfruit fiber on biofilm formation, growth, and gastrointestinal survival of probiotic yeast and bacteria A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN Food Science by Beverly Yuen Thesis Committee: Dr. Yong Li, Chairperson Dr. Kacie Ho Dr. Chin Nyean Lee Acknowledgements I would like to give my biggest acknowledgment to my principle investigator, Dr. Yong Li. Dr. Li has been immensely generous with his patience, flexibility, and guidance throughout my research. I would also like to acknowledge Dr. Chin Nyean Lee and Dr. Kacie Ho, my committee members, for their assistance with my thesis and graduate studies. I would like to give my next appreciation to Dr. Rajesh Jha for spectrophotometer use. My next gratitude is to current and past lab members for their guidance and friendships inside and outside lab. My final gratitude goes to my parents, Chan Cheong Yuen and Young Fei Lin, for their financial support. ii Abstract A healthy gut microbiome is critical for digestion and immunity. Probiotics help balance the gut microflora and compete against pathogens for binding sites. Probiotics are live microorganisms that provide health benefits upon ingestion. Prebiotics are non-digestible oligosaccharides that selectively stimulate the survival and growth of probiotics in the colon. Many foods, such as breadfruit, contain fibers that have the potential to be prebiotics. The overall objective of this thesis was to increase the growth, biofilm formation, and gastrointestinal survival of probiotics with inulin, fructooligosaccharide (FOS), and breadfruit fiber. Water soluble breadfruit fiber was extracted from breadfruit and used as a prebiotic in this study. The acid tolerance at pH 2, tolerance to 0.3% bile salt, prebiotic activity score (PAS), and survival under simulated digestion were determined on Lactobacillus rhamnosus, Lactobacillus acidophilus, and Saccharomyces boulardii supplemented with the three prebiotics. The effects of prebiotics on biofilm formation of the probiotics and pathogenic Escherichia coli O157:H7 were also tested. S. boulardii showed a significantly higher survival under acidic conditions than L. rhamnosus and L. acidophilus, and the optimum concentration of breadfruit fiber for the survival of L. rhamnosus was 2.5% and 3.0%. The addition of breadfruit fiber showed a similar effect to glucose on the acid survival of L. acidophilus. Three tested probiotics showed high tolerance to bile salt. Moreover, 1.0% of inulin, FOS, and breadfruit fiber was the optimum concentration for biofilm development of S. boulardii. The addition of breadfruit fiber was as effective as glucose in helping L. rhamnosus form biofilm. With S. boulardii, inulin and FOS exhibited a higher PAS than breadfruit fiber. The PAS of breadfruit fiber was approximately 0.4 for S. boulardii and L. rhamnosus and 0.2 for L. acidophilus. After 24 h of simulated digestion, the addition of breadfruit fiber increased the survival of S. boulardii, L. rhamnosus and L. acidophilus by 0.37, iii 5.45, and 5.59 log CFU/mL, respectively, compared to the negative control. These findings reveal the potential of S. boulardii to be marketed with prebiotics for individuals with gastrointestinal disorders. Moreover, breadfruit fiber is a possible prebiotic alternative to inulin and FOS in Hawai’i due to its sustainability and effectiveness with probiotics. iv Table of Contents Acknowledgements..........................................................................................................................ii Abstract...........................................................................................................................................iii Table of Contents.............................................................................................................................v List of Tables.................................................................................................................................vii List of Figures...............................................................................................................................viii Chapter 1 Introduction.....................................................................................................................1 Chapter 2 Literature Review............................................................................................................5 2.1 Digestive system and gut health....................................................................................5 2.2 Probiotics.......................................................................................................................6 2.2.1 Lactobacillus acidophilus...............................................................................8 2.2.2 Lactobacillus rhamnosus................................................................................9 2.2.3 Saccharomyces boulardii..............................................................................10 2.3 Pathogens.....................................................................................................................12 2.4 Prebiotics......................................................................................................................14 2.3.1 Synbiotics with probiotics and prebiotics.....................................................15 2.3.2 Inulin.............................................................................................................16 2.3.3 Fructooligosaccharides.................................................................................17 2.5 Foods with prebiotic potential.....................................................................................18 1.4.1 Breadfruit......................................................................................................19 1.4.1.1 Resistant starch in breadfruit.........................................................19 2.6 Summary......................................................................................................................20 Chapter 3 Materials and Methods..........................................................................................................................................22 3.1 Extraction of water-soluble dietary fiber from breadfruit................................22 v 3.2 Growth of probiotic and pathogenic strains.....................................................23 3.3 Acid resistance of yeast and bacteria...............................................................23 3.4 Bile resistance of yeast and bacteria................................................................23 3.5 Biofilm formation of microbial strains............................................................24 3.6 Determination of prebiotic activity score........................................................25 3.7 Simulated digestion of probiotic strains..........................................................25 3.8 Statistical analysis............................................................................................26 Chapter 4 Results............................................................................................................................................27 4.1 Extraction of water-soluble dietary fiber from breadfruit................................27 4.2 Acid resistance of probiotic strains..................................................................27 4.3 Bile resistance of probiotic strains...................................................................31 4.4 Biofilm formation of microbial strains............................................................35 4.5 Determination of prebiotic activity score........................................................38 4.6 Simulated digestion of probiotic strains..........................................................49 Chapter 5 Discussion......................................................................................................................................53 Chapter 6 Conclusion.....................................................................................................................................62 References......................................................................................................................................64 vi LIST OF TABLES Table 1 Electrolyte stock solutions of digestion fluids..................................................................26 Table 2: Optimum concentration of prebiotic for each probiotic..................................................59 vii LIST OF FIGURES Figure 1 A schematic of some mechanism of actions of probiotics in the intestine, such as antagonistic effects of bacteriocins, modulation of immune cells, and competing for binding sites against pathogens.............................................................................................................................8 Figure 2 The survival of S. boulardii in YNB broth and a pH 2 containing HCL and different concentrations of inulin..................................................................................................................27 Figure 3 The survival of S. boulardii in YNB broth and a pH 2 containing HCL and different concentrations of FOS.........................,,.........................................................................................28 Figure 4 The survival
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