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Influence of Operating Conditions on Reuterin Production by Resting Cells of Lactobacillus Reuteri DPC16

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Influence of Operating Conditions on Reuterin Production by Resting Cells of Lactobacillus Reuteri DPC16 Influence of Operating Conditions on Reuterin Production by Resting Cells of Lactobacillus Reuteri DPC16 Yuanze Sun A thesis submitted to the Faculty of Auckland University of Technology in partial fulfilment of the requirements for the degree of Master of Science, Auckland University of Technology, 2020 Influence of Operating Conditions on Reuterin Production by Resting Cells of Lactobacillus Reuteri Dpc16 Approved by: Supervisors: Dr. Noemi Gutierrez-Maddox (Primary Supervisor) AUT University Dr. Anthony N Mutukumira (Secondary Supervisor) Massey University I Table of Contents Table of Contents ............................................................................................................................................... II List of figures ...................................................................................................................................................... V List of tables ....................................................................................................................................................... VI Attestation of Authorship .............................................................................................................................. VII Abstract .............................................................................................................................................................. VIII Acknowledgment .............................................................................................................................................. IX Abbreviations ....................................................................................................................................................... X Chapter 1 General Introduction.................................................................................................................... 1 Chapter 2 Literature Review ......................................................................................................................... 2 2.1 Probiotics ....................................................................................................................................................... 2 2.1.1 Definition of probiotics ................................................................................................................ 2 2.1.2 Taxonomy of probiotics .............................................................................................................. 3 2.1.3 Mechanisms of action of probiotics ....................................................................................... 6 2.1.4 Applications of probiotics ........................................................................................................ 10 2.1.5 Safety and risk of probiotics ................................................................................................... 11 2.1.6 Probiotics market size .............................................................................................................. 12 2.2 Lactobacillus reuteri ............................................................................................................................... 13 2.2.1 History of L. reuteri .................................................................................................................... 13 2.2.2 Morphology and genome structure of L. reuteri ............................................................. 13 2.2.3 Safety and application of L. reuteri ...................................................................................... 16 2.2.4 The metabolic pathways of L. reuteri ................................................................................. 17 2.2.5 The mechanisms of action of L. reuteri ............................................................................. 21 2.2.6 Previous studies of L. reuteri DPC16 ................................................................................. 23 2.3 Reuterin ....................................................................................................................................................... 26 2.3.1 General information about reuterin ..................................................................................... 26 2.3.2 Mechanism of antimicrobial action of reuterin ................................................................ 28 2.3.3 Applications of reuterin ............................................................................................................. 29 2.3.4 Safety of reuterin ........................................................................................................................ 30 2.4 Production of reuterin ............................................................................................................................ 31 2.4.1 Chemical method........................................................................................................................ 31 2.4.2 Bacterial species used to produce reuterin ...................................................................... 33 2.4.3 Glycerol dehydratase enzyme ............................................................................................... 35 2.4.4 The structure of coenzyme Vb12 glycerol dehydratase ............................................... 37 2.4.5 Factors affecting glycerol dehydratase .............................................................................. 39 2.4.6 Current status of reuterin production using biosynthesis ........................................... 40 II 2.4.7 The significance of this project .............................................................................................. 41 2.4.8 Main research content .............................................................................................................. 42 Chapter 3 Materials and Methods ............................................................................................................ 44 3.1 Experimental materials ......................................................................................................................... 44 3.1.1 Bacterial strain and media ...................................................................................................... 44 3.1.2 Experimental reagents and equipment .............................................................................. 44 3.2 Experimental methods .......................................................................................................................... 45 3.2.1 Activation of L. reuteri DPC16 cells to prepare seed culture .................................... 45 3.2.2 Preparation of L. reuteri DPC16 glycerol seed cultures ............................................. 45 3.2.3 Time course of L. reuteri DPC16 batch fermentation .................................................. 45 3.2.4 Dry cell weight (DCW) measurement ................................................................................. 46 3.2.5 Standard curve between absorbance values and viable cell count ....................... 46 3.2.6 Reuterin production from glycerol ........................................................................................ 46 3.3 Analytical methods .................................................................................................................................. 47 3.3.1 Preparation of standard acrolein solution and tryptophan solutions ...................... 47 3.3.2 Determination of reuterin, 1,3-propanediol (1,3-PD), and glucose using HPLC ....................................................................................................................................................................... 48 3.3.3 Statistics method to analyze data ........................................................................................ 49 Chapter 4 Experimental results ................................................................................................................. 50 4.1 The growth of Lactobacillus reuteri DPC16 in batch culture ................................................. 50 4.1.1 The growth curve of Lactobacillus reuteri DPC16 ......................................................... 50 4.1.2 The standard curve of dry cell weight versus absorbance ........................................ 50 4.1.3 The standard curve of viable cell counts of DPC16 versus absorbance ............. 51 4.2 Reuterin production using resting-cells .......................................................................................... 52 4.2.1 Influence of biomass concentration on reuterin production....................................... 53 4.2.2 Influence of pH on reuterin production............................................................................... 56 4.2.3 Influence of glycerol concentration on reuterin production ........................................ 59 4.2.4 Influence of temperature on reuterin production ............................................................ 62 4.2.5 Influence of incubation time on reuterin production...................................................... 65 4.2.6 Influence of culture age on reuterin production of DPC16......................................... 68 4.2.7 Statistical analysis for reuterin production ........................................................................ 70 Chapter 5 Discussion ...................................................................................................................................
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