Studies on the microbiological quality and associated decontamination of reconstituted infant milk formulae and enteral feeds commercially available in Ireland / by Phillipa Noelle Haughton.

Project funded by the Department of Agriculture Food and Fisheries.

Master of Science thesis by research, 2008.

Includes bibliographical references.

Contents Page Acknowledgements i Contents ii 1.0 Abstract 1 2.0 Introduction 3 2.1 Infant milk formula 4 2.1.1 Pathogenic microorganisms associated with Infant Milk Formula 6 2.1.1.1 Enterobacter sakazakii 6 2.1.1.2 Bacillus cereus 7 2.2 Enteral nutrition 9 2.2.1 Contamination of enteral feeds 10 2.3 Control of microbial contamination of hospital prepared feeds 11 2.4 Thermal processing 11 2.4.1 Limitations to thermal processing 12 2.5 Pulsed Power Technologies 13 2.6 Pulsed Electric Fields 13 2.6.1 Mechanism of inactivation by pulsed electric fields 14 2.6.2 PEF Parameters influencing microbial inactivation 14 2.6.2.1 Electrical parameters 14 2.6.2.2 Treatment medium properties 16 2.6.2.3 Microorganism characteristics 17 2.6.3 Quality of PEF treated foods 18 2.7 Pulsed Ultraviolet Light 19 2.7.1 Mechanism of inactivation by PUV 19 2.7.2 Factors affecting microbial inactivation by PUV 20 2.7.3 Current Applications and Quality of Foods Treated by PUV 20 2.8 Hurdle Technologies 21 iii 3.0 Materials and Methods 23 3.1 Microorganisms 23 3.1.1 Microbial test strains 23 3.1.2 Preparation of fresh bacteria and yeast broth cultures 24 3.1.3 Enumeration of samples 24 3.2 Preparation of Feeds 25 3.2.1 Reconstitution of infant milk formulae 25 3.2.2 Preparation of enteral feeds 26 3.3 Bacteriological Analysis of Feeds 26 3.3.1 Infant milk formula 26 3.3.2 Recovery of Enterobacter sakazakii from infant milk formula 26 3.3.3 Identification of isolated Bacillus. spp from infant milk formula 27 3.3.4 API 50 CHB biochemical identification strips 28 3.3.5 Detection of Bacillus cereus enterotoxin using the Bacillus cereus enterotoxin Reverse-Passive Latex Agglutination Assay Kit (BCET-RPLA). 30 3.3.5.1 Preparation of B. cereus isolates from IMF for enterotoxin production 30 3.3.5.2 Assay principle 30 3.3.5 Enteral feeds 31 3.4 Growth of Microorganisms in Enteral Feeds 32 3.4.1 Preparation of microorganisms for growth studies in enteral feeds. 32 3.4.2 Confirmation of viability of microorganisms suspended in IMF and enteral feeds. 32 3.5 Thermal Inactivation Studies 33 3.5.1 Preparation of cells for thermal inactivation 33 3.6 Pulsed Ultraviolet Light System 33 3.6.1 Use of PUV for microbial inactivation studies 33 3.6.2 Preparation of liquid samples for PUV Inactivation 35 3.6.3 Treatment of liquid samples by PUV 35 3.6.4 PUV inactivation of microorganisms on agar media 35 3.7 Microbial Inactivation by Electroporation 36 3.7.1 Samtech® Pulsed Electric Field System (PEF) 36 3.7.2 Conductivity and pH of feeds 37 3.7.3 Preparation of samples for PEF treatment and calculation of flow rate 37 3.7.4 PEF treatment of samples 38 3.7.5 Bio-Rad® Gene Pulser Xcell 38 iv 3.7.6 Preparation of samples for treatment with Gene Pulser X-Cell 39 3.7.7 Treatment of samples 40 3.8 Statistical Analysis 40 4.0 Results 42 4.1 Microbiological Analysis of Feeds 42 4.1.1 Microbiological analysis of infant milk formula and enteral feeds 42 4.1.2 Isolation and identification of B. cereus from infant milk formula 44 4.1.3 Detection of Bacillus cereus Enterotoxin 45 4.2 Growth rate of microorganisms inoculated into Enteral Feeds 46 4.3 Confirmation of viability of microorganisms suspended in IMF and enteral feeds 47 4.4 Thermal Inactivation of Microorganisms 48 4.5 Inactivation of Microorganisms using Pulsed Ultraviolet Light 53 4.5.1 PUV inactivation of microorganisms in liquids 53 4.5.2 PUV inactivation of microorganisms on surfaces 56 4.6 Electroporation of microorganisms with the Biorad®Gene Pulser Xcell and Samtech® PEF system Microbial Inactivation with Pulsed Electric Fields 60 5.0 Discussion 67 5.1 Microbiological Analysis of Infant Milk Formula and Enteral Feeds 67 5.2 Growth of Microorganisms in Enteral Feeds 69 5.3 Microbial Inactivation 70 5.3.1 Thermal inactivation of microorganisms 70 5.3.2 Inactivation of Microorganisms by Pulsed UV Light 71 5.3.2.1 PUV inactivation of liquid samples 71 5.3.2.2 PUV inactivation of bacteria on surfaces 73 5.3.3 Inactivation of microorganisms by electric field pulses 73 5.3.3.1 Studies investigating the influence of process parameters 74 5.4 Conclusion 78 6.0 References 80 7.0 Appendix 97 7.1 Media 97 7.1.1 Broths 97 v 7.1.2 Agar 97 7.1.3 Diluents and sterilising solutions 98 7.2 Equipment 98 7.2.1 Centrifuge 98 7.2.2 Microscope 98 7.2.3 Digital Camera 99 7.2.4 Autoclave 99 7.2.5 pH Meter 99 7.2.6 Other Equipment