GOAT MILK OLIGOSACCHARIDES PURIFICATION AND SELECTED BIFIDOBACTERIA CARBOHYDRATE UTILISATION Caroline Thum, PhD student
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1 GOAT MILK OLIGOSACCHARIDES PURIFICATION AND SELECTED BIFIDOBACTERIA CARBOHYDRATE UTILISATION Caroline Thum, PhD student 04/06/2011
2 Introduction Maternal diet Fermentable oligosaccharides Breast feeding in utero Maternal intestinal microbiota Vaginal delivery
3 Work motivation Fermentable oligosaccharides 1. Improved immune response; 2. Growth stimulation of healthy bacteria in intestinal flora; 3. Preventing adhesion of pathogens to intestinal epithelial tissues; 4. Increased absorption of minerals; 5. Improved Produced glucose homeostasis. and extracted from plants Synthetically produced from lactose Bifidobacteria Lactobacillus sp Clostridium perfringens Escherichia coli Staphylococcus sp.
4 Project objective Evaluate the effect of goat milk oligosaccharides (GO) on dam s intestinal microbiota, milk composition and subsequent offspring s intestinal tissue development and maturation Aims: 1. Purify oligosaccharides from goat liquid whey. 2. Evaluate human bifidobacterial strains for their ability to ferment GO during in vitro batch culture.
5 Purification of oligosaccharides from goat liquid whey 2- Centrifuge 1- Pasteurised goat whey Fat + Proteins Protein + Lactose + Oligosaccharides 3- Ultrafiltration (10kDa) 4- β-galactosidase (>95% hydrolyses) 5- Centrifuge Proteins Lactose + Oligosaccharides Monosaccharides Monosaccharide + Oligosaccharides 6- Adsorption in Activated carbon Oligosaccharides + monosaccharides
6 Evaluation of human bifidobacterial strains for their ability to ferment GO during in vitro batch culture Isolation Bifidobacteria isolated from faeces of 4 breast-fed babies. Cultivation Grow in anaerobic MTPY media. Identification Bifidobacteria strains identified by 16S rrna: RAPD Baby Strain Fermentation 4 Bifidobacterium bifidum 1 1 Bifidobacterium breve 2 5 Bifidobacterium longum subsp longum 3 3 Bifidobacterium breve 1 Bifidobacterium longum subsp longum 4 5 Bifidobacterium breve Total 19 isolates
7 Evaluation of human bifidobacterial strains for their ability to ferment GO during in vitro batch culture Isolation Bifidobacteria isolated from faeces of 4 breast-fed babies. Cultivation Grow in anaerobic MTPY media. Identification Bifidobacteria strains identified by 16S rrna: RAPD Genetically Fermentation differentiation among isolates Primer Name Sequence (5 to 3 ) P2 GAT CGG ACG G P15 CTG GGC ACG A P16 TCG CCA GCC A P17 CAG ACA AGC C PER1 AAG AGC CCG T CC1 AGC AGC GTG G CORR1 TGC TCT GCC C Method extracted from J. Krizova et al, Folia Microbiol, 53 (2),
8 RAPD B. breve
9 RAPD B. bifidum & B. longum
10 Evaluation of human bifidobacterial strains for their ability to ferment GO during in vitro batch culture Isolation Bifidobacteria isolated from faeces of 4 breast-fed babies. Cultivation Grow in anaerobic MTPY media. Identification Bifidobacteria strains identified: 4 Bifidobacterium bifidum 7 Bifidobacterium longum subsp longum 8 Bifidobacterium breve RAPD 6 different PCR profiles 1 Bifidobacterium bifidum 2 Bifidobacterium longum subsp longum 3 Bifidobacterium breve Fermentation
11 Sugar fermentation Defined, semi-synthetic medium No carbohydrate (No CHO) or carbohydrates supplemented at 1% w/v Anaerobic environment Abs 600nm AGR Bif. bifidum Glucose Oligofructose Inulin Lactulose No CHO Time (hours) AGR Bif. longum AGR Bif. longum Abs 600nm Glucose Oligofructose Inulin Lactulose No CHO Abs 600 nm Glucose Oligofructose Inulin Lactulose No CHO Time (hours) Time (hours)
12 Sugar fermentation AGR Bif. breve AGR Bif. breve Abs 600 nm Time (hours) Glucose Oligofructose Inulin Lactulose Abs 600nm No CHO Time (hours) Glucose Oligofructose Inulin Lactulose No CHO AGR Bif. breve ABS 600nm Glucose Oligofructose Inulin Lactulose No CHO Time (hours)
13 Future work Process goat liquid whey to obtain enough GO needed to in vitro and in vivo studies. Growth and fermentation profiles of all selected strains in milk oligosaccharide supplemented media. The best bacterial fermentors of purified GO will be inoculated into germ free mice to evaluate their intestinal colonisation and the milk composition of the lactating dams.
14 Acknowledgments Supervisors Dr. Don Otter Dr. Adrian Cookson Dr. Jolon Dyer Dr. Ali Hodgkinson Dr. Nicole Roy Professor Warren McNabb Funding AgResearch Fellowship Riddet Institute (CoRE project)
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