In vitro and in vivo fermentation behaviour of prebiotic carbohydrates
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1 Nov 30th, 2017 Zwolle In vitro and in vivo fermentation behaviour of prebiotic carbohydrates Fangjie Gu & Henk Schols Laboratory of Food Chemistry
2 Introducing prebiotic fibres Resistant to human GI digestion Fermentable by the large intestinal microbiota Selectively stimulate beneficial microbes Substances - Isomalto/malto-polysaccharides (IMMPs) - Human milk oligosaccharides (HMOs)
3 IMMP as dietary fibre or prebiotic Enzyme-modified starch α-1,4-glycosidic linkages àα-1,6- glycosidic linkages Variable level of α-1,6-linkage levels (van de Zaal et al Carbohydr Polym 2017 in press)
4 Fermentation of IMMP 96 using human inoculum - 12 h delay of microbial utilization of α-1,6-linked glucose - Full utilization of IMMPs within 48 h Analyzed by HPAEC-PAD
5 Fermentation of IMMP 27 using human inoculum - Direct utilization of α-1,4-linked glucose by microbiota - Presence and utilization of α-1,4-linked glucose postponed the complete utilization of α-1,6 glc chains Analyzed by HPAEC-PAD
6 In vitro fermentation behavior of IMMPs q Isomalto-oligosaccharides were gradually released during in vitro fermentation. q The presence of α-1-4 linked glucose in IMMPs postponed the bacterial utilization of α-1,6-linked glucose. q Shorter α-1-6 linked glucose chains speed up the fermentation. q Fermentation of IMMPs promoted the growth of bifidobacteria and lactobacilli. q IMMPs are slowly-fermentable fibres with prebiotic potential. q Poster q In vivo study on mice has been performed (collaboration with other SPs).
7 Fate of HMOs in Infant GI metabolisation Breast milk HMOs as substrate Source of bifidobacteria Faeces e.g. LNT contains Lacto-N-biose First time acquiring bacteria Glycosidase Fucosidase Sialidase Encode GI bacteria establishment and growth Bifidobacteria B. infantis; B. bifidum; B. breve; Bacteroides
8 Milk and faecal samples q KOALA cohort Maastricht University 146 pair of mother-infant Milk and faecal samples 1 month after birth q BINGO onderzoek Radboud University 80 pair of mother-infant Milk and faecal samples at 3 time points (2wk, 6wk, 12wk)
9 1 month-old infants showed different consumption patterns Type A: complete consumption Analyzed by PGC-UPLC-MS Intensity Milk Faeces Indication linkage type Time (min)
10 1 month-old infants showed different consumption patterns Type B: selective consumption Analyzed by PGC-UPLC-MS Intensity Neutral Milk Sialylated Faeces Indication linkage type Time (min)
11 1 month-old infants showed different consumption patterns Type C: Non-selective and low consumption Analyzed by PGC-UPLC-MS Intensity Milk Faeces Indication linkage type Time (min)
12 Preference to sialylated HMO isomers q Linkage type: α 2-3 linked sialic acid is more utilized than α 2-6 q Differences for individual HMO structures > > > ~ 3 SL LST a 6 SL LST b LST c More prefered
13 Preference to fucosylated HMOs Mono-fucosylated or multi-fucosylated FL Milk Analyzed by PGC-UPLC-MS LNFP Faeces Milk Faeces Mono-fucosylated HMOs were mostly utilized MFLNH Milk Faeces
14 Preference to fucosylated HMOs Mono-fucosylated or multi-fucosylated LNDFH Milk Analyzed by PGC-UPLC-MS DFLNH Faeces Milk Faeces Di- and tri- fucosylated HMOs were largely retained MFLNH Milk Faeces
15 Fermentation of HMOs by 1-mon-old infants q Huge inter-individual differences were found in the HMO composition among different mother milk samples q Huge inter-individual differences were found in HMO utilisation in digestive tract of baby q The consumption patterns can be clustered according to the level and selectivity of utilization of HMOs by infant gut microbiota q Not easy to relate HMO utilization to milk type, faecal microbiota composition or characteristics recorded for mother and child q One time point limits interpretation and correlations with essential parameters
16 Variation of HMOs in milk over lactation time Concentrations (µg/ml) 4500, , , , , , , ,00 500,00 0,00 All milk groups - concentrations fucosylated sialylated non-fucosylated and non-sialylated 2wk 6wk 12wk Relative amout 80,0% 70,0% 60,0% 50,0% 40,0% 30,0% 20,0% 10,0% 0,0% HMO concentration decreased over time Fucosylated HMOs increased in relative amounts in time All milk groups-relative amounts fucosylated sialylated non-fucosylated and non-sialylated 2wk 6wk 12wk
17 Remaining HMOs in infant faeces HMO concentrations in faeces Concentrations (µg/ml) 1000,00 900,00 800,00 700,00 600,00 500,00 400,00 300,00 200,00 100,00 0,00 2wk 6wk 12wk Indication linkage type HMOs decreased over time LNDFHI mostly retained
18 Remaining HMOs in infant faeces µg/ml 2500, , , ,00 500,00 0,00 Concentrations fucosylated sialylated non-fucosylated and nonsialylated 2wk 6wk 12wk Trend over time: similar pattern as for milk HMOs Relative amounts 100,0% 80,0% 60,0% 40,0% 20,0% 0,0% fucosylated sialylated non-fucosylated and non-sialylated 2wk 6wk 12wk
19 Effects of external factors on GI microbiota q Delivery mode - First time acquiring bacteria Vaginal delivery: vaginal microbiota, e.g. bifidobacteria; Caesarean section: mother skin or environment. q Feeding type Growth of bacteria Breast-feeding exclusively; Infant formula introduced
20 Faeces: HMO digestion patterns (Secretor) classified by multi-stages and similarity LST a LST b LST c 3SL 6SL LNnH LNH LNT DFL LNDFH I LNFP V LNFP I LNFP II LNFP III 2FL 3FL Vaginally delivered and exclusively breast-fed babies (V-eB); Vaginally delivered and combination-fed babies (V-Comb); Caesarean section delivered babies (Cs) Faecal HMOs absolute amount mg/g # w6 007w12 019w12 087w6 023w12 087w12 025w12 101w12 033w12 140w12 019w6 020w12 020w6 087w2 140w6 023w6 039w6 039w2 019w2 007w2 101w6 033w2 015w12 021w12 020w2 018w12 099w12 018w6 023w2 021w6 021w2 018w2 140w2 039w12 101w2 099w2 099w6 015w2 015w6 20
21 Faeces: HMO digestion patterns (Secretor) classified by multi-stages and similarity LST a LST b LST c 3SL 6SL LNnH LNH LNT DFL LNDFH I LNFP V LNFP I LNFP II LNFP III 2FL 3FL Vaginally delivered and exclusively breast-fed babies (V-eB); Vaginally delivered and combination-fed babies (V-Comb); Caesarean section delivered babies (Cs) Faecal HMOs absolute amount mg/g #023 #021 stage I-a // slow digestion 007w6 007w12 019w12 087w6 023w12 087w12 025w12 101w12 033w12 140w12 019w6 020w12 020w6 087w2 140w6 023w6 039w6 039w2 019w2 007w2 101w6 033w2 015w12 021w12 020w2 018w12 099w12 018w6 023w2 021w6 021w2 018w2 140w2 039w12 101w2 099w2 099w6 015w2 015w6 21
22 Faeces: HMO digestion patterns (Secretor) classified by multi-stages and similarity LST a LST b LST c 3SL 6SL LNnH LNH LNT DFL LNDFH I LNFP V LNFP I LNFP II LNFP III 2FL 3FL Vaginally delivered and exclusively breast-fed babies (V-eB); Vaginally delivered and combination-fed babies (V-Comb); Caesarean section delivered babies (Cs) Faecal HMOs absolute amount mg/g #021 stage I-a #023 stage I-a à I-b à II, general digestion process 007w6 007w12 019w12 087w6 023w12 087w12 025w12 101w12 033w12 140w12 019w6 020w12 020w6 087w2 140w6 023w6 039w6 039w2 019w2 007w2 101w6 033w2 015w12 021w12 020w2 018w12 099w12 018w6 023w2 021w6 021w2 018w2 140w2 039w12 101w2 099w2 099w6 015w2 015w6 22
23 Faeces: HMO digestion patterns (Secretor) classified by multi-stages and similarity LST a LST b LST c 3SL 6SL LNnH LNH LNT DFL LNDFH I LNFP V LNFP I LNFP II LNFP III 2FL 3FL Vaginally delivered and exclusively breast-fed babies (V-eB); Vaginally delivered and combination-fed babies (V-Comb); Caesarean section delivered babies (Cs) #101 from disordered to normal digestion Faecal HMOs absolute amount mg/g #021 stage I-a #023 stage I-a à I-b à II, general digestion process 007w6 007w12 019w12 087w6 023w12 087w12 025w12 101w12 033w12 140w12 019w6 020w12 020w6 087w2 140w6 023w6 039w6 039w2 019w2 007w2 101w6 033w2 015w12 021w12 020w2 018w12 099w12 018w6 023w2 021w6 021w2 018w2 140w2 039w12 101w2 099w2 099w6 015w2 015w6 23
24 Fermentation of HMOs over lactation time Milk Concentrations of breast milk HMOs decreased during lactation Fucosylated HMOs showed an increase in relative amount Faeces The concentrations of faecal HMOs showed similar decreases as milk HMOs LNDFH I remained in the highest concentration in infant faeces till later time points External factors (feeding type, delivery mode) affect the HMO digestion mainly in the early time points
25 General conclusions Methods are present to evaluate the prebiotic potential of individual fibers by in vitro and in vivo fermentation. High-throughput analytical methods (PGC-UPLC-MS, HPAEC and NMR) are optimized to determine HMO profiles of mother milk and infant faecal samples. The problem of under-estimation of 3 - Fucosyllactose has been solved. The involvement of large sample number of both mother milk and infant faeces, as well as different time points over lactation time, lead to more comprehensive observations. The correlations between HMO fermentation profiles and microbiota data are studied by collaboration with Klaudyna from SP4.
26 Acknowledgement Wageningen University: Klaudyna Borewicz, Hauke Smidt, Bernadette Richter, Peter H. van der Zaal, AVEBE Food Innovation Center: Piet Buwalda Groningen University: Geralt A. ten Kate, Sander S. van Leeuwen UMCG: Rima Mistry, Uwe Tietge Maastricht University: Ilja Arts, John Penders, Carel Thijs Radboud University: Christine Hechler, Roseriet Beijers, Carolina de Weerth CCC3 Partners:
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