Primary cell wall - architecture Xyloglucan Xylan Galactan? - PDF document

9/20/2017 Physiciochemical characteristics of pectin as dietary fiber and its role in colonic health Henk A. Schols Laboratory of Food Chemistry Primary cell wall - architecture � Xyloglucan � Xylan � Galactan? McCann et al. 1992 J. microsc., 166, 123-136 1

9/20/2017 Pectin plays an important role as: � Cell Wall component ● Determine (partly) texture of tissues ● Change during ripening of e.g. fruits (endogenous enzymes) ● Change during processing due to depolymerization, de- esterification, solubilization, etc. ● Present in by-products from agro industry � Ingredient for food industry ● Modification during extraction and down-stream processing ● Thickener and gelling agent ● Stabilizer in fruit and milk beverages � Health promoting component ● Lowering cholesterol levels ● Dietary Fiber & fermentation ● Immuno modulating Schematic structure of pectic polysaccharides 2

9/20/2017 Occurrence and proportion of the various structural elements of native pectin in apple, sugar beet and soybean soybean meal sugar beet pulp apple total polysaccharide (w/w % dm) 16 67 20 pectic substances (% of total PS) 59 40 42 structural element (% of pectic substances) homogalacturonan 0 29 36 xylogalacturonan 21 <1 4 rhamnogalacturonan II 4 4 10 rhamnogalacturonan backbone 15 8 4 arabinan 60 46 27 arabinogalactan I 12 20 arabinogalactan II 0 0 + Same building blocks!! However, ‘always’ different fine-structure and different relative amounts! Pectin as food ingredient � HM Pectin: sugar-acid gel ● Sugar lowers water activity and increase chain-chain interaction (= gel) ● Low pH reduces acidity – less interaction – more chain- chain interaction � LM Pectin ● Calcium-pectate gel ● Low sugar jams 3

9/20/2017 Parameters influencing food applications of pectins COOH COOH COOCH 3 COOH COOCH 3 COOCH 3 COOH COOH COOH COOCH 3 O O O O O O O O H O O H H H H H H H H H H H H H H H H H H O O H O O O O O O O O OH H OH H OH H OH H H OH H OH H H H H OH H OH H H H OH H OH H H H H H H OH H OH H OH H OH H OH H OH H OH H OH H OH H OH � Raw material used � Extraction conditions used � Down stream modification (HM vs LM pectin, amidation) � Molecular weight � Degree of methyl esterification (and acetyl groups if present) � Distribution of methyl esters (and acetyl groups if present) � Calcium-sensitivity � Solubility Homogalacturonan key parameters: Level and distribution of methyl esters Random Sequential Blockwise Combi Pectin functionalities are strongly determined = Galacturonic acid (GalA) = methyl-GalA by level and distribution of the methyl esters 4

9/20/2017 Characterisation of pectins using enzymatic fingerprinting Use of polygalacturonase (PG) and pectine lyase (PL) to degrade pectins into diagnostic oligomers PG: Release of saturated galacturonic acid oligomers PL: Release of unsaturated galacturonic acid oligomers Homogalacturonan: Finger printing of methyl ester groups using PG Random Sequential Blockwise Combi = Galacturonic acid (GalA) = methyl-GalA = site of attack endo-PG 5

9/20/2017 MALDI TOF MS of random esterified DM70 pectin digested by endo-PG 9 5 9 6 3 1 10 6 10 7 11 7 12 8 11 6 4 2 10 5 11 8 13 8 12 7 13 9 14 10 5 2 4 1 6 3 3 0 5 3 7 4 6 4 8 4 8 5 7 3 7 5 6 2 8 6 4 2 = GalA 4 2 methyl HILIC-Iontrap-MS n method: methyl-esterified pectin oligomers 5 1 = GalA 5 1 methyl 6

9/20/2017 HILIC-Iontrap-MSn method is also capable to separate and identify complex mixtures of oligomers after PG and PL digestion of Sugar Beet Pectin RT: 5.00 - 50.00 SM: 7B 4 10 45000 3 00 40000 35000 Saturated Unsaturated 30000 Intensity 4 11 25000 6 21 20000 4 10 4 20 2 00 15000 6 11 5 11 4 21 10000 5 21 8 31 7 21 8 22 5 20 5000 9 32 0 5 10 15 20 25 30 35 40 45 Time (min) Annotation : 4 10 = 4 galacturonic acid unit; 1 methyl group. 0 acetyl groups HILIC-MS/MS fragmentation enables the structure elucidation of SBP oligomers 5 11 ( m/z 953) (759) (583) (365) Z 4 + 1Me+ 1Ac Z 3 + 1Me + 1Ac Z 2 + 1Me Z 1 COOH COOMe COOH COOH COOH O HO O O O O O O HO O HO OH O HO OH HO O - OA c HO OH H OH H H 0,2 A 5 0,2 A 4 0,2 A 3 + 1Ac 0,2 A 2 (717) (893) (485) C 1 C 3 + 1Ac C 4 + 1Me+ 1Ac (309) C 2 (587) (777) (369) Z 3 + 1Me + 1Ac B 583 100 Precise annotation of methyl ester and O-2 and O-3 acetyl 90 group position by ring cleavage MS/MS fragmentation Z 2 + 1Me C 3 + 1Ac 80 365 O-3 587 70 C 2 C 4 + 1Me+ 1Ac O-2 Relative Abundance 777 369 60 50 40 Z 4 + 1Me + 1Ac 30 0,2 A 5 759 0,2 A 2 Z 2 0,2 A 3 893 20 [M- H] -1 – H 2 O 351 Z 3 + 1Me 309 485 0,2 A 4 541 10 935 481 717 291 0 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z 7

9/20/2017 Introduction of descriptive parameters Degree of Blockiness and Degree of Hydrolysis PG PL DB abs DH PG DH PL GalA Methylester O-2 Acetyl O-3 Acetyl DB does quantify unsubstituted mono-, di- and tri GalA oligomers as released by PG DH PG does also include PG released methyl esterified segments of the pectin DH PL does quantify PL released highly methyl esterified / acetylated oligomers from the pectin Pectin is a dietary fibre Official AACC definition of dietary fibre � Dietary fibre is the edible parts of plants or analogous carbohydrates that are resistant to digestion and adsorption in the human small intestine with complete or partial fermentation in the large intestine. Dietary fibre includes polysaccharides, oligosaccharides, lignin and associated plant substances. Dietary fibres promote beneficial physiological effects including laxation, and/or blood cholesterol attenuation, and/or blood glucose attenuation. 8

9/20/2017 Batch fermentation of DF + Simple, easy to prepare, ‘one bottle’ fermentation. + Buffer-regulated pH + Possibility to do fermentation with small amount of substrate. + Fast, a lot of substrates can be tested simultaneously. - Change of pH during fermentation - Accumulation of fermentation products - Depletion of substrates Monitoring in vitro fermentation (human fecal inoculum) � Optical Density � pH curve � Short Chain Fatty Acids � Remaining dietary fibers (after hydrolysis) � NEED TO ANALYSE INDIVIDUAL MOLECULES Soy pectin Optical Density of various substrates pH curve of soy pectin SCFA profiles of Oligofructose 8.0 70 7.5 (different buffer systems) 7.5 60 7.0 Concentration 7.0 50 6.5 (umol/ml) 6.5 pH 40 6.0 6.0 pH 5.5 30 5.5 5.0 20 5.0 4.5 10 4.5 4.0 0 4.0 0 10 20 30 40 50 0 10 20 30 40 50 Time (h) Time (h) 9

9/20/2017 In vivo piglet trial Soluble pectin supplementation to diet autoclaved citrus pectin soybean meal LMP/HMP Soy pectin 3% 3% + + + + + wheat AX wheat AX + + wheat based diet wheat based diet + TiO 2 Soy bean meal TiO 2 0.25% 0.25% Sample and data collection Digesta present in: � Ileum � Proximal colon ( pCol ) � Mid colon ( mCol ) � Distal colon ( dCol ) Analysis of : � Dietary Fibre + fragments � Short Chain Fatty Acids � Digestibility marker � Microbiota composition Gastrointestinal tract of pig (Stevens et al., 1998). 10

9/20/2017 Constituent monosaccharide composition of total NSP in the digesta Constituent monosaccharide composition of total NSP in the digesta � Starch remains more dominantly present in pectin diets � Digestibility depends on pectin methyl esterification and complexity � Different dietary fibers are fermented differently � For quan4ta4ve data → diges4bility marker was added 11

9/20/2017 Apparent digestibility of pectin � Pectin is highly fermentable. � Fermentation profile is different for LM and HM pectin � Although same soy pectin structures are present in CONT and aSBM, pec4ns present are differently fermented → soluble vs insoluble soy pec4n Apparent digestibility of starch and Dietary Fibre (NSP) � Starch digestion is slightly lower in presence of pectin � Digestibility depends on pectin methyl esterification � Also Dietary Fibre (NSP) fermentation is postponed to distal part of colon in the presence of pectin 12

9/20/2017 Apparent digestibility of glucose and xylose (for cellulose and cereal xylan respectively) � Cellulose and Xylan fermentation is postponed to the more distal part of colon in the presence of pectin � Differences in apparent fermentability are smaller for the feces! Summary: w/w percentage of digested starch, pectin and NSP in different sections DF Diet CONT: control diet; LMP: low methyl-esterified pectin enriched diet; HMP: high methyl-esterified pectin enriched diet; aSBM: autoclaved soybean meal enriched diet. 13

9/20/2017 Concentration of SCFAs in digesta of piglets fed with different diets Relative abundance of microbiota at the genus level Lactobacillus Prevotella Microbiota pattern, typical for plant eaters U(Ruminococcaceae) Megasphaera 14

9/20/2017 Relative abundance of microbiota at the genus level Lactobacillus ↓ Prevotella ↑ U(Ruminococcaceae) ↑ Megasphaera ↑ Link between microbiota, SCFAs and fiber digestibility 15