Type of intrinsic resistant starch type 3 determines in vitro fermentation by pooled adult faecal inoculum.

Resistant starch (RS) results in relatively high health-beneficial butyrate levels upon fermentation by gut microbiota. We studied how physico-chemical characteristics of RS-3 influenced butyrate production during fermentation. Six highly resistant RS-3 substrates (intrinsic RS-3, 80-95 % RS) differing in chain length (DPn 16-76), Mw distribution (PI) and crystal type (A/B) were fermented in vitro by pooled adult faecal inoculum. All intrinsic RS-3 substrates were fermented to relatively high butyrate levels (acetate/butyrate ≤ 2.5), and especially fermentation of A-type RS-3 prepared from polydisperse α-1,4 glucans resulted in the highest relative butyrate amount produced (acetate/butyrate: 1). Analysis of the microbiota composition after fermentation revealed that intrinsic RS-3 stimulated primarily Lachnospiraceae, Bifidobacterium and Ruminococcus, but the relative abundances of these taxa differed slightly depending on the RS-3 physico-chemical characteristics. Especially intrinsic RS-3 of narrow disperse Mw distribution stimulated relatively more Ruminococcus. Selected RS fractions (polydisperse Mw distribution) obtained after pre-digestion were fermented to acetate and butyrate (ratio ≤ 1.8) and stimulated Lachnospiraceae and Bifidobacterium. This study indicates that especially the α-1,4 glucan Mw distribution dependent microstructure of RS-3 influences butyrate production and microbiota composition during RS-3 fermentation.

Digestibility of resistant starch type 3 is affected by crystal type, molecular weight and molecular weight distribution.

Resistant starch type 3 (RS-3) holds great potential as a prebiotic by supporting gut microbiota following intestinal digestion. However the factors influencing the digestibility of RS-3 are largely unknown. This research aims to reveal how crystal type and molecular weight (distribution) of RS-3 influence its resistance. Narrow and polydisperse α-glucans of degree of polymerization (DP) 14-76, either obtained by enzymatic synthesis or debranching amylopectins from different sources, were crystallized in 12 different A- or B-type crystals and in vitro digested. Crystal type had the largest influence on resistance to digestion (A >>> B), followed by molecular weight (Mw) (high DP >> low DP) and Mw distribution (narrow disperse > polydisperse). B-type crystals escaping digestion changed in Mw and Mw distribution compared to that in the original B-type crystals, whereas A-type crystals were unchanged. This indicates that pancreatic α-amylase binds and acts differently to A- or B-type RS-3 crystals.

The influence of α-1,4-glucan substrates on 4,6-α-d-glucanotransferase reaction dynamics during isomalto/malto-polysaccharide synthesis.

Starch-based isomalto/malto-polysaccharides (IMMPs) are soluble dietary fibres produced by the incubation of α-(1 → 4) linked glucans with the 4,6-α-glucanotransferase (GTFB) enzyme. In this study, we investigated the reaction dynamics of the GTFB enzyme by using isoamylase debranched starches as simplified linear substrates. Modification of α-glucans by GTFB was investigated over time and analysed with 1H NMR, HPSEC, HPAEC combined with glucose release measurements. We demonstrate that GTFB modification of linear substrates followed a substrate/acceptor model, in which α-(1 → 4) linked glucans DP ≥ 6 functioned as donor substrate, and α-(1 → 4) linked malto-oligomers DP < 6 functioned as acceptor. The presence of α-(1 → 4) linked malto-oligomers DP < 6 resulted in higher GTFB transferase activity, while their absence resulted in higher GTFB hydrolytic activity. The information obtained in this study provides a better insight into GTFB reaction dynamics and will be useful for α-glucan selection for the targeted synthesis of IMMPs in the future.

Enzymatic fingerprinting of isomalto/malto-polysaccharides.

In this study, we present an enzymatic fingerprinting method for the characterization of isomalto/malto-polysaccharides (IMMPs). IMMPs are produced by the modification of starch with the 4,6-α-glucanotransferase (GTFB) enzyme and consist of α-(1→4), α-(1→6) and α-(1→4,6) linked glucoses. Enzymes were used separately, simultaneously or in successive order to specifically degrade and/or reveal IMMP substructures. The enzymatic digests were subsequently analysed with HPSEC and HPAEC to reveal the chain length distribution (CLD) of different IMMP substructures. The presence of amylose in the substrate resulted in the formation of linear α-(1→6) linked glycosidic chains (13.5 kDa) in the former amylopectin fraction. The length of these chains indicates that GTFB transferase activity on amylopectin is more likely to elongate single amylopectin chains than to provide an even distribution. Enzymatic fingerprinting also revealed that the GTFB enzyme is capable of introducing large (20 kDa) linear α-(1→6) linked glycosidic chains in the α-glucan substrate.

Isomalto/malto-polysaccharide structure in relation to the structural properties of starch substrates.

Isomalto/malto-polysaccharides (IMMPs) are soluble dietary fibres produced by the enzymatic modification of starch with 4,6-α-glucanotransferase (GTFB). The structure, size, and linkage distribution of these IMMPs has remained largely unknown, since most structural information has been based on indirect measurements such as total α-(1→6) content, iodine staining and GTFB hydrolytic activity. This study provides a deeper understanding of IMMP structure in relation to its respective starch substrate, by combining preparative fractionation with linkage compositition analysis. IMMPs were produced from a variety of amylose-rich and amylose-free starches. The extent of modification was investigated per IMMP molecular weight (Mw)-fraction, distinguishing between linear α-(1→6) linkages introduced by GTFB and starch's native α-(1→4,6) branching points. It emerged that the amount of α-(1→6) linkages was consistently higher in IMMP low Mw-fractions and that GTFB activity was limited by native α-(1→4,6) linkages. The presence of amylose turned out to be a prerequisite for the incorporation of linear α-(1→6) linkages in amylopectin.