Starch: Image contrast in AFM

Starch is the major carbohydrate consumed by mankind. Partial breakdown of starch in the stomach and the small intestine contributes to blood glucose levels. The remaining resistant starch (RS) is fermented in the colon into short chain fatty acids, mainly butyrate. RS is claimed to be beneficial in reducing the risk of colon cancer.

At IFR we have produced a systematic description of the molecular basis of starch gelation and retrogradation, identifying the roles played by the starch polysaccharides amylose and amylopection. Understanding starch structure in foods provides a basis for interpreting starch digestion and colonic fermentation.

We are studying starch structure in order to identify ways of controlling starch digestion. This includes work on the effects of genetic mutations on granule structure and on the mechanisms of enzymatic digestion of crystalline starch.

As part of these studies we are using AFM to probe the ultra-structure of starch granules. In order to image the internal structure of starch granules we have to cut the granules open. This is done by encasing the starch in a block of non-penetrating resin. The top surface of the block is honed down with a glass knife to produce a surface consisting of starch granules with cut surfaces exposed.

 

AFM images of these samples show the presence of the granules but no internal structure is revealed.

AFM images of un-wetted, cut faces of pea starch granules encased in Araldite.
(a) Topography and (b & c) 'error-signal' mode images.
The scan sizes are: (a) & (b) 80 x 80 µm, and (c) 8.7 x 8.7 µm.

However, wetting the sample either through cutting sections, or simply wetting the surface of the cut block, reveals the internal structure of the granule.

Effect of wetting on the images of pea starch encased in Araldite
(a) Topography, (b) 'error-signal' mode and (c) left-shaded topography images.
Scan sizes are all 35.5 x 35.5 µm.

Higher-resolution AFM topography image of a pea starch granule showing the blocklet structure within the granule

We suggest that the contrast is induced by selective hydration of regions of the sample leading to softening and swelling of these areas.

Further, we believe that the amorphous amylose within the granule preferentially absorbs water and swells exposing the harder semi-crystalline amylopectin; present as blocklet structures within the granule. These blocklets are visible in higher-resolution AFM images of the granule structure.

Further Reading

Parker M. L. , Kirby A. R. & Morris V. J.
In situ imaging of pea starch in seeds
Food Biophysics. 3 66-76 2008.

Ridout M. J., Parker M. L., Hedley C. L., Bogracheva T. & Morris V. J. Atomic force microscopy of pea starch granules: granule architecture of the rug3, rug4, rug5 and the lam mutant
Carbohydrate Polymers. 65 (1) 64-74 2006.

Morris VJ, Ridout MJ & Parker ML.
AFM of starch: Hydration and Image Contrast.Progress in Food Biopolymer Research 1 (2005) 28-42.

Ridout MJ, Parker ML, Hedley CL, Bogracheva TY & Morris VJ.
Atomic Force Microscopy of pea starch granules: granule architecture of the rug3, rug4, rug5, and the lam mutants. Carbohydrate Polymers 65 (2006) 64-74.

Ridout MJ, Parker ML, Hedley CL, Bogracheva TY & Morris VJ.
Atomic Force Microscopy of Pea Starch: Origins of Image Contrast. Biomacromolecules 5 (2004) 1519-1527.

Ridout MJ, Parker ML, Hedley CL, Bogracheva TY & Morris VJ.
Atomic force microscopy of pea starch granules: Granule architecture of wild-type parent, r, and rb single mutants, and the rrb double mutant. Carbohydr. Res. 338 (2003) 2135 – 2147.

Ridout MJ, Gunning AP, Wilson RH, Parker ML & Morris VJ.
Using AFM to image the internal structure of starch granules. Carbohydrate Polymers. 50 (2002) 123-132.

Bogracheva TY, Cairns P, Noel T, Hulleman S, Wang TL, Morris VJ, Ring SG & Hedley CL.
The effect of mutant genes at the r, rb, rug3, rug4, rug5 and lam loci on the granular structure and physico-chemical properties of pea seed starch.. Carbohydrate Polymers 39 (1999) 303-314.

Bogracheva TY, Morris VJ, Ring SG & Hedley CL.
The granular structure of C-type pea starch and its role in gelatinisation. Biopolymers 45 (1998) 323-332.

Cairns P, Bogracheva T, Ring SG, Hedley CL & Morris VJ.
Determination of the polymorphic composition of smooth pea starch. Carbohydrate Polymers 32 (1997) 275-282.

Botham RL, Cairns P, Faulks RM, Livesey G, Morris VJ & Ring SG.
A physico-chemical characterisation of Barley carbohydrates resistant to digestion in a Human Ileostomate. Cereal Chemistry 74 (1997) 29-33.

Botham RL, Ring SG, Noel TR & Morris VJ.
A study on the “in vivo “digestibility of retrograded starch. Carbohydr. Polym. 29 (1996) 347-352.

Cairns P, Morris VJ, Botham RL & Ring SG.
Physicochemical studies of resistant starch ‘in vivo “and “in vitro “. J. Cereal Sci. 23 (1996) 265-275.

Botham RL, Cairns P, Morris VJ, Ring SG, Englyst HN & Cummings JH.
A physicochemical characterisation of starch resistant to digestion in the human small intestine. Carbohydrate Polymers 26 (1995) 85-90.

Cairns P, Sun L, Morris VJ & Ring SG.
Physical chemical studies using amylose as an “in vitro “model for resistant starch. J. Cereal Sci. 21 (1995) 37-47.

Cairns P, Laloup V, Miles MJ, Ring SG & Morris VJ.
Resistant starch: An X- ray diffraction study into the effects of enzymic hydrolysis on amylose gels in vitro. J. Cereal Sci. 12 (1990) 203-206.

Berry CS, I’ Anson KJ, Miles MJ, Morris VJ & Russell PL.
Physical chemical characteristics of resistant starch. J. Cereal Sci. 8 (1988) 203-206.

Ring SG, Colonna P, I’ Anson KJ, Kalichevsky M, Miles MJ, Morris VJ & Orford PD.
Gelation and crystallisation of amylopectin. Carbohydrate Research 162 (1987) 277-293.

Orford PD, Ring SG, Carrroll V, Miles MJ & Morris VJ.
The effect of concentration and botanical source on the gelation and retrogradation of starch. J. Sci. Food & Agr. 39 (1987) 169-177.

Ring SG, Colonna P, Miles MJ, Morris VJ & Turner R.
Spherulitic crystallisation of short chain amylose. International Journal of Biological Macromolecules 9 (1986) 158-160.

Miles MJ, Morris VJ & Ring SG.
Gelation of amylose. Carbohydrate Research 135 (1985) 257-269.

Miles MJ, Morris VJ, Orford PD & Ring SG.
The roles of amylose and amylopectin in the gelation and retrogradation of starch. Carbohydrate Research 135 (1985) 271-281.