The Microscopic Structure of Starch Grains Food Microscopy Part I

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Author: Dr. Robert Berdan

Jewel-like Potato starch in polarizing microscope

Potato starch grains appear jewel-like in a polarizing microscope with a quartz wedge compensator 200X

© Robert Berdan

Introduction

Light microscopy is widely used in food science both in research and quality control. Brightfield, polarizing, and fluorescence microscopy are often used. This article will focus on starch grains which form an important food and industrial source. I first examined starch grains with a polarizing microscope as a teenager in 1978 in my garage laboratory and was impressed by their jewel-like appearance. I attempted to understand how the molecules of glucose were organized in the granule and why the granules displayed a Maltese cross. Since then researchers have learned more about starch and light microscopy continues to be an important tool in understanding its structure.

Starch is the most widespread and abundant storage carbohydrate in plants. Starch is used for nutrition, feedstock, to produce paste, glues, and thickeners, and more recently for the production of bioethanol.  Identification of starch grains by microscopy can be used to determine if cereal grains have been added to ground meat. Starch grains found in a persons’ stomach after death can also be used by Forensic scientists to determine the time of death and starch residues on ancient tools are used in archeology to determine the diet of ancestral peoples (M. Mozdy 2016).    

Starch grains vary in size, shape, and form and can be used to identify the plants they originated from. They vary in size from about 1 to 200 microns (micron = 0.001 mm), some are circular in shape while others are guitar–pick shaped and ellipsoid. Plants convert sugar to starch in chloroplasts within their leaves and then transport the starch after converting it to disaccharides to other parts of the plants where the sugars are stored as starch in roots, seeds, and tubers. The starch is stored in amyloplasts which may contain one or more starch granules.

The reason plants store glucose as starch is that as starch it is no longer osmotically active otherwise it would draw water into the plant cells which would swell. Starch is essentially a stored form of energy that can be used later. Before we can use starch as food,  we need to remove the starch granule layer which is done by mechanical, enzymatic means or by heating. The dissolution of starch into a paste by heating is called gelatinization. Prior to humans learning to cook food, starch was of limited use to our ancestors and could not be used nutritionally. 

Potatoes

Russet Potato cultivar with sprouts is a tuber filled with starch grains.

Photo by Zoofaris- Wikipedia

 

Table of Contents:

  1. What is the structure of Starch Grains?
  2. Growth Ring formation in Potato Starch granules
  3. Maltese Cross pattern of Starch Grains
  4. Summary

Microscopic Identity and Structure of Starch Grains

The morphological features of starch granules from different plants are characteristic and can often be used to identify the plant species (A.R. Cortella and M.L. Pochettino 1994) though we have a poor understanding of what controls the shape and size of the granules. Starch can be detected by staining with Lugol’s iodine (potassium iodide and iodine in water) which turns the surface of the granules dark blue (C. Goedecke 2016).  Amylose binds most of the Iodine, about 20X more than Amylopectin. Amylopectin in the absences of amylose displays a reddish-brown color (T. Coultate 2015).

Potato starch grains within amyloplasts

Above photomicrograph shows potato starch grains within amyloplasts. Note the starch grains vary in size and shape. Amyloplasts are plastids that produce and store starch within internal membrane compartments. Brightfield microscopy 100X

Hilum

All starch grains have a hilum which is the point around which layers of protein are deposited and represents the center of the Maltese cross in polarized light microscopy. In tubers, bulbs, and rhizomes, the hilum is often off-center, with growth rings emanating outward (M. Mozdy 2016).

Starch granules are made up of two types of starch; amylopectin which can make up from 30 to 70% of the granule and amylose which can make up the remainder. Amylose is a linear sugar with very little branching.

Sugars in starch

Starch is made up of two main sugars 20-30% amylose which is linear and 70-80% amylopectin which is a branched molecule. Both sugars bind Iodine, but amylose binds 20X more of it. Diagram from C. Goedecke (2016). Diagram used under Creative Commons Attribution License.  

Potato starch stained with a dye

Potato starch grains stained with Lugol’s iodine brightfield microscopy. A blue colouration indicates the grains are positive for starch - amylose and amylopectin. 200X Of the two sugars Amylopectin is a higher molecular weight polysaccharide that is highly branched and forms a semicrystalline structure. The sugars are deposited in layers within the granules. The layers can be observed by both light and scanning electron microscopy (S. Jagadeesan et al. 2020).

Growth Rings

Starch granules from higher plants contain alternating zones of semicrystalline and amorphous material known as growth rings which are visible by light microscopy. The growth rings indicate how the starch grains are assembled and give the grains a semicrystalline characteristic due to amylopectin.  The factors regulating growth ring formation are not understood (M. Pilling and A.M. Smith 2003).  Growth rings can sometimes be enhanced by staining with rhodamine or safranin and viewing the grains by fluorescence microscopy whereas Fast green or Acid Fuchsin is useful for localizing proteins within the granules by brightfield microscopy (W. Blaszczak and G. Lewandowicz 2020).

Maltese Cross

Potato starch viewed between crossed polarizers

Potato starch grains viewed between crossed polarizers. Motic BA310 polarizing microscope 400X.

© Robert Berdan

Note the dark cross called a Maltese cross that is seen in crystals and starch grains. These regions are transparent to the crossed polarized light and one is seeing the background through the crystals. The bright areas are birefringent. When compensators are added these quadrants become colored due to light interference.

The semicrystalline properties of starch grains make them easy to identify using a polarized light microscope. Birefringence is the property of some materials to split polarized light into two different vectors. Theoretically, positive birefringence indicates that the molecules within the starch granule have a radial orientation. Crystallinity also varies from about 15 to 45% and is due primarily to amylopectin (S. Perez et al. 2009). The black cross may be distorted in shape due to the ellipsoid shape of the starch grains (K.A. McMahon (2004). In perfectly round crystals the cross appears symmetrical (e.g. corn starch – see photo further below).

The bright regions of the granules represent those parts of the crystal that are anisotropic and break the polarized light into two vectors that travel at different velocities through the starch grains.  When the light rays emerge from the crystal they are out of phase and interference produces bright regions and if the starch grain or crystal is thick or the difference in the two refractive indices are sufficiently large some colors are removed from white light due to destructive interference and only the remaining colors are seen. For example, if blue is removed by destructive interference we see yellow.  Using wave plates, also called compensators in polarizing microscopy, that light path difference between the light emerging from the starch crystals can be amplified we observe colors (see articles on Birefringence by R. Berdan, 2021). When the starch granules are broken down by heating and gelatinization occurs, the Maltese cross disappears and Iodine staining is reduced or disappears.  During gelation, I have noticed some potato starch grains exhibit branched crystal-like structures after observation by phase-contrast microscopy and the crystals are presumably amylopectin.

Banana Starch

Banana starch grains within amyloplasts. Polarized light microscopy with a full wave retardation compensator 200X. Note their oblong shape.

© Robert Berdan

Corn starch from the box

Corn starch straight from the box A) Polarized light and a full-wave compensator B) Polarized light both 200X. Note the starch grains are smaller and more circular in shape than potato starch grains. Cornstarch is made by removing the protein and fiber of the corn kernel, leaving only the starchy center called the endosperm. Corn starch is used as a thickening agent in liquid-based foods (e.g., soup, sauces, gravies), usually by mixing it to form a paste or slurry.

Summary

Light microscopy plays an important role in the analysis and research of plant starch. In the future researchers are trying to modify the properties of the starch so it can be used in industry to produce biodegradable products and be more easily digested. Altering starch molecules may be beneficial for persons with diabetes and other digestive disorders. Studies are underway to engineer vaccines by fusing starch binding proteins to known antigens using new methods to manufacture bulk commodities of starch using Glyconanotechnology (E.C. O’Neill and R.A. Field 2015). Some historians believe that potatoes might be responsible for changing the course of world history and even the evolution of the human brain (J.J. Provost et al. 2016).

For those with access to or own a microscope with polarizing filters, you owe it to yourself to examine some potato or plant starch granules.  Simply squash a small piece of raw potato on a microscope slide and it will reveal millions of jewel-like structures with a polarized light microscope. Most of the pictures in this article were taken with the Motic BA310 Pol microscope.   

References

1. Sueng (2020) Amylose in starch: towards an understanding of biosynthesis, structure and function. Tansley reviews. New Phytologist 228: 1490-1504. Doi: 10.1111/nph 16858

2. Blaszczak and G. ZLewandowicz (2020) Light Microscopy as a Tool to Evaluate the Functionality of Starch in Food. Foods 9, 670.

3. Jagadeesan, I. Govindaraju and N. Mazumder (2020) An Insight into the Ultrastructural and Physiochemical Characterization of Potato Starch: a Review. American J. of Potato Res. 97: 464-476.

4. Goedecke (2016) Why does Iodine turn Starch Blue?https://www.chemistryviews.org/details/education/10128441/Why_Does_Iodine_Turn_Starch_Blue.html

5. Mozdy (2016) In the Tiny World of Starch Grains, Bigger is Better. https://nhmu.utah.edu/BLOG/2016/08/08/TINY-WORLD-STARCH-GRAINS-BIGGER-BETTER

6. J.J.Provost, K.L. Colabroy, B.S. Kelly, Mark.A.Wallert (2016) The Science of Cooking – Understanding the Biology and Chemistry Behind Food and Cooking. John Wiley & Sons. Page 231-254.

7. C. O’Neill and R. A. Field (2015) Frontiers in Bioengineering and Biotechnology – Mini Review. Underpinning Starch Biology with in vitro Studies on Carbohydrate-Active Enzymes and Biosynthetic Glycomaterials. Frontiers in Bioengineering & Biotechnology Vol 13, Article 136.

8. Coultate (2015) Food: The Chemistry of its Components. Royal Society of Chemistry; 6th edition.

9. C. Zeeman, J. Kossmann and A. M. Smith (2010) Starch: It’s Metabolism, Evolution, and Biotechnological Modification in Plants. Annu. Rev. Plant Biol. 61: 209-234.

10. Perez, P.M. Baldwin and D.J. Gallant (2009) Chapter 5 Structural Features of Starch Granules In Starch 3rd Edition Chemistry and Technology Food Science and Technology. 149-192.

11. K.A. McMahon (2004) Practical Botany – The Maltese Cross in Tested Studies for Laboratory Teaching, Vol 25 (M.A. O’Donnell ed).  Pages 352-357.

12. Pilling and A.M. Smith (2003) Growth Ring Formation in the Starch Granules of Potato Tubers. Plant Physiology 132:365-371.

13. A.R. Cortella and M.L. Pochettino (1994) Starch Grain analysis as a Microscopic Diagnostic Feature in the Identification of Plant Material.  Economic Botany 48: 171-181.

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