Microscopic Analysis of Starch Grains and its Applications in the Archaeology of the Stone Age

Archaeobotanical micro-residuals are today a major focus in artefactual and bioarchaeological investigations. Though starch grains analysis may be regarded as marginal, it can be a useful analysis for archaeological research, being a method suitable for the investigation of stone artefacts and ceramic vessels. Soil samples and dental calculus can also be examined. Through the use of various extraction methods it is possible to answer questions of diet composition and purpose of stone tool use. As documented in recent studies examining the composition of the human diet, starch grain research should be one of the main areas of archaeobotanical investigation. Its applicability can be seen in studies where it is useful to define the role of plants in human subsistence. New evidence of plant use in archaeological contexts in the Stone Age, beginning in the Palaeolithic and ending in the Neolithic, has been presented in recent papers. Current archaeological studies, including those using starch grain analyses, have particularly indicated the higher ratio of plants in the diet during the Palaeolithic period. IANSA 2018 ● IX/1 ● 83–93 Jaromír Kovárník, Jaromír Beneš: Microscopic Analysis of Starch Grains and its Applications in the Archaeology of the Stone Age 84 However, damaged starch grains can hinder the use of this particular technique. The results from starch grain analysis are suitable as complementary analyses to other techniques, such as palynology, phytolith analysis or plant macroremains (García-Granero et al., 2015; Pestle, Laffoon, 2018). The examination of starch grains has improved along with improvements in microscopic technique. Antonia van Leeuwenhoek (1632–1723) was the first scientist to publish an illustration of starch grains. This Dutch scientist and microscopist engaged in the observation of natural materials and created a record of the starch grains of common species of plants such as wheat, barley, rye, oats, beans, peas, rice and corn (Hogg, 1854; Britannica, 2016). The work of Fritzche continued that of Leeuwenhoek. He also recognized the potential of the heterogeneity of starch grains and its use for determining the genus and species of plants. It was only a short step towards the creation of taxonomic keys and atlases (Torrence, Barton, 2006). The German botanist and cofounder of cell theory, Matthias Jakob Schleiden (1804–1881), created a key with his own classification based on starch shape and hilum position. Karl Wilhelm von Nägeli (1817–1891) continued the study of the structure of starch (Britannica, 2016; Torrence, Barton, 2006). This Swiss botanist built on the work of J. M. Schleiden and created a modificaton of the starch-grain sorting system (Britannica, 2016), among others we could mention, such as Henry Figure 1. Illustration of Poaceae starch grain (Reichert, 1913). IANSA 2018 ● IX/1 ● 83–93 Jaromír Kovárník, Jaromír Beneš: Microscopic Analysis of Starch Grains and its Applications in the Archaeology of the Stone Age 85 Kraemer (1868–1924) (Kraemer, 1907). Edward Tyson Reichert was a physiologist from Philadelphia who created a comprehensive work summarizing the knowledge of the study of starches (Figure 1), and described their properties and use for starch grain identification (Reichert, 1913; Torrence, Barton, 2006). These works found use in everyday life, especially in the pharmaceutical and food industries, where there was a need to check the quality and origin of food and plant products from medicinal plants. For example, in 1978, William Sedgwick Saunders (London’s Medical Officer for Health) inspected the flour sold in London to prevent it being mixed with gypsum (Stevenson, 2014). Subsequently, starch atlases were created to recognize individual species by noting differences in starch grains (Loy et al., 1992). Starch analysis has then been put into use in archaeological research over the last twenty-five years. In 2006, Robin Torrence and Huw Barton published a comprehensive account of starch analyses in archaeology (Torrence, Barton, 2006). One of the most prominent results of starch analysis connected with Stone Age artefacts can be traced to 2007, when the first results of the human plant diet at the Palaeolithic Gravettian site were published, describing the important role of plants in the Palaeolithic diet (Aranguren et al., 2007). Before Palaeolithic people were usually regarded as only hunters of large animals. In the last thirty years, we can see the expansion of starch studies in archaeology: as described in a recent account by Barton and Torrence (2015). In this paper, we summarize some basic knowledge of starch analyses from the Palaeolithic, Mesolithic and Neolithic period. Our main focus is on the starch grain itself, its biology and mode of identification of stone implements in particular. A special section summarizes some of the results of starch analyses at specific archaeological sites and with certain objects. 1.1 Starch Starch is a polysaccharide used as a reserve energy store in the majority of autotrophic plants. The exception is presented by the families Asteraceae, Campanulaceae, Liliaceae, and others, which store inuline as their reserve polysaccharide. Starch is a ready source of glucose for plants, suitable for long storage. It is a composition of two homopolysaccharides (amylose and amylopectine), originating from α-D-glukopyranose. Amylose and amylopectine occur in a weight ratio of 1:3. In some crop plants, cultivars have been bred with an elevated one or the other component of starch (amylose or amylopectine) (Prugar, 2008). Amylose is a linear homoglycan consisting of up to 4500 (more often 1000–2000) glucose units. Amylopectine is a multiple-branched polysaccharide consisting of chains of 50,000–100,000 D-glucose units. Amylose is a linear α-D(1-4)-glucane of disaccharide maltose; the branching of the chain is limited to approximately ten loci per molecule (Velíšek, 1999; Bemiller, Whistler, 2009). Starch is synthesised in the green parts of the plant – in the chloroplasts. There, small starch grains, about 1 μm in diameter, which are called temporary or transitory starch, are created. These are further used or transported. Starch is further stored in special organelles – amyloplasts. Major quantities of starch are stored in reserve organs in specialized cells of the seeds, roots and tubers (Bemiller, Whistler, 2009). Premature fruits also contain starch, but with the ripening process the starch content decreases and in ripe fruits the starch hardly occurs. However, there are exceptions, such as bananas, where high amounts of starch are contained in the fruit (Velíšek, 1999). Starch is stored in amyloplasts in the form of starch grains, which are species-specific and differ in shape, size and polysaccharide ratio. These starch grain characteristics are, for the most part, given genetically, but are also influenced by external influences (Selvam, 2013). According to the crystallinity level of the granules, the starch can be divided into four forms, designated A, B, C and V. The variability is due to the internal spatial arrangement of the molecules. The most stable is form A, which occurs in cereals, and the least stable is form B, which is found in root crops and potatoes. The C form is characteristic of leguminous seeds (it is a mixture of starch form A and B), whereas gelatinized starches occur in the V form. From a chemical point of view, starch grains can also contain small quantities of other substances that occur in plant cells, such as proteins and lipids (Velíšek, 1999; Ahmed et al., 2016; Bemiler, Whister, 2009). 1.2 Starch grain Starch granules occur in various shapes and sizes: round, kidney-shaped, oval-elongated and polygonal shapes are common. Granules can be separated or coagulated into aggregates. Starch grains also differ in size. It is possible to distinguish several structures and formations, all of which are used in the identification process. For example, the visibility and position of the hilum is observed: whether it is located in the centre of the starch granule or off-centre (Lentfer et al., 2002). Sometimes lamellas are visible. These are concentric lighter and darker stripes on the granule that are circular from the centre towards the starch edge (Czaja, 1969). They are more recognizable in larger starch grains and are connected to the gradual growth of starch grains. They can be divided into crystalline (a denser part) and semi-crystalline (a softer part with darker colouring). On the surface of starch grains, fissures or other superficial structures can be distinguished. Furthermore, the bevelling of the starch grain can be considered (Gott et al., 2006, Bemiller, Whistler, 2009). The effect of outside, natural or anthropogenic, influences on the starch granule can lead to its injury or even destruction (starch modification, swelling, gelatinization). Starch can be damaged mechanically (e.g. broken in the course of milling). A limiting factor for its preservation can be temperatures above 50°C, when gelatinization takes place in the presence of water. The starch grain starts to deteriorate with exposure to enzymes, the effect of the amylase enzymatic group. Other harmful influences are, for example, long water exposure, low temperatures and charring (carbonization) (Messner et al., 2008; Lentger, 2012). IANSA 2018 ● IX/1 ● 83–93 Jaromír Kovárník, Jaromír Beneš: Microscopic Analysis of Starch Grains and its Applications in the Archaeology of the Stone Age 86 2. Methodology of starch analysis When the starch grain has a certain optical appearance, the starch can be identified with optical microscopy in polarized light by observing the extinction cross on the starch granule (Figure 2). Further approaches to starch identification encompass chemical methods and staining methods (Haslam, 2004). Lugol’s solution stains the starch grains dark blue (Reichert, 1913). Using CongoRed colouring can be useful for ident