Detailed studies in both archaeobotany and the food sciences have emphasised the range of changes that can occur when native starch is exposed to a range of physical, chemical and biological environments. A number of methodological studies have focused on these taphonomic issues, highlighting the changes that can affect native starch biochemistry and morphology under differing conditions both prior to and after deposition, and how these factors can influence the identification and quantification process (Babot 2003; Crowther 2009, 2012; Haslam 2004; Henry et al. 2009; Weston 2009).
Plant processing
Archaeobotanical research targets plant remains that are found within archaeological, and therefore anthropogenic, contexts. Most starch remains found within domestic deposits such as occupation floors or middens derive from economic or supplementary plant taxa that have been processed in some way. Roasting, charring, boiling, milling, pounding, freezing, dehydrating, and rehydrating are all examples of cultural food processing techniques that can affect the morphology and chemistry of starch granules (Babot 2003; Crowther 2009; Henry et al. 2009; Laurence 2013:34-36; Messner and Schindler 2010). The effects of various cooking techniques on starch have been by far the most researched of these processing techniques. Experiments have been tailored to assess the range of the conditions under which starch either melts or gelatinises when exposed to water and/or heat.
The thermal conversion of starch is defined when “…starch is converted from an ordered, semi-crystalline to a disordered, amorphous state during cooking…” (Crowther 2012,
36 2009:23). Gelatinisation can occur when starch is exposed to both moisture and increased temperature, causing swelling and a loss of birefringence (Banks and Greenwood 1975:259-67; Messner and Schindler 2010). Species-specific water-dependent temperature thresholds affect the nature and timing of starch thermal conversion (Crowther 2009; Henry et al. 2009:917), but complete and uninhibited gelatinisation of all granules can only occur when more than 60-65% water is present. Within most Pacific cultigens the temperature range for gelatinisation under excess moisture is around 60-85˚C (Moorthy 2002). Partial gelatinisation is possible when these conditions are not met and the amylopectin crystallites only partly melt, resulting in an incomplete loss of birefringence and limited morphological change. Swelling is also reversible up to a point when loss of optical properties begins, which occurs within a population of starch grains over a relatively narrow temperature range of 5-10˚C (Banks and Greenwood 1975:260).
Melting of starch crystallites occurs when granules are dry-heated, through charring or roasting at temperatures above 220˚C. Starch heated in the absence of moisture will thermally degrade through breaking down into smaller glucose units and eventually carbonise before becoming structurally disordered (Crowther 2012, 2009:26). Experimentation has demonstrated the visible changes that can occur during these types of cooking techniques. Babot (2003) roasted samples of corn (Zea mays L.) kernels and quinoa (Chenopodium quinoa Willd.), reporting that these granules displayed various combinations of flat relief, weak birefringence, deformation of the extinction cross, clumping and slight gelatinisation, but not all grains showed gelatinisation features. Babot concluded that the water content at time of cooking and the heating temperature were responsible for the damage (Babot 2003:73). Similarly, the effects of charring were evident in changes in granule birefringence, swelling and clumping, but were dependent on grain size (Babot 2003:74). Starch size is related to hydration, where water molecules inhabit the crystalline regions of the amyolopectin molecules (Torrence and Barton 2006). Smaller starch granules have less capacity for swelling, while gelatinisation occurs more quickly for larger granules unless these granules have a high ratio of amylase to amylopectin which buffers gelatinisation (Banks and Greenwood 1975:260; Crowther 2012; Saul et al. 2012:3484).
Studies also demonstrated that gelatinization is not as simple as dry heat versus wet heat cooking, or charring and roasting versus boiling. Consideration must be given to the role that heat-transfer mechanisms and micro-climates have in the cooking process (Messner and Schindler 2010). In earth ovens the thermal agent is heated rock. When these are placed in direct contact with starchy organs, the oven is instantaneously heated through trapping hot moist air which emanates from the food and is then vaporised when this comes in contact with the rocks, resulting in complete breakdown of starch within the oven (2010:334). In contrast, when the external heat source is in the form of fire on top of the earth oven that increases in temperature gradually, the moisture is slowly evaporated from the organ and starch is damaged but can still
37 survive cooking. Messner and Schindler (2010) therefore argue that “…the environmental conditions generated by the heat transfer systems were of greater influence to starch degradation than temperature alone.” Different water-uptake mechanisms in different wet cooking methods also affect the rate and nature of gelatinization. For example, boiling increases water absorption at a faster rate, but steaming allows more uniform absorption over time (Crowther 2009:28-29). These factors, and the type and order of cooking procedures followed can greatly affect the archaeological visibility of particular activities. Two of the main methods for cooking starchy foods in the Pacific throughout prehistory have been by steaming in earth ovens and boiling, so these studies’ findings are particularly relevant to this study.
Other studies have demonstrated that the temperature during charring can significantly alter the nature of starch damage. Low temperatures up to 220ºC will not commonly cause any morphological changes, but higher temperatures up to 350ºC can cause complete loss of structure or fusion of granules similar to those observed within boiled samples at lower temperatures (Valamoti 2008). Similarly the location of starch within an organ that has been cooked whole at any temperature will limit the amount of damage. For example, granules in the centre of grains are more likely to survive any cooking method as these are protected from the effects of liquid and heat (2008:269). Some alteration of morphology, such as slight swelling, is still possible.
Crowther (2012, 2009) reviews the potential for starch survival within charred and carbonised residues on ceramics, and argues that these are ideal contexts for recovering non- gelatinised granules as evidence of cooked foods under particular charring conditions. The temperature must not exceed starch thermal degradation temperature, but this depends on the intensity of the heat source, the distance of the vessel from that source and the location on the vessel where the granules are located. Secondly, desiccation and carbonisation must occur relatively quickly before all starch granules undergo gelatinisation. Starch granules of underground storage organs (USO) such as roots and tubers are less likely to survive charring without gelatinisation due to the high water content within these organs, biasing the archaeological record towards cereals and legumes where these organs are utilised (2012).
Further studies have looked beyond the morphological changes that can occur within starch granules exposed to charring, and instead attempted to assess whether the survival of starch granules within charred residues reflect the intensity of plant use. Raviele (2011) replicated charred maize residue construction under controlled conditions, where the variables tested were the ratio of plant extract to water and meat, and the type of maize (Zea mays) (whole green cob, whole green kernel, lightly pounded dried kernel, whole dried kernel and ground maize flour).The overall results did not demonstrate a trend for increasing abundance with increasing proportions of maize in residues, suggesting differential survival of starch during
38 charring. Despite this, it was noted that dried kernels and cob had higher abundances of starch survival, most likely a result of high starch production within maize at this time of organ development (Raviele 2011:2711). Similarly, Saul and colleagues (2012) experimentally replicated charring conditions for einkorn (T. monococcum) and acorn (Quercus sp.) in ceramic pots at more than 100ºC for three hours. These experiments indicated that starch can survive repeated charring episodes in relatively high quantities (184 granules mg-1 and 608 mg-1
respectively).
Other food processing techniques such as parching, fermentation, freezing and milling can also cause distinctive damage to native starch granules. Henry and others (2009) tested different cooking techniques on both ground and whole legumes and grains. One of these tests involved parching three millilitres of each sample for three minutes in a muffle furnace at 200ºC. The authors noted a significant degree of heterogeneity within the sample, but argued that parching caused the most distinctive damage to granules (Henry et al. 2009:918). Most granules appeared encrusted with small particles that were either small starch granules or other organic material, which does not occur through any other cooking technique. Other common morphological changes associated with parching were the definition of lamellae and development of deep radial fissures (Henry et al. 2009:921). Within the same study, starch granules were exposed to yeast to replicate fermentation. Whole samples displayed surprisingly little change except for a possible extra ‘arm’ within the extinction cross; however, granules within the ground samples often displayed signs of ‘hollowing out’ at the hilum. (2009:921).
Effects of freezing on starch granules from several cereals, tubers and legumes has also been tested, and indicate that this process changes both the chemical properties of the starch granules, specifically a loss of birefringence, and also the physical appearance of the grains in terms of flat relief, fragmenting, fissuring, breaking and bursting (Babot in Beck and Torrence 2006:66-67; Babot 2003:74). Milling involves the application of friction to plant parts and their by-products, and is a technique most commonly used for separating the starchy endosperm of grains from the pericarp (Henry et al. 2009:916). Experimentation has revealed the extensive damage that milling can have on starch granules, which includes truncation, incompleteness, fracturing, collapsing, and bursting (Babot 2003:76). Some damage also can occur at the hila, with fissuring and large open vacuoles. In contrast, short term pounding using stone implements has been demonstrated to have very limited affect on starch granules aside from the disaggregation of compound granules (Robertson, in Beck and Torrence 2006:68-69).
Damage caused by these various food processing techniques can render starch unrecognisable as such in archaeological contexts. Studies have therefore attempted to use other chemical tests to identify the presence of cooked starch residues on artefacts or in sediments. Congo Red (empirical formula C32H22N6O6S2Na2) stain has been used in the agricultural and
39 food sciences as a contrast stain for cellulose, starch and amyloid fibrils, but the potential of this stain to test for cooked or damaged starch in archaeological contexts has only been recognised within the last two decades (Cortella and Pochettino 1994; Lamb and Loy 2005; Lamb 2003; Reichert 1913; Weston 2009). The stain only binds to proteins in alkaline or acid buffer conditions, so at a neutral pH only starch and cellulose will be stained. Unaltered or undamaged starch is hydrophobic and so does not take up the stain, but starch that has become disordered allows Congo Red to react with the amylase content of these granules resulting in a red discolouration with an orange-red or green-gold glow in cross-polarised light (Lamb and Loy 2005:1434). Trypan blue has been used in much the same way, penetrating the outer layer of damaged starch granules to stain the exposed interior blue (Barton 2007:1734).
Some researchers have argued that these stains are not reliable indicators of the presence of starch within archaeological contexts, and have subsequently experimented with thermally stable α-amylase, an enzyme that degrades the chemical linkages contained within starch (Hardy et al. 2009). These tests involve exposing a sample of extracted residue to α- amylase and observing the degradation of objects tentatively identified as starch (2009:251). Alternatively a sub-sample can be used as a control by adding α-amylase, and comparing this to the remaining analysed portion of extracted residue (Saul et al. 2012). If starch is thought to be present in the remaining sample but not visible within the control, then this is a good indication that the identification of starch within an archaeological sample is correct. However, this can be problematic as it is not possible to confirm the identification of individual granules.
Starch in sediments
Soil properties such as pH levels, temperature, texture and moisture content, in conjunction with soil constituents including enzymes, bacteria, fungi and earthworms, play the greatest role in influencing starch degradation and movement after deposition (Barton and Matthews 2006; Haslam 2004:1721). These factors are particularly relevant in tropical climates such as Tonga which generally have low rates of organic preservation (Hather 1994). A large array of studies has been carried out under varying soil conditions, either as individual variables or in conjunction with one another. These experimental studies been utilised by archaeologists and archaeobotanists to further our understanding of the extent to which morphological and chemical changes can occur after deposition within archaeological contexts.
One of the most common causes of starch degradation within tropical sediments is through hydrolysis, whereby starch is chemically broken down by polysaccharidases (enzymes) produced by bacteria and fungi. When plant and animal cellular material decays, these enzymes are released into the soil in significant quantities and are present in almost every soil type encountered throughout the world (Cheshire et al. 1974). Two classes of enzyme-producing bacteria are recognised: autochthonous, which grow slowly and predominate when there is little
40 oxidisable substrate and zymogenous, which respond to substrate addition by rapidly increasing, with the majority dying out after substrate exhaustion (Burns 1982; Haslam 2004). It is therefore possible for inactive enzymes and the bacteria that produce them to exist in the soil prior to starch deposition. The presence of these enzymes, and their potential to remain inactive in the soil substrate, limits the potential for starch survival in archaeological contexts.
To gauge the extent and rate of starch degradation, several studies have replicated hydrolysis in vitro (Mellon et al. 2002; Steup et al. 1983). Results indicate that the majority of both transitory and reserve starch is degraded within the first few days of exposure to enzymatic digestion, with rates of decomposition after this following an asymptotic curve (Haslam 2004:1720, Barton 2009). Within this generalised trend there are species-specific differences in enzyme attack patterns and rates, depending upon factors such as granule structure and size, amylose to amylopectin ratios and crystal types (Haslam 2004: 1720; MacGregor 1980; Zhang et al. 2005). Haslam (2004) compiled these studies into a table that archaeobotanists can use to understand and interpret possible biases in the archaeological record. When ranked, taxonomic patterning suggests that the rate of enzymatic degradation increased when granule size and amylose content decreased. Some species such as bananas (Musa and Eumusa spp.) are resistant to enzymatic degradation unless gelatinised (Zhang et al. 2005:144). Once gelatinisation begins the rate of degradation increases as enzymes have easier access to the more susceptible amorphous cores of the granules of most taxa. The temperature at which gelatinisation occurs does not appear to be a significant factor affecting the rate of starch degradation for each taxa. However, as discussed previously, the amount of moisture in the soil regulates the extent of swelling and gelatinisation and thus has more impact (Haslam 2004). One variable that does seem to affect the presence of microbial, enzyme and fungal activity in soils is depth. Enzymatic degradation of starch is much more likely to occur closer to the surface than at midpoints or near basal deposits, where activity is correlated with the presence of other organic matter (Haslam 2004:1721; Taylor and Belton. 2002). The rate of starch degradation is also seen on artefacts, where starch abundance at 108 weeks is already representative of those in archaeological contexts (Barton 2009:135).
Other taphonomic factors that affect starch preservation in sediments include temperature, pH and moisture content of the sedimentary matrix. These variables have the potential to damage starch through causing gelatinisation or damaging the biochemical properties of granules; however, these rarely directly affect starch individually, and so should be considered in conjunction with enzymatic hydrolysis (Haslam 2004). It is the interplay between these variables and the presence of microbial and enzyme activity that cause starch degradation. As mentioned earlier, temperature and moisture cause swelling and shrinking of starch, enabling various enzymes access to the interior of the granule. The same principles described in relation to heating and cooling within food processing techniques is applicable to soil conditions.
41 Hydrolysis can also occur in both acidic and alkaline soils, but is slower than enzymatic decomposition (2004:1721). Some enzymes often favour different pH levels, such as invertase which is more active in more alkaline soils, whereas amylase can affect starch in any conditions (Dick et al. 2000).
Quantitative studies of starch preservation (Barton 2009; Haslam 2009; Therin 1998) have made observations of displacement of starch within sediments over time. Therin (1998) attempted to replicate the downward displacement of starch grains in various sandy matrices using groundwater. Variables tested included sediment particle size, irrigation rate and starch grain size, monitored over a two-month period. The results indicated that there is limited downward movement of starch grains, but within this overall trend patterning related to granule and particle size. Larger starch grains have less chance of becoming mobile, but if they do move, these granules move slowly and have less chance of becoming trapped. Smaller sand particles also correlate with less starch mobility, but an increase in irrigation rate will increase starch movement. Haslam (2009) attempted to take this experimentation a step further, and investigate both upward and downward movement of starch, as well as lateral movement. These parameters mirrored those used by Therin (1998), but only one sand matrix was tested with larger particles of 250-500µm. The sediment was autoclaved for 30 mins at 120ºC to eliminate fungi and bacteria then placed into a PVC pipe ‘cross’ with a small amount of starch (0.1g) in the centre, and placed upright (Haslam 2009:95). The experiment was irrigated at a rate of 160ml twice a day from the top of the set-up for a period of 30 days. The sediment was then sampled at 27 locations within the cross and processed for starch extraction, revealing that around 16% of starch moved downward up to 6cm although the highest number of starch was still found at the centre (2009:98). Very small numbers of starch also moved laterally up to 12cm, and upwards 2cm from the centre. The implications of these experiments are that researchers need to assess factors such as sediment compaction, particle size and local rainfall when interpreting the potential movement of starch in sediments, along with other taphonomic issues such as bioturbation and human activities.
Researchers have sought to provide solutions to taphonomic issues through investigating the types of conditions that are favourable for starch preservation (Barton 2009; Haslam 2009, 2004; Laurence 2013). The presence of heavy metals within soils or high clay content can neutralise enzymes and thus limit the effect that bacteria and fungi can have on starch granules within these deposits (Haslam 2004). Additionally, soil aggregates, or Particulate Organic Matters (POMs), and residues on artefacts can form a protective barrier for starch through limiting surface area accessible for enzyme digestion. Artefact surfaces can also provide a micro-environment that protects starch from the effects of temperature, moisture and downward displacement through groundwater. Studies of starch residues on bone, wood, lithic and ceramic artefacts indicate that the preservation of these residues in soils is possible.
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