Identification of starch granules on ground stone tools exposed to fire

Starch Extraction and Identification All samples were processed for starch following standard lab procedures (see Louderback et al., 2015). Large ground stone tools were surface sampled for ~5 min with a sonicating toothbrush and DH2O ; smaller tools were sampled after sonicating in a DH2O bath for 3 min. Each sample was rinsed through an Endecott mesh sieve (125µm) with DH2O and sample liquid <125µm was retained in a 50mL test tube. Sample extract >125µm was discarded. The sample liquid (presumably containing starch) was centrifuged for 3minutes at 3,000 rpm. The supernatant was decanted and the sample pellet was transferred to a sterile 15mL tube with DH2O , mixed with a vortex, centrifuged for 3minutes at 3,000 rpm, and decanted. Samples were resuspended with ~7mL of heavy liquid (lithium heteropolytungstate; specific gravity 2.2), vortex-mixed, and centrifuged for 15 minutes at 1,000rpm. Heavy liquid separates the lighter organic material, including starch granules, from the heavier content. The lighter organics were collected using a pipette and transferred to a new, sterile 15mL tube. Residual heavy liquid was rinsed from the organic matter with ~10mL of DH2O , mixed with a vortex, and centrifuged for 3 minutes at 3,000rpm; twice. The samples were decanted and rinsed with ~7mL of acetone, vortex-mixed, and centrifuged for 3 minutes at 3,000rpm. After decanting one final time, processed samples were left to dry overnight before mounting on glass slides for microscopy observation. Starch granules were measured and described based on a set of established criteria, including maximum length through the hilum (µm), hilum position, two-dimensional shape, clarity of the extinction cross, and the presence or absence of surface features such as fissures and pressure facets (Brown and Louderback 2020; Holst et al., 2007; ICSN, 2011; Joyce et al 2021; Louderback and Pavlik, 2017; Musaubach et al., 2013; Piperno et al., 2004, 2009; Reichert, 1913; Torrence and Barton, 2016). These criteria were recorded as absent (0) or present (1) and expressed as a percentage of the occurrence. Slides were scanned with a transmitted brightfield microscope using polarizing filters and Nomarski optics (Zeiss Axioskop Imager M1, Zeiss International, Göttingen, Germany). Observations were obtained using randomly generated X and Y coordinates on the microscope stage. All starch granules observed within each field of view were measured and described. Images and measurements at 400X were captured under polarized light (POL) with a digital camera (Zeiss AxioCam MRc5) using Zen Core 3.1 imaging and measurement software. The presence of surface features was imaged and recorded in differential interference contrast (DIC) micrographs. Identification and Quantification of Treated Starch The relative proportions and arrangements of amylose and amylopectin affect both granule morphology and functionality (Vamadevan and Bertoft, 2015). They can also cause various mechanical and chemical changes, such as granule swelling (gelatinization), pasting, and loss of birefringence in response to different food processing methods (Cai et al., 2014; Crowther, 2012; Di Poala et al., 2003; Gong et al., 2011; Mason, 2009; Wang et al., 2014). Exposing starch granules to heat can cause morphological damage such that granules may be difficult to identify. Granules exposed to high temperatures have been shown to enlarge in size (swell) and gelatinize, losing their birefringence cross, amylose layers, and surface characteristics before dispersing away entirely (Crowther, 2012; Singh et al., 2002; Vamadan and Bertoft, 2015). Congo Red dye (empirical formula C32H22N6O6S2Na2) reacts with granules when amylose layers are broken down (usually due to cooking or some other form of damage), staining them orange to vivid red. Undamaged starch granules (with intact amylose layers), however, are hydrophobic and, therefore, do not react with Congo Red (Lamb and Loy, 2005). To measure the percent or degree of damage to starch granules, milled and burned samples were treated with a Congo Red solution following a protocol similar to Lamb and Loy (2005). Dried residue samples were resuspended with 25µL of Congo Red and absorbed the stain for 15 minutes before diluting with 100µL of DH2O (1:4 ratio). Slides were prepped with 25µL of the hydrated sample and a cover slip was applied but not affixed with fingernail polish (experimentation found that clear fingernail polish interfered with Congo Red stain). The liquified stained samples dried within ~45 minutes, therefore, all observations were photographed immediately. Microscope observations on slides from the milled and burned (close proximity) samples were obtained using randomly generated X and Y coordinates. Samples exposed directly to flame, however, produced fewer measurable granules, so observations were collected by scanning the entire slide. Size distributions from the control, milled, and burned samples were statistically analyzed with a Kolmogorov-Smirnov (K-S) test to determine any significant difference in the distribution of median lengths. Boxplot-stripcharts were generated in R Statistical Software (v4.0.2; R Core Team 2020) to show the quartile summary variation

淀粉提取与鉴定
所有样本均遵循标准实验室流程进行淀粉提取处理（详见Louderback等人，2015年研究）。大型磨制石器工具采用超声牙刷结合去离子水（DH₂O）对其表面进行约5分钟的采样；小型工具则先在去离子水浴中超声处理3分钟后再进行采样。每一份样本均通过孔径125μm的Endecott网筛用去离子水冲洗，截留粒径<125μm的样本悬浮液至50mL试管中，弃去粒径>125μm的萃取物。
将疑似含有淀粉的样本悬浮液以3000转/分钟离心3分钟，倾去上清液后，将沉淀转移至含去离子水的无菌15mL试管中，涡旋混匀后再次以3000转/分钟离心3分钟，随后倾去上清液。将样本重悬于约7mL重液（杂多钨酸锂，比重2.2）中，涡旋混匀后以1000转/分钟离心15分钟。该重液可将包括淀粉颗粒在内的较轻有机质与较重杂质分离。随后用移液枪收集较轻的有机质组分，转移至新的无菌15mL试管中。用约10mL去离子水冲洗有机质以去除残留重液，涡旋混匀后以3000转/分钟离心3分钟，重复该步骤两次。倾去上清液后，用约7mL丙酮冲洗样本，涡旋混匀并以3000转/分钟离心3分钟。最后一次倾去上清液后，将处理好的样本置于室温过夜晾干，随后封片以待显微镜观察。
淀粉颗粒的观测与描述基于一系列公认标准，包括沿脐点方向的最大长度（单位：μm）、脐点位置、二维形态、消光十字清晰度，以及是否存在裂隙、压力面等表面特征（Brown & Louderback, 2020；Holst等, 2007；ICSN, 2011；Joyce等, 2021；Louderback & Pavlik, 2017；Musaubach等, 2013；Piperno等, 2004, 2009；Reichert, 1913；Torrence & Barton, 2016）。上述特征以“存在（1）”或“不存在（0）”进行记录，并以出现百分比的形式呈现。
采用配备偏振滤光片与Nomarski微分干涉相衬光学系统的透射式明场显微镜（Zeiss Axioskop Imager M1，蔡司国际，德国哥廷根）对载玻片进行扫描。观测点位通过显微镜载物台随机生成的X、Y坐标确定，对每个视野内观察到的所有淀粉颗粒均进行测量与描述。采用Zeiss AxioCam MRc5数码相机搭配Zen Core 3.1成像与测量软件，在偏振光（POL）模式下以400倍放大倍率采集图像与测量数据。表面特征的成像与记录则采用微分干涉差（DIC）显微照片。
处理后淀粉的鉴定与定量
直链淀粉与支链淀粉的相对比例及排布方式会同时影响淀粉颗粒的形态与功能特性（Vamadevan & Bertoft, 2015）。在不同食品加工方式作用下，二者还可引发多种机械与化学变化，例如淀粉颗粒溶胀（糊化）、糊化以及双折射性丧失（Cai等, 2014；Crowther, 2012；Di Poala等, 2003；Gong等, 2011；Mason, 2009；Wang等, 2014）。
将淀粉颗粒置于高温环境中会造成形态损伤，导致其难以被识别。已有研究表明，经高温处理的淀粉颗粒会发生体积增大（溶胀）与糊化，逐渐丧失其消光十字、直链淀粉层与表面特征，最终完全分散（Crowther, 2012；Singh等, 2002；Vamadevan & Bertoft, 2015）。
刚果红染料（分子式：C₃₂H₂₂N₆O₆S₂Na₂）可在直链淀粉层被破坏时（通常由烹饪或其他形式的损伤导致）与淀粉颗粒结合，将其染为橙色至亮红色。而未受损伤的淀粉颗粒（直链淀粉层完整）具有疏水性，因此无法与刚果红结合（Lamb & Loy, 2005）。
为测定淀粉颗粒的损伤百分比或损伤程度，本研究参照Lamb与Loy（2005）的实验方案，采用刚果红溶液对研磨与灼烧后的样本进行处理。将干燥后的残留样本重悬于25μL刚果红溶液中，染色15分钟后用100μL去离子水稀释（稀释比例1:4）。取25μL水化后的样本制备载玻片，加盖盖玻片但不使用指甲油封固（预实验发现透明指甲油会干扰刚果红染色效果）。染色后的液态样本约45分钟内即可干燥，因此所有观测均需即时完成拍摄。对于研磨与近距离灼烧的样本，其载玻片的显微镜观测点位通过随机生成的X、Y坐标确定；而直接暴露于火焰的样本可观测到的可测量淀粉颗粒数量较少，因此需通过扫描整个载玻片完成观测。采用Kolmogorov-Smirnov（K-S）检验对对照组、研磨组与灼烧组样本的粒径分布进行统计分析，以确定各组中位长度分布是否存在显著差异。采用R统计软件（版本4.0.2；R核心团队, 2020）生成箱线图-条形图，以展示四分位数汇总的变异情况。