Parchment browning and the Dead Sea Scrolls – Part I: Artificial aging

Highlights

• Artificial aging shows impact of environmental conditions on parchment and collagen.

• Chemical changes to collagen are minimal, despite parchment's extreme color change.

• Color migration under certain conditions indicates pigment formation.

• Pigments may be melanoidins formed via Maillard reaction.

• Parchment fiber structure compacting could also be contributing to the color change.

Abstract

The Dead Sea Scrolls, a two-thousand-year-old parchment corpus of manuscripts, thought to be the most important discovery of the 20th century including the first known copies of the Hebrew Bible, have darkened in parts to the point that the text is illegible. This motivated our research to try to understand the browning of parchment mechanisms. The scrolls cannot be analyzed by any destructive methods, so alternatives had to be sought. Here we describe a series of artificial aging experiments using modern parchment, prepared especially for our project, according to traditional methods, demonstrating how the different types of browning observed on the scrolls relate to environmental conditions as well as post finding treatments. Even the most extreme browning could be caused by prolonged exposure to water. This was in complete variance from controlled relative humidity exposure, where even elevated temperatures over a full five months caused only mild browning. We also confirm browning occurs at room temperature and within short periods when parchment is wet. We noted greater darkening of the flesh side in short term experiments, while browning was similar on both sides in conditions where water or humidity levels were maintained over a longer period. Artificial aging also confirmed the damage to the lower ends of many scrolls was likely caused by capillary actions when parchment was standing in water. That experiment also showed a dark tide-line phenomenon at the wet-dry interface, similar to that observed in paper substrates. Using analytical methods including FTIR, SEM, Light microscopy, UV–Vis and Fluorimetry we found the browning was always associated with denaturation, or loss of structure, mostly due to water and heat. This results in a flattening of the parchment structure which could be altering how light is reflected. However, in one artificial aging experiment the color was clearly seen to migrate and disperse over the parchment, after its initial formation. We believe this means the color is due to dark pigments; therefore, the main mechanism we suggest is melanoidin pigment formation due to a Maillard type reaction between amino acids and sugars or oxidized lipids, which are perhaps freed to react due to denaturation and hydrolysis. The results collected here can serve as an important step towards a full mechanistic understanding of parchment browning.

Introduction

The Dead Sea Scrolls (DSS) are a large collection of manuscripts which were found in caves on the western shore of the Dead Sea, mostly written on parchment. The majority dating to the 1st century BCE-70 CE, include partial copies of all Books of the Hebrew Bible, (except for the Book of Esther) as well as many other religious texts written at the time when both Judaism and Christianity were formalizing as we know them today [[1], [2], [3]]. Other corpuses from other sites in the Judean Desert range from the 4th century BCE to the 11th century CE including historical documents from the Second Jewish Revolt against the Romans in 132–136 CE.

Thus, the Dead Sea Scrolls are one of the most important archaeological finds of the 20th century, and definitely the most important find in Israel; accordingly their preservation is imperative. Unfortunately, the most obvious form of degradation involves the scrolls darkening or browning, in some cases to such an extent that the text can no longer be read by the naked eye. This means the documents have lost their main functionality. Fig. 1 shows examples of scrolls, beginning with the Psalms scroll which is in a relatively excellent condition, but still showing a typical degradation of the lower end. Next, we have a fragment from the Book of Genesis, which has darkened more extensively, luckily the text can still be read using near infra-red (NIR) light. Finally, we have the most degraded scroll fragment, which hardly looks like parchment. It is a small fragment with no text or association with any specific scroll. This fragment was provided, together with a few others, for analysis and will be discussed in the second part of this article. This first part focuses on the use of modern parchment, prepared by traditional methods, in artificial aging experiments to understand the process of parchment browning and degradation. In the early stages of our project, we focused on using non-destructive analytical methods in the hopes that we could then apply the same methods to the scrolls; however we eventually did utilize the artificially aged parchment for a few more destructive tests.

Parchment is processed animal skin, usually goat, sheep or calf. Most parchment that survived to this day is of European manufacture, from the middle-ages and up to the 18th century when paper became more common. The European manufacturing methods are well known; typically the skin was flayed, cured in salt, and then soaked in a lime bath. This loosened the hair, which could then be easily scraped off along with any remaining flesh. The skin was then stretched on a frame to dry. There are many possible modifications, such as adding enzymes (or plant matter as a source of enzymes) to help soften the parchment. In traditional Jewish parchment preparation barley flour is often added and there is an important final step which is lightly tanning the parchment. Tannins are obtained by extracting plant galls, or other tree parts rich in tannins, and this extract could be either sprinkled on the hair side (the writing side) or the parchment could be briefly soaked in the extract solution [4,5]. This tanning means Jewish parchment can be viewed as a hybrid between leather and European parchment. During the preparation many components from the skin are cut or washed away. Most research on parchment has focused only on the main component which remains; collagen.

Collagen is a group of three protein chains twisted in a triple helix formation. The main degradation pathways are hydrolysis, oxidation, and denaturation [6]. Hydrolysis can break off side groups or break the backbone of the collagen molecules thus weakening the strength of the collagen matrix or parchment. Denaturation is the loss of the tertiary structure – at the end of the process, instead of the collagen triple helix a random arrangement of the same collagen molecules remains, this is gelatin. Oxidation can introduce a variety of changes depending on the location and nature of the oxygen introduced. There is an extensive body of research about parchment chemistry and its degradation, including two main European Union funded projects, the first focused on microanalysis methods [7] and the second on damage assessment [8]. Despite these and many other outstanding papers published, no research was ever done on the browning process of the DSS. One study on the DSS did note color differences and found much greater conversion to gelatin in darker areas [9]. Others have noted that parchment darkening corresponds to other forms of degradation, but without noting specific correlations [10], or found no correlation between visible appearance and damage to collagen [11]. Gonzalez et al. found they could not explain the browning through changes to collagen and suggested that an “independent product” was forming through Maillard reactions and causing browning [12].

Following this research survey and preliminary experiments it was possible to formulate several main theories regarding parchment browning, some more likely than other. First, a common guess when discussing parchment browning is oxidation: this is due to the main roll oxidation plays in browning of paper and other plant matter; however, both previous research and our preliminary results showed significant browning in conditions where oxidation was negligible. Considering the minimal changes observed in FTIR spectra of our preliminary experiments, and the fact that color could not be extracted by a variety of solvents attempted, the second theory was structural in nature: The collagen fibers are translucent and arranged in a layered structure in in-tact parchment. Possibly the degradation causes the layers to flatten and fibers compact and this changes how light is reflected, causing an apparent color change even without chemical changes. Finally, the last theory involves formation of strong brown pigments, with a high absorption coefficient, such that an amount undetected by analysis methods such as FTIR, could still cause a large color change. The pigments must also be strongly attached to the collagen matrix and therefore not easily extractable. Melanoidins are such a pigment family.

Melanoidins are generally described as, brown nitrogen containing macromolecules formed via Maillard reaction between amino-acids and reducing sugars. Most of the research on melanoidins comes from the food industry [13] and often deals with compounds formed at relatively high temperatures, such as during cooking of meat [14]. Research on simple model systems has however proved that these compounds do form slowly even at room temperature [15]. They form in collagen aging in human skin and collagen-based tissues such as eye lenses [16,17]. Unfortunately, only a few specific melanoidins have ever been isolated and properly characterized [18], these are the compounds that survive the strong acid hydrolysis necessary to free them from the collagen matrix and are not necessarily representative of the whole class.

We suggest that melanoidins may not have previously been researched as a cause of browning in parchment because the source of sugar is not clear. In skin there are proteoglycans that can then react with amino acids. These were assumed to wash out in the parchment preparation, because they are not covalently bonded to the collagen, but only attached by hydrogen bonds which would be disrupted by the water [19]. If a small amount of proteoglycans do survive, they may be enough to initiate the browning reaction. In the case of parchment prepared by Jewish tradition there are other possible sources: The flour added to help soften the parchment contains sugars. Also, the plant extracts used to lightly tan the parchment could be a source of reducing sugars and the phenols in the tannins could be an additional cause of browning. Moreover, Zamora & Hidalgo show that lipids and their oxidation products can work in conjunction with carbohydrates and can in fact initiate the melanoidin browning reaction instead of sugars [20]. Lipids definitely do survive the parchment preparation process and were studied previously by us [21] and other working with similarly prepared parchment [22]. Other studies have quantified lipids in parchment and studied their influence on parchment degradation [23,24]. The paper by Možir et al. reviewed 28 parchment samples of various origins and found all contained lipids. They also summarized multiple papers discussing how lipids in parchment may oxidize to form reactive carbonyl compounds that will then react with the proteins in the parchment [25], potentially initiating the browning reaction.

Besides the plant extracts added to the parchment [26,27], the DSS have various other additives on some of fragments. When the collection was first discovered in the 1950s many different materials were applied to the scrolls in attempts to unroll and read them. We have discussed the history of the scrolls and their treatment in a previous paper [21]. While some of those additives have undoubtedly caused extreme darkening of the parchment, since parts of the DSS were very dark already when discovered, as seen in images from the time, we decided to focus on untreated parchment and the sort of environmental conditions they could have been exposed to during their years hidden away in the Qumran caves. However, it is worth noting that in an experiment studying the effect of various additives on parchment (too be published in the future), significant browning was caused by added oils: olive oil in particular, but also castor oil.

Considering the tremendous volume of human history written on parchment, an improved understanding of the parchment browning process is extremely important. The series of artificial aging experiments demonstrates how different environmental conditions influence collagen and parchment degradation. This will help not only with the preservation of the DSS and other parchment documents, but all collagen based artifacts in museums and archives around the world. Also, collagen based tissues are relevant in many fields besides archaeology, including medical and food science, so perhaps our research may be extrapolated to those fields as well.