Characterization of the secondary structure, renaturation and physical ageing of gelatine adhesives

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Despite this decline, gelatine, derived from denatured and partially hydrolysed collagen, has gained popularity in various applications. This study focuses on gelatinous glue samples derived from animal bone and hide tissues, examining their secondary structure and thermal properties to identify structure-property correlations. Infrared spectroscopy analysis has revealed differences in the secondary structures, with hide glues exhibiting more β-structures than bone glues, indicating a higher degree of aggregation. Thermogravimetric analysis and differential scanning calorimetry also have highlighted differences between hide and bone glues, showing that the latter are more hydrolysed. Furthermore, the calorimetric curves have showed different values of denaturation enthalpy thus indicating a different degree of gelatine renaturation. Additionally, the calorimetric analysis has demonstrated the physical ageing of gelatinous glue samples, a key factor in maintaining adhesive properties for long-term use under specific storage conditions. In a context prioritizing the use of waste biomass over fossil fuels, understanding the properties of gelatine in glues is crucial for enhancing their performance and promoting their adoption as sustainable alternatives to non-renewable adhesives. Physical sciences/Chemistry Physical sciences/Materials science Gelatine animal glue gelatine adhesives secondary structure thermal analysis physical ageing Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Animal glues have been used for centuries as adhesives in a variety of applications [ 2 , 3 ]. The earliest evidence of its use dates back to the Palaeolithic period, where traces of animal glue have been found in cave paintings [ 4 ]. In later years, the use of animal glues became an integral part of various industries and everyday life [ 5 , 6 ]. In the 20th century, the demand for animal glue declined due to the advent of synthetic adhesives. However, the application fields for gelatine, which is based on partially hydrolysed and denatured collagen like animal glues, have expanded rapidly. While animal glue remained the adhesive of choice for traditional bookbinders, luthiers and restorers[ 7 , 8 ], gelatine is used in many different fields. Due to its gelling, emulsifying, and film-forming capabilities, gelatine is a key component of the food and cosmetic industries [ 9 – 11 ]. Gelatine is also widely used in the medical and tissue engineering fields as hydrogel, nanomicrosphere containers, nanofibers, pharmaceutical additives, haemostasis, and for the encapsulation of different drug products [ 12 – 15 ]. Moreover, gelatine is used in gelatine-based films for food bio-packaging. Recent studies have concentrated on creating active packaging films by incorporating essential oils or plant extracts, which impart antimicrobial and antioxidant properties, thereby enhancing the shelf life of food products [ 16 – 20 ]. The glue production process from animal tissue involves the hydrolysis and denaturation of native triple helix of collagen to obtain water-soluble gelatine, mainly in a random coil conformation [ 21 – 25 ]. The amino acid composition of gelatine may differ slightly from that of collagen as the manufacturing process may result in chemical modification in the amino acids [ 25 ]. For example, the alkaline process employed for gelatine extraction results in the deamination of glutamine and asparagine to the corresponding glutamic and aspartic acids [ 26 ]. Consequently, the primary structure of gelatine closely resembles that of collagen, with glycine, proline, and hydroxyproline as the main amino acids. However, its secondary and higher-order structures are significantly different. A partial recovery of the collagen structure occurs during cooling and drying, where the helices partially reform. This process is called renaturation or gelation process [ 25 ]. The structural order achieved depends on molecular weight and on several factors including chemical composition (mainly Pro and Hyp content) of the gelatine polypeptides. The molecular weight distribution of the gelatine molecules depends on the production process, which causes molecular degradation [ 27 ] as well as the type of tissue and the age of the animals. As reported by Elharfaoui et al. [ 27 ], the extraction of highly cross-linked collagen from old tissue results in the formation of high molecular weight gelatine molecules. However, these molecules are highly branched and the presence of many low molecular weight helices make the renaturation very improbable. Guo et al. [ 28 ] reported that gelatine can be organised in different ways depending on the length of the α-chains, which also affects the stability and reversibility of these structures. Different types of reorganisation at a higher structural level can occur involving one or more helices linked by hydrogen bonds [ 28 ] which lead to the formation of gel junction zones which act as sites for the formation of a three-dimensional network. The structural order of the network depends on molecular weight and chemical composition (mainly Pro and Hyp content) of gelatine molecules. Long, high molecular weight chains and high Pro and Hyp content promote highly ordered network [ 14 , 29 ]. Literature reports that the content of native-like triple helixes formed during the renaturation process is important as it affects the thermal, mechanical and adhesive properties of the glues [ 30 – 32 ]. The degree of renaturation and the structural order achieved from gelatine also depends on the storage conditions, in particular temperature and humidity [ 33 – 35 ]. Obas et al. [ 33 ] have reported that in high-solids confectionary gel made with gelatine, the amount of renatured structures is reduced when the gelatine sample is stored at or above the gelling temperature (20°C). Furthermore, Dai et al [ 34 ]. have observed that the amount of structural gelatine in film samples is strictly dependent on the drying temperature with the degree of renaturation decreasing as the temperature increases. Recently, Mosleh et al.[ 36 ] have reported on the effect of hygrothermal ageing on the properties of various gelatine glues films from mammalian and fish tissues, showing how humidity and temperature affect the microstructure of the gelatine and thus the mechanical properties of the glue. Generally, the presence and amount of triple-helix-like structures are determined through X-ray diffraction analysis and differential scanning calorimetry as the enthalpy of denaturation is positively correlated with the amount of triple-helix-like structures [ 31 , 33 , 36 – 39 ]. The few studies reported in the literature directly correlate the amount of triple helices present with the mechanical and adhesion properties. In particular, a greater quantity of these structures leads to a reduction in the degree of swelling, an increase in gel strength (higher degree of Bloom) and an increase in Young's modulus [ 30 , 31 ]. It is known in literature that β-structures have an influence on the properties of silk protein and gelatin-electrospun materials [ 40 , 41 ], but the effect of β-structures on the mechanical properties of adhesives has not been investigated. Therefore, this aspect warrants attention and should be considered in the study of gelatine-based adhesives. Further insights can be gained from the calorimetric analysis of polymers and biopolymers. In particular, from the enthalpy relaxation superimposed on the glass transition, it is possible to obtain insights into physical ageing. Physical aging in polymers and biopolymers refers to the gradual changes in the physical properties of a polymeric material over time due to its molecular structure and the environmental conditions it is exposed to. This process is related to the relaxation of the polymer's chains toward a more thermodynamically stable state. As polymers age, they may experience various physical changes that can impact their mechanical, thermal, and optical properties [ 42 , 43 ] Physical ageing is commonly observed when an amorphous polymer is rapidly cooled below its glass transition temperature ( t g ) and then stored at a temperature below t g [ 42 ]. It is understood that an amorphous polymer in the glassy state is in a non-equilibrium condition, whereas in the rubbery state, above the glass transition temperature (Tg), it reaches an equilibrium state. Consequently, if the polymer is stored at a temperature below its t g , it will gradually tend to relax towards a state of thermodynamic equilibrium. Physical ageing affects the thermodynamic and mechanical properties of the polymer [ 42 ]. The identification of this phenomenon is crucial in material studies, particularly in the pharmaceutical and food area, as it offers valuable insights into optimal storage conditions, shelf life, and how material properties may evolve over time due to factors such as temperature, humidity, and irradiation. Therefore, although this phenomenon has primarily been studied in synthetic [ 42 , 44 – 47 ] and conjugated polymers [ 48 ], recent interest in examining the physical aging of biopolymers has grown. These studies have allowed the observation of physical ageing in carbohydrates [ 49 , 50 ] and several protein materials [ 51 – 55 ]. Literature reports that in synthetic glues physical ageing affects their properties causing a reduction in adhesion strength, an increase in brittle, a loss of structural integrity with the formation of creep, and a loss of flexibility[ 56 – 58 ]. Recently it has been reported that physical ageing can affect also the properties of gelatine glue. Enrione et al. [ 59 ]has conducted a study of physical aging of a model gelatine film, which was prepared obtained by dissolving salmon gelatine in water and then drying it, using the same procedure applied in the production of animal glue. The results have showed that during the ageing of the model gelatine film the material changes its mechanical properties becoming stiffer. Physical ageing causes also a modification in porosity and density. All these can decrease the adhesive properties over time [ 36 , 60 ]. In this study, we have examined the structural characteristics of commercial collagen, gelatine A, gelatine B and a subset of gelatine-based animal glues derived from hide and bone tissues, among those characterized by Ntasi et al. [ 1 ]. This investigation focused on animal origin, degree of deamination of glutamine and asparagine, the molecular weights of the acid-soluble collagen fraction, backbone cleavage and the amount of 2,5-diketopiperazines produced during thermal degradation. The samples investigated in this work are reported in Table 1 that also includes a summary of the main results of Ntasi et al. [ 1 ] on the same samples. We used thermogravimetric analysis (TGA), to compare their thermal degradation profiles, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) to obtain information on their secondary structures, including helix, β-structures and random coil content, and differential scanning calorimetry (DSC) to determine temperature and denaturation enthalpy, glass transition temperature (a useful parameter for the application of protein biomaterials), and enthalpy relaxation related to physical ageing. 2. Materials and Methods The animal glue samples were provided by Museo Nacional del Prado (Madrid) and restoration workshop of the University Suor Orsola Benincasa (Naples). Table 1 reports the samples’ list including sample name, and a summary of the main results for each sample obtained by Ntasi et al. [ 1 ], such as taxonomy, molecular weight (MW) of acid soluble collagen (ASC) obtained by SDS-PAGE, backbone cleavage %, and the area of the pyrolytic signals related to all 2,5-diketopiperazines (DKPs) detected by Py-GC/MS. According to this, the samples were classified as Hide Pure (HP), Hide Mixed (HM), Bone Mixed (BM). Standard gelatine A from porcine skin (G2500, MW = 50–100 kDa) and gelatine B from bovine skin (G 9382, MW = 40–50 kDa) and standard collagen derived from calf skin (Bornstein and Traub Type I) were purchased from Sigma-Aldrich (Milan, Italy). Table 1 Animal glue’s molecular features from Ntasi et al. 1 : taxonomy, MW (kDa) of ACS, backbone cleavage percent and area of DKPs. Sample name Taxonomy MW (kDa) of ACS Backbone Cleavage percent Sum of the normalized areas of all DKPs *10 − 4 Deamidation degree* Hide Pure (HP) glues HP3 Bos taurus ≥ 30 25% 144 +++ HP4 Bos taurus ≥ 40 ~ 10% 146 +++ HP6 Bos taurus ≥ 60 25% 157 +++ HP8 Bos taurus ≥ 50 ~ 20% 239 ++ Hide Mixed (HM) glues HM1 Bos taurus/Sus scrofa ≥ 50 (with some fraction at lower molecular weigth) ~ 35% 165 ++ HM4 Bos taurus/Oryctolagus cuniculus ≥ 50 (with traces at lower molecular weigth) ~ 20% 126 ++ HM5 Oryctolagus cuniculus/Sus scrofa ≥ 70 (with traces at lower molecular weigth) ~ 25% 156 + Bone Mixed (BM) glues BM1 Bos taurus/Sus scrofa ~ 25; ~ 60; ≥100 ~ 60% 207 ++ BM2 Bos taurus/Sus scrofa/Equus asinus ~ 25;~ 60; ≥100 ~ 70% 197 ++ BM3 Bos taurus/Sus scrofa/Equus asinus ~ 25;~ 60; ≥100 ~ 45% 181 ++ BM4 Bos taurus/Sus scrofa/Equus asinus ~ 25;~ 60; ≥100 ~ 30% 214 ++ BM5 Bos taurus/Sus scrofa/Ovis aries/Equus asinus ~ 25;~ 60; ≥100 ~ 80% 174 ++ *+++ corresponds to deamidation % of asparagine > 80% and glutamine > 60%; ++ corresponds to deamidation % of asparagine between 50% and 70% and glutamine between 1% and 30%; + corresponds to deamidation % of asparagine and glutamine < 10%. 2.1 Thermogravimetric analysis TG measurements were conducted on collagen, gelatine A and B and on glue samples using TA Instruments Thermobalance model Q5000IR, from 25°C to 900°C, at a heating rate of 10°C/min. The thermogravimetric analysis was carried out under nitrogen flow (25 mL/min). Approximatively 5 mg of sample were weighted and put in platinum crucibles. The instrument was mass calibrated using certified mass standards in the range of 0-100 mg and temperature calibrated using five reference materials (Alumel, Ni, Ni83%Co17%, Ni63%Co37%, Ni37%Co63%). TA Universal Analysis 200 ver. 4.5A was employed for data treatment. 2.2 Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy ATR-FTIR investigation of the protein secondary structure in the collagen, gelatine A and B and animal glue samples was performed using a Perkin-Elmer Frontiers FTIR spectrophotometer, equipped with a universal Attenuated Total Reflectance (ATR) accessory and a triglycine sulphate TGS detector. All the spectra were recorded in the 4000 − 600 cm − 1 range in ATR mode after background acquisition. For each sample, 128 scans were recorded, averaged, and Fourier transformed to generate a spectrum with a nominal resolution of 4 cm − 1 . Spectrum software (Perkin-Elmer) and a written-in house LabVIEW program for peak fitting were employed to run and process spectra, respectively[ 61 – 63 ]. A straight baseline passing through the ordinates at 1800 cm − 1 and 1480 cm − 1 was subtracted before processing the curves, and the spectra were normalized in the 1700 − 1600 cm − 1 region. This approach was taken to avoid artefacts in absorptions near the limits of the region examined. The second derivatives of the amide I band of the spectra examined were then analysed to determine the initial data (number and position of the Gaussian components) required for the deconvolution procedure. The amide I band was chosen for structural analysis because of the very low contribution of the amino acid side chain absorptions present in this region [ 64 , 65 ], and its higher intensity compared to the other amide modes (amide II at 1540 cm − 1 and Amide III between 1350 − 1190 cm − 1 ). Based on the infrared assignment of amide components, assuming that the extinction coefficient is the same for all the secondary structures, the secondary structure composition can be obtained from the FTIR spectra. The percentages of the different secondary structures were estimated by expressing the amplitude value of the bands assigned to each of these structures as a fraction of the total sum of the amplitudes of the amide I components. Although the general validity of the above assumption about extinction coefficients remains to be verified, there are studies in the literature that show good correlation between the distribution of secondary structures obtained by FTIR and X-ray crystallography approaches, suggesting that the assumption made is reasonable [ 62 ]. 2.3 Differential scanning calorimetry DSC analyses were performed by TA Instruments Discovery DSC model 250 under nitrogen flow (50 mL/min). The instrument was temperature calibrated with indium. For each sample, about 5 mg was weighted and hermetically sealed into aluminium DSC pans. An empty pas was used as a reference. Collagen, gelatine A and B, and glue samples were subjected to heating/cooling/heating cycle in the temperature range of 10–130°C at heating rate of 10°C/min and cooling rate of 50°C/min. Physical ageing study was carried out on glue samples consisting of one type of collagen belonging to the same animal species, Bos taurus , i.e. HP3, HP4, HP6, HP8 samples and on one sample derived from bone tissue, namely BM5. The procedure was the following. The samples were heated/cooled/reheated under the same conditions as described above, and after the second heating scan, the samples were cooled to 40°C (temperature below the glass transition temperature) at 50°C/min, held at 40°C (annealing temperature) for up to 3 hours and heated to 130°C at 10°C/min. The samples were then cooled to 40°C at 50°C/min, held at 40°C for up to 6 hours and heated to 130°C at 10°C/min. The sequence was repeated with the sample held at 40°C for up to 12 h. The same procedure was repeated at an annealing temperature of 30°C only on HP6 in order to confirm the presence of physical ageing. This procedure was inspired by Farahnaky et al. [ 51 ]. For each calorimetric method used, a baseline was recorded and subtracted from the calorimetric curve of each sample. TRIOS v5.1.1 was employed for data treatment. 3. Results 3.1 Thermogravimetric analysis The thermal degradation of collagen, gelatine standards and glue samples was investigated by TGA under nitrogen flow. Figure 1 shows thermogravimetric (TG), and differential thermogravimetric (DTG) curves obtained for all samples. All the samples exhibit the same thermal degradation profile, which primarily consists of two steps. The first mass loss (10–12% w/w) below 60°C is due to moisture evaporation and the second (65–75% w/w) centred at 300–320°C is due to protein pyrolysis resulting in the formation of 2,5-diketopiperazines (DKP), the most abundant of which in the glue samples are cyclo(Pro-Gly) and cyclo(Pro-HyP), 1 and of several aromatic and nitrogen-containing compounds, such as pyrrole, indole, phenol, and the respective alkylated compounds [ 66 , 67 ]. The residue at 900°C is in the range of 15–20% w/w, as generally reported in literature for proteins. Table S3 shows the percentage mass loss and the temperature peak determined for each sample. Standard collagen showed an additional degradation step below 200°C. As reported in the literature, this is due to the evaporation of the strongly H-bonded water involved in the stabilization of the triple helix [ 68 – 70 ]. By observing the shape of the DTG related to water loss, gelatine standards exhibited a broader signal in over a wide temperature range (from 30°C to 200°C) indicating the presence of structural water. The DTG curves of glue samples suggest that HP3, HP6 and HM5 contain more structural water than the standard gelatine, while the other samples have little to none. Different degree of hydrolysis of collagen and the different animal species can affect the thermal degradation [ 1 , 71 – 73 ]. By considering the t endset and t onset (Table S4), a slight difference was observed for gelatine standards, as gelatine A has t onset higher than gelatine B that indicates higher thermal stability, but t endset lower than gelatine B, i.e. a lower amount of thermostable cross-linked/aggregated structures. This result agrees with the literature and depends on the different production process used. Gelatine A undergoes an acid pretreatment whereas gelatine B undergoes a basic pretreatment. This leads to gelatine with different properties and, in general, gelatine A is less covalently cross-linked than gelatine B [ 15 ], probably due to the protonation of groups involved in cross-linking formation. A slight shift of the maximum towards lower temperatures was observed for animal glue samples, suggesting the presence of more hydrolysed molecules compared to gelatine and collagen. Furthermore, looking at the t endset in Table S4, BM sample completed its degradation at a lower temperature than the other samples. This result further suggest a higher degree of hydrolysis, a lower degree of aggregation and the presence of molecules with a lower molecular weight in BM samples, as reported by Ntasi et al. [ 1 ], who identified highly hydrolysed collagen in BM samples through the SDS-PAGE, backbone cleavage percentage and Py-GC/MS analysis (Table 1 ). Another factor to consider is the percentage of deamidation. Notably, hide glues exhibit higher levels of deamidation compared to bone glues 1 . This increased deamidation can result in a higher number of charges, potentially leading to the formation of more thermally stable aggregates. Regarding the HP and HM samples, no clear difference in thermal degradation was observed. Both HP and HM samples have a t onset lower than that of collagen and gelatine and a t endset lower than collagen but equal to or higher than that of gelatine, indicating the presence of more hydrolysed molecules as well as thermostable aggregates. 3.2 Secondary structure of gelatine in animal glue samples ATR-FTIR analysis was carried out on animal glue samples and on collagen, gelatine A and B to investigate the protein secondary structures. Figure S1 shows the FTIR spectra, which display the typical features of protein materials, i.e. the amide I band in the 1700 − 1600 cm − 1 region (corresponding to C = O stretching vibration), the amide II in the 1600 − 1480 cm − 1 region (corresponding to out-of-phase combination of the NH in plane bend and the CN stretching vibration) and the amide III in the 1350 − 1190 cm − 1 region (corresponding to in-phase combination of the NH bending and the CN stretching vibration) [ 17 ]. The amide I frequency is strongly affected by the protein conformation [ 17 ], consequently the amide I shift observed in the animal glue samples clearly indicates a change in the protein structure. To gain a deeper understanding of the gelatine conformation, a curve-fitting method was applied to deconvolve the complex amide I band, allowing for the determination of the different percentages of secondary structures. [ 61 – 63 ]. Characteristic components of the Amide I band are assigned to the following secondary structures: helix (1650–1660 cm − 1 ), random (1640–1660 cm − 1 ), collagen native triple helix (1660–1666 cm − 1 ), antiparallel β-sheets (two components around 1620 cm − 1 and 1690 cm − 1 ), parallel β-sheets (one component around 1630 cm − 1 ), β-turns (1670–1685 cm − 1 ) [ 65 ]. Table 2 show the secondary structure percentages, the wavenumber in cm − 1 and half height bandwidth (HHBW), based on the results of the peak fitting of Amide I band. The assignment of bands to a precise secondary structure is challenging as different bands often overlap, as in the case of helix and random coil structures. Because of the wide bandwidth of the bands assigned to helices and random structures in the FTIR spectra of these samples (Table 2 ), in this work was not possible to discriminate between these two structures in all the samples. The assignment to β-sheet structures in Table 2 includes parallel and antiparallel β-sheets and β-turn [ 65 , 74 ]. The results obtained on the standard of collagen indicate that the collagen is not in its native form, as a significant amount of β-structures are present. The same results can be observed for gelatine standards. The high amount of helix/random structures testify the semicrystalline nature of gelatine, i.e. a crystalline domain consisting of the triple-helix structures and an amorphous domain composed mainly of the random structures and single helix chains, as already reported in the literature [ 25 , 75 , 76 ]. When comparing the results of animal glues, the HP and BM1 samples exhibit a secondary structure like that of the collagen and gelatine standards. HM samples appear to be the only ones without random structures and, along with BM samples, exhibit a lower amount of helix/random structures compared to the others. Table 2 Secondary structure percentages, wavenumber (cm − 1 ) and half height bandwidth (HHBW) of the Amide I component and of the collagen and gelatine standard and glue samples. Sample Random and helices* βstructures (β-sheets/ β-sheet (p + ap)/ β-turn) Collagen 1650 (33) (random) 21% 1659 (50) (helix) 23% 1611 (53) 36% 1626 (17) 20% Gelatine A 1652 (60) 51% 1618 (42); 1693 (28) 49% Gelatine B 1651 (28) 42% 1619 (44) 43% 1685 (39) 15% HP3 1659 (51) 41% 1622 (29); 1693 (26) 58% HP4 1654 (59) 48% 1618 (45); 1693 (28) 53% HP6 1659 (45) 30% 1622 (54); 1689 (36) 70% HP8 1660 (59) 40% 1621 (54); 1687 (21) 61% HM1 1666 (43) (helix) 23% 1627 (60); 1689 (30) 77% HM4 1668 (49) (helix) 26% 1626 (61); 1689 (24) 74% HM5 1668 (51) (helix) 30% 1624 (61);1691 (22) 70% BM1 1659 (60) 42% 1620 (53) 55% 1688 (22) 3% BM2 1660 (41) 29% 1625 (52) 59% 1687 (33) 12% BM3 1657 (16) 7% 1629 (75) 83% 1682 (36) 10% BM4 1656 (33) 21% 1628 (46) 56% 1679 (40) 23% BM5 1661 (42) 28% 1625 (53);1696 (7) 61% 1687 (32) 11% * Where not specified this band includes random and helix structures because the peaks assigned to these two components in collagen are too close (1650–1660 cm − 1 and 1640–1660 cm − 1 ) 65 and they may not be separated by the peak deconvolution procedure. 3.2 Differential scanning calorimetry Calorimetric analysis was performed on glue samples to obtain information on the thermal stability of the protein structures and to compare them with those of gelatine and collagen standards. The calorimetric curves obtained are reported in Fig. 2 . The calorimetric curve of the collagen standard showed in the first heating scan a broad endothermic signal between 36°C and 121°C with a maximum at 82°C accounting for 18 J/g as enthalpy value and ascribable to collagen unfolding [ 77 ]. In the second heating scan, the only signal observed was the glass transition occurring at almost 0°C. This result is not expected, as the literature reports a glass transition temperature for tendon collagen of around 40–45°C [ 78 , 79 ]. No information is provided about the processes undergone by standard collagen, but this difference may suggest that standard collagen is not in its native form and has undergone a treatment that partially altered its structure, as also observed in the secondary structure analysis. The unfolding enthalpy of collagen reported in literature is in the range 40–50 J/g [ 35 , 77 ]. Our lower value could be due to a partial denaturation of collagen, as indicated by the β-sheets content. Considering the proximity of β- sheets and native collagen helix in the Ramachandran plot [ 80 ], collagen is unlikely to return to its native shape, but rather it could give rise to β- sheet structures, which are notoriously very stable. The calorimetric curve of both gelatine A and B showed two distinct endothermic signals during the first heating scan. The first signal (6 J/g and 12 J/g, respectively) at about 70°C is an overshooting overlapped to the glass transition. The second signal (11 J/g and 3 J/g, respectively) at 95°C corresponds to the unfolding of the gelatine structure, which is associated with the degree of renaturation of the gelatin. A higher amount of reformed native-like triple helix structure would result in a higher enthalpy value [ 35 , 77 ]. The results indicate that gelatine A exhibits a higher degree of renaturation compared to gelatine B. This difference may be attributed to variations in their covalent cross-linking levels, as well as the conditions (temperature and humidity) during the renaturation process. The presence of a higher number of covalent bonds in gelatine B may hinder the reorganisation and formation of triple helix-like structures, resulting in a lower degree of renaturation. The calorimetric profiles of all glue samples in the first heating scan were more similar to that of gelatine than collagen, displaying two endothermic signals: the first in the temperature range 50–80°C, overlapping the glass transition, the second, in the temperature range 80–110°C (Fig. 2 ). In the second heating scan all the analysed samples exhibited only the signal related to glass transition (Figure S2 in supporting information). The signal superimposed to the glass transition was better investigated in order to confirm it to be associated with structural relaxation associated with physical ageing of the sample as reported in literature for synthetic polymers, some proteins and gelatines [ 35 , 51 , 81 , 82 ]. The study of physical ageing was performed on glue samples of the same animal origin (HP3, HP4, HP6, HP8) to reduce the variability associated with different animal sources and on one bone glue sample (BM5) to confirm the presence of this phenomenon also in the bone glues. To study the physical ageing, after the first heating up to 130°C, the samples were kept at 40°C (temperature below the t g ) for different periods of time (3 h, 6 h, 12 h, 24 h) as specified in the experimental section and heated to 130°C in order to observe the enthalpy relaxation signal. Since the extent of physical ageing depends on the annealing temperature, the experiment was repeated on sample HP6 also at the annealing temperature of 30°C. Specifically, as the annealing temperature increases, both the peak temperature and the relaxation enthalpy value also increase [ 51 , 52 , 83 ]. The values of the enthalpy relaxation and the peak temperature calculated at each time point are reported in the table S1 . Figure 3 reports the DSC curves obtained for HP6 as example. The characteristics of this relaxation peak are compatible with the physical ageing phenomenon occurring in the glues, as described in the literature [ 51 ]: the signal intensity increases over time and the peak temperature increases linearly with the logarithm of the ageing time (Fig. 4 ). The same behaviour was observed for BM5 (Table S1 ). Regarding the glass transition, when applied to proteins, this term refers to the change in the dynamic properties of proteins that takes place at a specific temperature. It has been reported in the literature that at temperatures above the glass transition the dynamics of protein molecules are dominated by large scale motions of groups of atoms, whereas at lower temperatures vibrational motions predominate. The temperature at which this change in molecular dynamics occurs is influenced by the degree of hydration of the protein and the intrinsic temperature dependence of the motions [ 84 ]. All the glue samples have the same moisture content (around 10%, as proved by TGA data reported below) and the t g values, obtained from the first heating scan, range from 52°C (i.e., HP8) to 73°C. (i.e., HM1), with the gelatine A and B values almost in the middle at 62°C and 68°C, respectively, without any clear correlation with the animal source (Figure S2 and Table S2a). In general, t g is lower in the second heating scan compared to the first one, except for HP4 and HP8, which exhibited nearly the same temperature. As reported in [ref 35] and by Tseretely and Smirnova [ 85 ], this is due to the release of bounded water during the denaturation process, which acts as a plasticiser by lowering the t g . The values of the heat capacity change (∆ C p ) associated with the glass transition, are in the range 0.65–1.3 J/K∙g and 0.53–1.3 J/K∙g calculated from the first and second heating scan, respectively. These values agree with the data reported in literature on amorphous gelatines and disordered crystalline gelatines [ 85 ] and they are slightly higher than those reported for other proteins [ 43 , 54 , 86 ]. All the glues (both from hide and from bone) show the relaxation enthalpy peak superimposed to the glass transition with values varying between 5 (i.e., HP4 and HP8) and 11 J/g (i.e., HM4). The value obtained for gelatine is 6 J/g as well as HM5 (Table S2). For the collagen standard, as well as BM3, BM4, and BM5 samples, it was not possible to determine the relaxation enthalpy associated with the glass transition due to the signal overlapping with other signals at higher temperatures. This signal is often overlooked and there are few studies in which the enthalpy relaxation has been determined in gelatinous samples. Enrione et al. [ 59 ], determined the enthalpy associated with physical ageing of fish gelatine film and obtained a value of 2.42 J/g after an ageing of 40 h at annealing temperature 5°C lower than t g , i.e., 29°C. The higher enthalpy relaxation values of the glue samples obtained in this study could be due to the annealing temperature used, which was 20–30°C lower than t g . In these conditions, the enthalpy relaxation can be higher because the sample is far away from equilibrium state. The unfolding peak in the hide glues is well separated in temperature from the relaxation enthalpy peak with a t peak varying between 89°C (i.e., HP8) and 100°C (i.e., HM1) and enthalpy values varying between 3 (i.e., HP3) and 11 J/g (i.e., gelatine and HP6), and almost absent in HP4 and HM4. These values are lower than those obtained for collagen, indicating a lower structural order due to the higher degree of hydrolysis and lower molecular weight of the gelatine molecules that hinder renaturation and thus the formation of structures similar to the native triple helix [ 85 ]. For bone glues is not possible to determine separate values for the relaxation enthalpy and for the unfolding one, because all the DSC peaks are partially superimposed. 4. Discussion and Conclusion TGA data clearly indicate that glue samples are more similar to gelatine than to the reference collagen. Therefore they didn’t show the degradation step at around 200°C that is reported to be related to the evaporation of the strongly H-bonded water involved in the stabilization of the triple helix [ 68 – 70 ]. In addition, they present an unfolding enthalpy that is less than half of that of collagen. These results confirmed those obtained by Ntasi et al. [ 1 ] reporting that glues are composed by hydrolysed collagen of lower molecular weight and also highlight that glues have a lower content of collagen triple helix. Some differences on structures also seem to exist depending on whether the samples are from bone (B) or tissue (H). HP and HM samples are less hydrolyzed than BM samples, have an average MW above 40 KDa (Table 1 ) and these properties appear to result in a well-defined denaturation peak although the low value of the denaturation enthalpy indicates a low content of collagen triple helix-like structures. In particular, the HP4 and HM4 samples appear to be those with a lower degree of structural order given their low enthalpy value (about 1 J/g). This is likely due to the conditions used during the drying process after extraction, the storage conditions and the possible presence of covalent cross-linking that hinders renaturation. BM3, BM4, and BM5 exhibited a calorimetric curve significantly different from the other samples. Specifically, the signal consists of several overlapping signals, likely due to the higher degree of hydrolysis in the gelatine, resulting in molecules with broader molecular weight distribution. This suggests that a well-defined three-dimensional network is unlikely to form, but rather several aggregates with lower structural order. In contrast, BM1 and BM2 displayed a denaturation signal similar to that of HP and HM samples, indicating a higher degree of renaturation compared to the other BM samples. FTIR results revealed a relative high content of β-structures in all the glue samples, gelatine and collagen. This highlights that is extremely difficult to obtain native collagen after the extraction process from tissue and it explains the low denaturation enthalpy obtained from collagen. In general the structural analysis of gelatine in the literature focuses on evaluating the triple helix content using X-ray diffraction, supported by DSC results [ 31 , 33 , 35 , 36 , 59 , 80 , 87 ]. Structure property studies link the mechanical properties of glues to the triple helix content, and vice versa [ 31 , 33 , 36 ]. However, β-structures can also play a role in the mechanical and adhesive properties of glues and gelatine. In fact, a few studies have reported that in silk protein toughness and tensile strength are positively correlated with β-sheet content [ 40 ], and in gelatin-electrospun materials stiffness increases with a higher β-sheet content [ 41 ]. Therefore, we believe that in gelatine-based adhesives, it is crucial to consider not only the amount of triple-helix-like structures but also the amount of β-sheet structures to explain the structure-dependent mechanical properties. Another observation in this study is the presence of physical ageing in all the glue samples analysed, regardless of animal origin or tissue type. As reported in the literature, the enthalpy relaxation value is influenced by storage conditions, including temperature and humidity. Since the glass transition temperature is higher than room temperature, physical aging will inevitably occur, and because it affects mechanical properties, it should be considered when using gelatin-based adhesives. Declarations Acknowledgements Museo Nacional del Prado (Madrid) and restoration workshop of the University Suor Orsola Benincasa (Naples) are acknowledged for providing the animal glues samples. CRediT authorship contribution statement Elena Pulidori: Conceptualization, Investigation, Data Curation, Writing - Original Draft. Celia Duce: Conceptualization, Writing - Review & Editing, Supervision, Funding acquisition. Emilia Bramanti: Conceptualization, Writing - Review & Editing, Supervision, Funding acquisition. Leila Birolo: Resources, Writing - Review & Editing, Funding acquisition. Brunella Cipolletta : Writing - Review & Editing. Laura Dello Ioio : Resources, Writing - Review & Editing. Ilaria Bonaduce: Writing - Review & Editing, Supervision, Funding acquisition. Funding This work was supported by MUR project PRIN 2022 PNRR ArtDECOW (project code P2022HWL7L). The project is financed by European Union-Next Generation EU, Mission 2, Component 1 (M2C1) CUP: I53D23005990001. 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Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 02 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 10 Feb, 2025 Reviews received at journal 04 Jan, 2025 Reviewers agreed at journal 03 Jan, 2025 Reviewers agreed at journal 25 Dec, 2024 Reviews received at journal 22 Dec, 2024 Reviewers agreed at journal 19 Dec, 2024 Reviewers invited by journal 19 Dec, 2024 Editor assigned by journal 19 Dec, 2024 Editor invited by journal 19 Dec, 2024 Submission checks completed at journal 19 Dec, 2024 First submitted to journal 18 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5670377","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":393310448,"identity":"7bdc12d2-f305-492a-a803-7c970c1fd342","order_by":0,"name":"Elena Pulidori","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Pulidori","suffix":""},{"id":393310449,"identity":"ca8fb308-ff90-41d9-94f6-175e8cd2e2a6","order_by":1,"name":"Celia Duce","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYFAC5oYDQALEANIGDDxgNn7ACNPClgDVQlAPYwMDRAuPAcxe/Br42xsbD/xgsE7sb+/5Jl1QcEeGgZ3/AF4tEmcONhzsYUhPnHHm7DbpGQbPCDvMQCKx4QAPw+HEDRK526R5DA4ToUX+YcPBPyAt8m+eEalFgrHhMMQWHjbitEicSWw4LGOQbjzjTJqxNQ/QL2zMzAZ4tfC3Hz788U2FtWx/++GHt3n+3LHn5z/4AL81EOfBWQcY2IhQjwIOkKphFIyCUTAKRgAAAB74P5d+fQn0AAAAAElFTkSuQmCC","orcid":"","institution":"University of Pisa","correspondingAuthor":true,"prefix":"","firstName":"Celia","middleName":"","lastName":"Duce","suffix":""},{"id":393310450,"identity":"d99e44a4-bb36-4d28-a65a-a373443ed53f","order_by":2,"name":"Emilia Bramanti","email":"","orcid":"","institution":"National Research Council","correspondingAuthor":false,"prefix":"","firstName":"Emilia","middleName":"","lastName":"Bramanti","suffix":""},{"id":393310451,"identity":"4c44c0d0-f485-4cb8-aa4f-0be989e99111","order_by":3,"name":"Leila Birolo","email":"","orcid":"","institution":"University of Naples Federico II","correspondingAuthor":false,"prefix":"","firstName":"Leila","middleName":"","lastName":"Birolo","suffix":""},{"id":393310452,"identity":"a6a51aa8-b6a8-417c-8592-835d0058f01e","order_by":4,"name":"Brunella Cipolletta","email":"","orcid":"","institution":"University of Naples Federico II","correspondingAuthor":false,"prefix":"","firstName":"Brunella","middleName":"","lastName":"Cipolletta","suffix":""},{"id":393310453,"identity":"696c24cb-f56d-48ae-b5e6-b88b295c6758","order_by":5,"name":"Laura Dello Ioio","email":"","orcid":"","institution":"Dello Ioio Restauri","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"Dello","lastName":"Ioio","suffix":""},{"id":393310454,"identity":"2c9d7772-a18c-4785-9f59-3d417d7cec80","order_by":6,"name":"Ilaria Bonaduce","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Ilaria","middleName":"","lastName":"Bonaduce","suffix":""}],"badges":[],"createdAt":"2024-12-18 14:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5670377/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5670377/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-04910-8","type":"published","date":"2025-07-02T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":72185228,"identity":"9349682b-84d1-44e5-9ce7-de4b3b4a1361","added_by":"auto","created_at":"2024-12-23 13:18:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2131028,"visible":true,"origin":"","legend":"\u003cp\u003eTG (a,c) and DTG (b,d) curves of collagen (red line), gelatine A and gelatine B (black line), and glue samples (green line for HP and HM samples, blue line for BM samples) obtained under 25 mL/min nitrogen flow and using 10 °C/min as heating rate.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5670377/v1/523733d1c1b8f771029074a4.png"},{"id":72185229,"identity":"50fd7932-1da9-4577-9f25-ead2f45a778c","added_by":"auto","created_at":"2024-12-23 13:18:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1437878,"visible":true,"origin":"","legend":"\u003cp\u003eDSC curves obtained during the first heating scan at 10 °C/min of collagen (red line), gelatine A and B (black line), hide-derived glues (green line), bone-derived glues (blue line).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5670377/v1/70cc6cdd87a7a8f840a8d9cd.png"},{"id":72185232,"identity":"b37935e8-a1d7-46eb-b99f-466539ad2fd5","added_by":"auto","created_at":"2024-12-23 13:18:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":775277,"visible":true,"origin":"","legend":"\u003cp\u003eDSC curves of HP6 before and after physical ageing: black line: first heating at 10 °C/min from 10 °C to 130 °C; grey line: second heating at 10 °C/min from 10 °C to 130 °C; green line: third heating at 10 °C/min from 10 °C to 130 °C carried out after 3 h of isothermal; purple line: fifth heating at 10 °C/min from 10 °C to 130 °C carried out after 12 h of isothermal at 40 °C; orange line: fifth heating at 10 °C/min from 10 °C to 130 °C carried out after 24 h of isothermal at 40 °C \u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FIgure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5670377/v1/248962e4e60ce4a9edf5c653.png"},{"id":72186399,"identity":"4ac616e1-82b2-4026-9767-39bd2e7d8431","added_by":"auto","created_at":"2024-12-23 13:26:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":801628,"visible":true,"origin":"","legend":"\u003cp\u003eDependence of enthalpy (a) and peak temperature (b) on the logarithm of the ageing time (min) for HP 6 sample aged at 30 °C (orange triangle) and 40 °C (blue circular dot).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5670377/v1/a5580ddb39121ffc86066dd1.png"},{"id":86179074,"identity":"67fbaf23-efae-4495-ad96-88ca85f0cc6f","added_by":"auto","created_at":"2025-07-07 16:15:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6111116,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5670377/v1/d882a205-cfa1-4d06-9d13-a3a577e20e4c.pdf"},{"id":72185249,"identity":"074ce16f-d8a5-4652-8932-99aadf787ad5","added_by":"auto","created_at":"2024-12-23 13:18:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":7818967,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5670377/v1/f424625987a655fa145f2245.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization of the secondary structure, renaturation and physical ageing of gelatine adhesives","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAnimal glues have been used for centuries as adhesives in a variety of applications [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The earliest evidence of its use dates back to the Palaeolithic period, where traces of animal glue have been found in cave paintings [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In later years, the use of animal glues became an integral part of various industries and everyday life [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the 20th century, the demand for animal glue declined due to the advent of synthetic adhesives. However, the application fields for gelatine, which is based on partially hydrolysed and denatured collagen like animal glues, have expanded rapidly.\u003c/p\u003e \u003cp\u003eWhile animal glue remained the adhesive of choice for traditional bookbinders, luthiers and restorers[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], gelatine is used in many different fields. Due to its gelling, emulsifying, and film-forming capabilities, gelatine is a key component of the food and cosmetic industries [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Gelatine is also widely used in the medical and tissue engineering fields as hydrogel, nanomicrosphere containers, nanofibers, pharmaceutical additives, haemostasis, and for the encapsulation of different drug products [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, gelatine is used in gelatine-based films for food bio-packaging. Recent studies have concentrated on creating active packaging films by incorporating essential oils or plant extracts, which impart antimicrobial and antioxidant properties, thereby enhancing the shelf life of food products [\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe glue production process from animal tissue involves the hydrolysis and denaturation of native triple helix of collagen to obtain water-soluble gelatine, mainly in a random coil conformation [\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe amino acid composition of gelatine may differ slightly from that of collagen as the manufacturing process may result in chemical modification in the amino acids [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For example, the alkaline process employed for gelatine extraction results in the deamination of glutamine and asparagine to the corresponding glutamic and aspartic acids [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Consequently, the primary structure of gelatine closely resembles that of collagen, with glycine, proline, and hydroxyproline as the main amino acids. However, its secondary and higher-order structures are significantly different. A partial recovery of the collagen structure occurs during cooling and drying, where the helices partially reform. This process is called renaturation or gelation process [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The structural order achieved depends on molecular weight and on several factors including chemical composition (mainly Pro and Hyp content) of the gelatine polypeptides. The molecular weight distribution of the gelatine molecules depends on the production process, which causes molecular degradation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] as well as the type of tissue and the age of the animals.\u003c/p\u003e \u003cp\u003eAs reported by Elharfaoui et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], the extraction of highly cross-linked collagen from old tissue results in the formation of high molecular weight gelatine molecules. However, these molecules are highly branched and the presence of many low molecular weight helices make the renaturation very improbable.\u003c/p\u003e \u003cp\u003eGuo et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] reported that gelatine can be organised in different ways depending on the length of the α-chains, which also affects the stability and reversibility of these structures. Different types of reorganisation at a higher structural level can occur involving one or more helices linked by hydrogen bonds [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] which lead to the formation of gel junction zones which act as sites for the formation of a three-dimensional network. The structural order of the network depends on molecular weight and chemical composition (mainly Pro and Hyp content) of gelatine molecules. Long, high molecular weight chains and high Pro and Hyp content promote highly ordered network [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLiterature reports that the content of native-like triple helixes formed during the renaturation process is important as it affects the thermal, mechanical and adhesive properties of the glues [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe degree of renaturation and the structural order achieved from gelatine also depends on the storage conditions, in particular temperature and humidity [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Obas et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] have reported that in high-solids confectionary gel made with gelatine, the amount of renatured structures is reduced when the gelatine sample is stored at or above the gelling temperature (20\u0026deg;C). Furthermore, Dai et al [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. have observed that the amount of structural gelatine in film samples is strictly dependent on the drying temperature with the degree of renaturation decreasing as the temperature increases. Recently, Mosleh et al.[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] have reported on the effect of hygrothermal ageing on the properties of various gelatine glues films from mammalian and fish tissues, showing how humidity and temperature affect the microstructure of the gelatine and thus the mechanical properties of the glue.\u003c/p\u003e \u003cp\u003eGenerally, the presence and amount of triple-helix-like structures are determined through X-ray diffraction analysis and differential scanning calorimetry as the enthalpy of denaturation is positively correlated with the amount of triple-helix-like structures [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The few studies reported in the literature directly correlate the amount of triple helices present with the mechanical and adhesion properties. In particular, a greater quantity of these structures leads to a reduction in the degree of swelling, an increase in gel strength (higher degree of Bloom) and an increase in Young's modulus [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is known in literature that β-structures have an influence on the properties of silk protein and gelatin-electrospun materials [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], but the effect of β-structures on the mechanical properties of adhesives has not been investigated. Therefore, this aspect warrants attention and should be considered in the study of gelatine-based adhesives.\u003c/p\u003e \u003cp\u003eFurther insights can be gained from the calorimetric analysis of polymers and biopolymers. In particular, from the enthalpy relaxation superimposed on the glass transition, it is possible to obtain insights into physical ageing. Physical aging in polymers and biopolymers refers to the gradual changes in the physical properties of a polymeric material over time due to its molecular structure and the environmental conditions it is exposed to. This process is related to the relaxation of the polymer's chains toward a more thermodynamically stable state. As polymers age, they may experience various physical changes that can impact their mechanical, thermal, and optical properties [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/p\u003e \u003cp\u003ePhysical ageing is commonly observed when an amorphous polymer is rapidly cooled below its glass transition temperature (\u003cem\u003et\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e) and then stored at a temperature below \u003cem\u003et\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. It is understood that an amorphous polymer in the glassy state is in a non-equilibrium condition, whereas in the rubbery state, above the glass transition temperature (Tg), it reaches an equilibrium state. Consequently, if the polymer is stored at a temperature below its \u003cem\u003et\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e, it will gradually tend to relax towards a state of thermodynamic equilibrium. Physical ageing affects the thermodynamic and mechanical properties of the polymer [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The identification of this phenomenon is crucial in material studies, particularly in the pharmaceutical and food area, as it offers valuable insights into optimal storage conditions, shelf life, and how material properties may evolve over time due to factors such as temperature, humidity, and irradiation. Therefore, although this phenomenon has primarily been studied in synthetic [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and conjugated polymers [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], recent interest in examining the physical aging of biopolymers has grown. These studies have allowed the observation of physical ageing in carbohydrates [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and several protein materials [\u003cspan additionalcitationids=\"CR52 CR53 CR54\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLiterature reports that in synthetic glues physical ageing affects their properties causing a reduction in adhesion strength, an increase in brittle, a loss of structural integrity with the formation of creep, and a loss of flexibility[\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Recently it has been reported that physical ageing can affect also the properties of gelatine glue. Enrione et al. [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]has conducted a study of physical aging of a model gelatine film, which was prepared obtained by dissolving salmon gelatine in water and then drying it, using the same procedure applied in the production of animal glue. The results have showed that during the ageing of the model gelatine film the material changes its mechanical properties becoming stiffer. Physical ageing causes also a modification in porosity and density. All these can decrease the adhesive properties over time [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we have examined the structural characteristics of commercial collagen, gelatine A, gelatine B and a subset of gelatine-based animal glues derived from hide and bone tissues, among those characterized by Ntasi et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This investigation focused on animal origin, degree of deamination of glutamine and asparagine, the molecular weights of the acid-soluble collagen fraction, backbone cleavage and the amount of 2,5-diketopiperazines produced during thermal degradation. The samples investigated in this work are reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e that also includes a summary of the main results of Ntasi et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] on the same samples. We used thermogravimetric analysis (TGA), to compare their thermal degradation profiles, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) to obtain information on their secondary structures, including helix, β-structures and random coil content, and differential scanning calorimetry (DSC) to determine temperature and denaturation enthalpy, glass transition temperature (a useful parameter for the application of protein biomaterials), and enthalpy relaxation related to physical ageing.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eThe animal glue samples were provided by Museo Nacional del Prado (Madrid) and restoration workshop of the University Suor Orsola Benincasa (Naples). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e reports the samples\u0026rsquo; list including sample name, and a summary of the main results for each sample obtained by Ntasi et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], such as taxonomy, molecular weight (MW) of acid soluble collagen (ASC) obtained by SDS-PAGE, backbone cleavage %, and the area of the pyrolytic signals related to all 2,5-diketopiperazines (DKPs) detected by Py-GC/MS. According to this, the samples were classified as Hide Pure (HP), Hide Mixed (HM), Bone Mixed (BM).\u003c/p\u003e \u003cp\u003eStandard gelatine A from porcine skin (G2500, MW\u0026thinsp;=\u0026thinsp;50\u0026ndash;100 kDa) and gelatine B from bovine skin (G 9382, MW\u0026thinsp;=\u0026thinsp;40\u0026ndash;50 kDa) and standard collagen derived from calf skin (Bornstein and Traub Type I) were purchased from Sigma-Aldrich (Milan, Italy).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnimal glue\u0026rsquo;s molecular features from Ntasi et al.\u003csup\u003e1\u003c/sup\u003e: taxonomy, MW (kDa) of ACS, backbone cleavage percent and area of DKPs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTaxonomy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW (kDa)\u003c/p\u003e \u003cp\u003eof ACS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBackbone Cleavage percent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSum of the normalized areas of all DKPs *10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDeamidation degree*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003eHide Pure (HP) glues\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBos taurus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBos taurus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e~\u0026thinsp;10%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHP6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBos taurus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHP8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBos taurus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e~\u0026thinsp;20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e239\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eHide Mixed (HM) glues\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBos taurus/Sus scrofa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;50 (with some fraction at lower molecular weigth)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e~\u0026thinsp;35%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHM4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBos taurus/Oryctolagus cuniculus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;50 (with traces at lower molecular weigth)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e~\u0026thinsp;20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHM5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eOryctolagus cuniculus/Sus scrofa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;70 (with traces at lower molecular weigth)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e~\u0026thinsp;25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eBone Mixed (BM) glues\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBos taurus/Sus scrofa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e~\u0026thinsp;25; ~ 60; \u0026ge;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e~\u0026thinsp;60%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBM2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBos taurus/Sus scrofa/Equus asinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e~\u0026thinsp;25;~ 60; \u0026ge;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e~\u0026thinsp;70%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBM3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBos taurus/Sus scrofa/Equus asinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e~\u0026thinsp;25;~ 60; \u0026ge;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e~\u0026thinsp;45%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBM4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBos taurus/Sus scrofa/Equus asinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e~\u0026thinsp;25;~ 60; \u0026ge;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e~\u0026thinsp;30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e214\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBM5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBos taurus/Sus scrofa/Ovis aries/Equus asinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e~\u0026thinsp;25;~ 60; \u0026ge;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e~\u0026thinsp;80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*+++ corresponds to deamidation % of asparagine\u0026thinsp;\u0026gt;\u0026thinsp;80% and glutamine\u0026thinsp;\u0026gt;\u0026thinsp;60%; ++ corresponds to deamidation % of asparagine between 50% and 70% and glutamine between 1% and 30%; + corresponds to deamidation % of asparagine and glutamine\u0026thinsp;\u0026lt;\u0026thinsp;10%.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Thermogravimetric analysis\u003c/h2\u003e \u003cp\u003eTG measurements were conducted on collagen, gelatine A and B and on glue samples using TA Instruments Thermobalance model Q5000IR, from 25\u0026deg;C to 900\u0026deg;C, at a heating rate of 10\u0026deg;C/min. The thermogravimetric analysis was carried out under nitrogen flow (25 mL/min). Approximatively 5 mg of sample were weighted and put in platinum crucibles. The instrument was mass calibrated using certified mass standards in the range of 0-100 mg and temperature calibrated using five reference materials (Alumel, Ni, Ni83%Co17%, Ni63%Co37%, Ni37%Co63%). TA Universal Analysis 200 ver. 4.5A was employed for data treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy\u003c/h2\u003e \u003cp\u003eATR-FTIR investigation of the protein secondary structure in the collagen, gelatine A and B and animal glue samples was performed using a Perkin-Elmer Frontiers FTIR spectrophotometer, equipped with a universal Attenuated Total Reflectance (ATR) accessory and a triglycine sulphate TGS detector. All the spectra were recorded in the 4000\u0026thinsp;\u0026minus;\u0026thinsp;600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range in ATR mode after background acquisition. For each sample, 128 scans were recorded, averaged, and Fourier transformed to generate a spectrum with a nominal resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Spectrum software (Perkin-Elmer) and a written-in house LabVIEW program for peak fitting were employed to run and process spectra, respectively[\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. A straight baseline passing through the ordinates at 1800 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1480 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was subtracted before processing the curves, and the spectra were normalized in the 1700\u0026thinsp;\u0026minus;\u0026thinsp;1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region. This approach was taken to avoid artefacts in absorptions near the limits of the region examined. The second derivatives of the amide I band of the spectra examined were then analysed to determine the initial data (number and position of the Gaussian components) required for the deconvolution procedure. The amide I band was chosen for structural analysis because of the very low contribution of the amino acid side chain absorptions present in this region [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], and its higher intensity compared to the other amide modes (amide II at 1540 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and Amide III between 1350\u0026thinsp;\u0026minus;\u0026thinsp;1190 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Based on the infrared assignment of amide components, assuming that the extinction coefficient is the same for all the secondary structures, the secondary structure composition can be obtained from the FTIR spectra. The percentages of the different secondary structures were estimated by expressing the amplitude value of the bands assigned to each of these structures as a fraction of the total sum of the amplitudes of the amide I components. Although the general validity of the above assumption about extinction coefficients remains to be verified, there are studies in the literature that show good correlation between the distribution of secondary structures obtained by FTIR and X-ray crystallography approaches, suggesting that the assumption made is reasonable [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Differential scanning calorimetry\u003c/h2\u003e \u003cp\u003eDSC analyses were performed by TA Instruments Discovery DSC model 250 under nitrogen flow (50 mL/min). The instrument was temperature calibrated with indium. For each sample, about 5 mg was weighted and hermetically sealed into aluminium DSC pans. An empty pas was used as a reference.\u003c/p\u003e \u003cp\u003eCollagen, gelatine A and B, and glue samples were subjected to heating/cooling/heating cycle in the temperature range of 10\u0026ndash;130\u0026deg;C at heating rate of 10\u0026deg;C/min and cooling rate of 50\u0026deg;C/min.\u003c/p\u003e \u003cp\u003ePhysical ageing study was carried out on glue samples consisting of one type of collagen belonging to the same animal species, \u003cem\u003eBos taurus\u003c/em\u003e, i.e. HP3, HP4, HP6, HP8 samples and on one sample derived from bone tissue, namely BM5. The procedure was the following. The samples were heated/cooled/reheated under the same conditions as described above, and after the second heating scan, the samples were cooled to 40\u0026deg;C (temperature below the glass transition temperature) at 50\u0026deg;C/min, held at 40\u0026deg;C (annealing temperature) for up to 3 hours and heated to 130\u0026deg;C at 10\u0026deg;C/min. The samples were then cooled to 40\u0026deg;C at 50\u0026deg;C/min, held at 40\u0026deg;C for up to 6 hours and heated to 130\u0026deg;C at 10\u0026deg;C/min. The sequence was repeated with the sample held at 40\u0026deg;C for up to 12 h. The same procedure was repeated at an annealing temperature of 30\u0026deg;C only on HP6 in order to confirm the presence of physical ageing. This procedure was inspired by Farahnaky et al. [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor each calorimetric method used, a baseline was recorded and subtracted from the calorimetric curve of each sample. TRIOS v5.1.1 was employed for data treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Thermogravimetric analysis\u003c/h2\u003e \u003cp\u003eThe thermal degradation of collagen, gelatine standards and glue samples was investigated by TGA under nitrogen flow. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows thermogravimetric (TG), and differential thermogravimetric (DTG) curves obtained for all samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll the samples exhibit the same thermal degradation profile, which primarily consists of two steps. The first mass loss (10\u0026ndash;12% w/w) below 60\u0026deg;C is due to moisture evaporation and the second (65\u0026ndash;75% w/w) centred\u003c/p\u003e \u003cp\u003eat 300\u0026ndash;320\u0026deg;C is due to protein pyrolysis resulting in the formation of 2,5-diketopiperazines (DKP), the most abundant of which in the glue samples are cyclo(Pro-Gly) and cyclo(Pro-HyP),\u003csup\u003e1\u003c/sup\u003e and of several aromatic and nitrogen-containing compounds, such as pyrrole, indole, phenol, and the respective alkylated compounds [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe residue at 900\u0026deg;C is in the range of 15\u0026ndash;20% w/w, as generally reported in literature for proteins. Table S3 shows the percentage mass loss and the temperature peak determined for each sample.\u003c/p\u003e \u003cp\u003eStandard collagen showed an additional degradation step below 200\u0026deg;C. As reported in the literature, this is due to the evaporation of the strongly H-bonded water involved in the stabilization of the triple helix [\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBy observing the shape of the DTG related to water loss, gelatine standards exhibited a broader signal in over a wide temperature range (from 30\u0026deg;C to 200\u0026deg;C) indicating the presence of structural water. The DTG curves of glue samples suggest that HP3, HP6 and HM5 contain more structural water than the standard gelatine, while the other samples have little to none.\u003c/p\u003e \u003cp\u003eDifferent degree of hydrolysis of collagen and the different animal species can affect the thermal degradation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR72\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBy considering the \u003cem\u003et\u003c/em\u003e\u003csub\u003eendset\u003c/sub\u003e and \u003cem\u003et\u003c/em\u003e\u003csub\u003eonset\u003c/sub\u003e (Table S4), a slight difference was observed for gelatine standards, as gelatine A has \u003cem\u003et\u003c/em\u003e\u003csub\u003eonset\u003c/sub\u003e higher than gelatine B that indicates higher thermal stability, but \u003cem\u003et\u003c/em\u003e\u003csub\u003eendset\u003c/sub\u003e lower than gelatine B, i.e. a lower amount of thermostable cross-linked/aggregated structures. This result agrees with the literature and depends on the different production process used. Gelatine A undergoes an acid pretreatment whereas gelatine B undergoes a basic pretreatment. This leads to gelatine with different properties and, in general, gelatine A is less covalently cross-linked than gelatine B [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], probably due to the protonation of groups involved in cross-linking formation.\u003c/p\u003e \u003cp\u003eA slight shift of the maximum towards lower temperatures was observed for animal glue samples, suggesting the presence of more hydrolysed molecules compared to gelatine and collagen. Furthermore, looking at the \u003cem\u003et\u003c/em\u003e\u003csub\u003eendset\u003c/sub\u003e in Table S4, BM sample completed its degradation at a lower temperature than the other samples. This result further suggest a higher degree of hydrolysis, a lower degree of aggregation and the presence of molecules with a lower molecular weight in BM samples, as reported by Ntasi et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], who identified highly hydrolysed collagen in BM samples through the SDS-PAGE, backbone cleavage percentage and Py-GC/MS analysis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Another factor to consider is the percentage of deamidation. Notably, hide glues exhibit higher levels of deamidation compared to bone glues\u003csup\u003e1\u003c/sup\u003e. This increased deamidation can result in a higher number of charges, potentially leading to the formation of more thermally stable aggregates.\u003c/p\u003e \u003cp\u003eRegarding the HP and HM samples, no clear difference in thermal degradation was observed. Both HP and HM samples have a \u003cem\u003et\u003c/em\u003e\u003csub\u003eonset\u003c/sub\u003e lower than that of collagen and gelatine and a \u003cem\u003et\u003c/em\u003e\u003csub\u003eendset\u003c/sub\u003e lower than collagen but equal to or higher than that of gelatine, indicating the presence of more hydrolysed molecules as well as thermostable aggregates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Secondary structure of gelatine in animal glue samples\u003c/h2\u003e \u003cp\u003eATR-FTIR analysis was carried out on animal glue samples and on collagen, gelatine A and B to investigate the protein secondary structures. Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e shows the FTIR spectra, which display the typical features of protein materials, i.e. the amide I band in the 1700\u0026thinsp;\u0026minus;\u0026thinsp;1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region (corresponding to C\u0026thinsp;=\u0026thinsp;O stretching vibration), the amide II in the 1600\u0026thinsp;\u0026minus;\u0026thinsp;1480 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region (corresponding to out-of-phase combination of the NH in plane bend and the CN stretching vibration) and the amide III in the 1350\u0026thinsp;\u0026minus;\u0026thinsp;1190 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region (corresponding to in-phase combination of the NH bending and the CN stretching vibration) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The amide I frequency is strongly affected by the protein conformation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], consequently the amide I shift observed in the animal glue samples clearly indicates a change in the protein structure.\u003c/p\u003e \u003cp\u003eTo gain a deeper understanding of the gelatine conformation, a curve-fitting method was applied to deconvolve the complex amide I band, allowing for the determination of the different percentages of secondary structures. [\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCharacteristic components of the Amide I band are assigned to the following secondary structures: helix (1650\u0026ndash;1660 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), random (1640\u0026ndash;1660 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), collagen native triple helix (1660\u0026ndash;1666 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), antiparallel β-sheets (two components around 1620 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1690 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), parallel β-sheets (one component around 1630 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), β-turns (1670\u0026ndash;1685 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e show the secondary structure percentages, the wavenumber in cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and half height bandwidth (HHBW), based on the results of the peak fitting of Amide I band.\u003c/p\u003e \u003cp\u003eThe assignment of bands to a precise secondary structure is challenging as different bands often overlap, as in the case of helix and random coil structures. Because of the wide bandwidth of the bands assigned to helices and random structures in the FTIR spectra of these samples (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), in this work was not possible to discriminate between these two structures in all the samples. The assignment to β-sheet structures in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e includes parallel and antiparallel β-sheets and β-turn [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results obtained on the standard of collagen indicate that the collagen is not in its native form, as a significant amount of β-structures are present. The same results can be observed for gelatine standards. The high amount of helix/random structures testify the semicrystalline nature of gelatine, i.e. a crystalline domain consisting of the triple-helix structures and an amorphous domain composed mainly of the random structures and single helix chains, as already reported in the literature [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. When comparing the results of animal glues, the HP and BM1 samples exhibit a secondary structure like that of the collagen and gelatine standards. HM samples appear to be the only ones without random structures and, along with BM samples, exhibit a lower amount of helix/random structures compared to the others.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSecondary structure percentages, wavenumber (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and half height bandwidth (HHBW) of the Amide I component and of the collagen and gelatine standard and glue samples.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRandom and helices*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβstructures\u003c/p\u003e \u003cp\u003e(β-sheets/ β-sheet (p\u0026thinsp;+\u0026thinsp;ap)/ β-turn)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCollagen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1650 (33) (random)\u003c/p\u003e \u003cp\u003e21%\u003c/p\u003e \u003cp\u003e1659 (50) (helix)\u003c/p\u003e \u003cp\u003e23%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1611 (53)\u003c/p\u003e \u003cp\u003e36%\u003c/p\u003e \u003cp\u003e1626 (17)\u003c/p\u003e \u003cp\u003e20%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGelatine A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1652 (60)\u003c/p\u003e \u003cp\u003e51%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1618 (42); 1693 (28)\u003c/p\u003e \u003cp\u003e49%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGelatine B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1651 (28)\u003c/p\u003e \u003cp\u003e42%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1619 (44)\u003c/p\u003e \u003cp\u003e43%\u003c/p\u003e \u003cp\u003e1685 (39)\u003c/p\u003e \u003cp\u003e15%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1659 (51)\u003c/p\u003e \u003cp\u003e41%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1622 (29); 1693 (26)\u003c/p\u003e \u003cp\u003e58%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1654 (59)\u003c/p\u003e \u003cp\u003e48%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1618 (45); 1693 (28)\u003c/p\u003e \u003cp\u003e53%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHP6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1659 (45)\u003c/p\u003e \u003cp\u003e30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1622 (54); 1689 (36)\u003c/p\u003e \u003cp\u003e70%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHP8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1660 (59)\u003c/p\u003e \u003cp\u003e40%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1621 (54); 1687 (21)\u003c/p\u003e \u003cp\u003e61%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1666 (43) (helix)\u003c/p\u003e \u003cp\u003e23%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1627 (60); 1689 (30)\u003c/p\u003e \u003cp\u003e77%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHM4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1668 (49) (helix)\u003c/p\u003e \u003cp\u003e26%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1626 (61); 1689 (24)\u003c/p\u003e \u003cp\u003e74%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHM5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1668 (51) (helix)\u003c/p\u003e \u003cp\u003e30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1624 (61);1691 (22)\u003c/p\u003e \u003cp\u003e70%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1659 (60)\u003c/p\u003e \u003cp\u003e42%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1620 (53)\u003c/p\u003e \u003cp\u003e55%\u003c/p\u003e \u003cp\u003e1688 (22)\u003c/p\u003e \u003cp\u003e3%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBM2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1660 (41)\u003c/p\u003e \u003cp\u003e29%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1625 (52)\u003c/p\u003e \u003cp\u003e59%\u003c/p\u003e \u003cp\u003e1687 (33)\u003c/p\u003e \u003cp\u003e12%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBM3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1657 (16)\u003c/p\u003e \u003cp\u003e7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1629 (75)\u003c/p\u003e \u003cp\u003e83%\u003c/p\u003e \u003cp\u003e1682 (36)\u003c/p\u003e \u003cp\u003e10%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBM4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1656 (33)\u003c/p\u003e \u003cp\u003e21%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1628 (46)\u003c/p\u003e \u003cp\u003e56%\u003c/p\u003e \u003cp\u003e1679 (40)\u003c/p\u003e \u003cp\u003e23%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBM5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1661 (42)\u003c/p\u003e \u003cp\u003e28%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1625 (53);1696 (7)\u003c/p\u003e \u003cp\u003e61%\u003c/p\u003e \u003cp\u003e1687 (32)\u003c/p\u003e \u003cp\u003e11%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e* Where not specified this band includes random and helix structures because the peaks assigned to these two components in collagen are too close (1650\u0026ndash;1660 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1640\u0026ndash;1660 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003csup\u003e65\u003c/sup\u003e and they may not be separated by the peak deconvolution procedure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Differential scanning calorimetry\u003c/h2\u003e \u003cp\u003eCalorimetric analysis was performed on glue samples to obtain information on the thermal stability of the protein structures and to compare them with those of gelatine and collagen standards. The calorimetric curves obtained are reported in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe calorimetric curve of the collagen standard showed in the first heating scan a broad endothermic signal between 36\u0026deg;C and 121\u0026deg;C with a maximum at 82\u0026deg;C accounting for 18 J/g as enthalpy value and ascribable to collagen unfolding [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. In the second heating scan, the only signal observed was the glass transition occurring at almost 0\u0026deg;C. This result is not expected, as the literature reports a glass transition temperature for tendon collagen of around 40\u0026ndash;45\u0026deg;C [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. No information is provided about the processes undergone by standard collagen, but this difference may suggest that standard collagen is not in its native form and has undergone a treatment that partially altered its structure, as also observed in the secondary structure analysis.\u003c/p\u003e \u003cp\u003eThe unfolding enthalpy of collagen reported in literature is in the range 40\u0026ndash;50 J/g [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Our lower value could be due to a partial denaturation of collagen, as indicated by the β-sheets content. Considering the proximity of β- sheets and native collagen helix in the Ramachandran plot [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e], collagen is unlikely to return to its native shape, but rather it could give rise to β- sheet structures, which are notoriously very stable.\u003c/p\u003e \u003cp\u003eThe calorimetric curve of both gelatine A and B showed two distinct endothermic signals during the first heating scan. The first signal (6 J/g and 12 J/g, respectively) at about 70\u0026deg;C is an overshooting overlapped to the glass transition. The second signal (11 J/g and 3 J/g, respectively) at 95\u0026deg;C corresponds to the unfolding of the gelatine structure, which is associated with the degree of renaturation of the gelatin. A higher amount of reformed native-like triple helix structure would result in a higher enthalpy value [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. The results indicate that gelatine A exhibits a higher degree of renaturation compared to gelatine B. This difference may be attributed to variations in their covalent cross-linking levels, as well as the conditions (temperature and humidity) during the renaturation process. The presence of a higher number of covalent bonds in gelatine B may hinder the reorganisation and formation of triple helix-like structures, resulting in a lower degree of renaturation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe calorimetric profiles of all glue samples in the first heating scan were more similar to that of gelatine than collagen, displaying two endothermic signals: the first in the temperature range 50\u0026ndash;80\u0026deg;C, overlapping the glass transition, the second, in the temperature range 80\u0026ndash;110\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the second heating scan all the analysed samples exhibited only the signal related to glass transition (Figure S2 in supporting information).\u003c/p\u003e \u003cp\u003eThe signal superimposed to the glass transition was better investigated in order to confirm it to be associated with structural relaxation associated with physical ageing of the sample as reported in literature for synthetic polymers, some proteins and gelatines [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe study of physical ageing was performed on glue samples of the same animal origin (HP3, HP4, HP6, HP8) to reduce the variability associated with different animal sources and on one bone glue sample (BM5) to confirm the presence of this phenomenon also in the bone glues. To study the physical ageing, after the first heating up to 130\u0026deg;C, the samples were kept at 40\u0026deg;C (temperature below the \u003cem\u003et\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e) for different periods of time (3 h, 6 h, 12 h, 24 h) as specified in the experimental section and heated to 130\u0026deg;C in order to observe the enthalpy relaxation signal. Since the extent of physical ageing depends on the annealing temperature, the experiment was repeated on sample HP6 also at the annealing temperature of 30\u0026deg;C. Specifically, as the annealing temperature increases, both the peak temperature and the relaxation enthalpy value also increase [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. The values of the enthalpy relaxation and the peak temperature calculated at each time point are reported in the table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e reports the DSC curves obtained for HP6 as example.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe characteristics of this relaxation peak are compatible with the physical ageing phenomenon occurring in the glues, as described in the literature [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]: the signal intensity increases over time and the peak temperature increases linearly with the logarithm of the ageing time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The same behaviour was observed for BM5 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding the glass transition, when applied to proteins, this term refers to the change in the dynamic properties of proteins that takes place at a specific temperature. It has been reported in the literature that at temperatures above the glass transition the dynamics of protein molecules are dominated by large scale motions of groups of atoms, whereas at lower temperatures vibrational motions predominate. The temperature at which this change in molecular dynamics occurs is influenced by the degree of hydration of the protein and the intrinsic temperature dependence of the motions [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll the glue samples have the same moisture content (around 10%, as proved by TGA data reported below) and the \u003cem\u003et\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e values, obtained from the first heating scan, range from 52\u0026deg;C (i.e., HP8) to 73\u0026deg;C. (i.e., HM1), with the gelatine A and B values almost in the middle at 62\u0026deg;C and 68\u0026deg;C, respectively, without any clear correlation with the animal source (Figure S2 and Table S2a). In general, \u003cem\u003et\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e is lower in the second heating scan compared to the first one, except for HP4 and HP8, which exhibited nearly the same temperature. As reported in [ref 35] and by Tseretely and Smirnova [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e], this is due to the release of bounded water during the denaturation process, which acts as a plasticiser by lowering the \u003cem\u003et\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe values of the heat capacity change (∆\u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e) associated with the glass transition, are in the range 0.65\u0026ndash;1.3 J/K∙g and 0.53\u0026ndash;1.3 J/K∙g calculated from the first and second heating scan, respectively. These values agree with the data reported in literature on amorphous gelatines and disordered crystalline gelatines [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e] and they are slightly higher than those reported for other proteins [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll the glues (both from hide and from bone) show the relaxation enthalpy peak superimposed to the glass transition with values varying between 5 (i.e., HP4 and HP8) and 11 J/g (i.e., HM4). The value obtained for gelatine is 6 J/g as well as HM5 (Table S2). For the collagen standard, as well as BM3, BM4, and BM5 samples, it was not possible to determine the relaxation enthalpy associated with the glass transition due to the signal overlapping with other signals at higher temperatures. This signal is often overlooked and there are few studies in which the enthalpy relaxation has been determined in gelatinous samples. Enrione et al. [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], determined the enthalpy associated with physical ageing of fish gelatine film and obtained a value of 2.42 J/g after an ageing of 40 h at annealing temperature 5\u0026deg;C lower than \u003cem\u003et\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e, i.e., 29\u0026deg;C. The higher enthalpy relaxation values of the glue samples obtained in this study could be due to the annealing temperature used, which was 20\u0026ndash;30\u0026deg;C lower than \u003cem\u003et\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e. In these conditions, the enthalpy relaxation can be higher because the sample is far away from equilibrium state.\u003c/p\u003e \u003cp\u003eThe unfolding peak in the hide glues is well separated in temperature from the relaxation enthalpy peak with a \u003cem\u003et\u003c/em\u003e\u003csub\u003epeak\u003c/sub\u003e varying between 89\u0026deg;C (i.e., HP8) and 100\u0026deg;C (i.e., HM1) and enthalpy values varying between 3 (i.e., HP3) and 11 J/g (i.e., gelatine and HP6), and almost absent in HP4 and HM4. These values are lower than those obtained for collagen, indicating a lower structural order due to the higher degree of hydrolysis and lower molecular weight of the gelatine molecules that hinder renaturation and thus the formation of structures similar to the native triple helix [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor bone glues is not possible to determine separate values for the relaxation enthalpy and for the unfolding one, because all the DSC peaks are partially superimposed.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion and Conclusion","content":"\u003cp\u003eTGA data clearly indicate that glue samples are more similar to gelatine than to the reference collagen. Therefore they didn\u0026rsquo;t show the degradation step at around 200\u0026deg;C that is reported to be related to the evaporation of the strongly H-bonded water involved in the stabilization of the triple helix [\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. In addition, they present an unfolding enthalpy that is less than half of that of collagen. These results confirmed those obtained by Ntasi et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] reporting that glues are composed by hydrolysed collagen of lower molecular weight and also highlight that glues have a lower content of collagen triple helix. Some differences on structures also seem to exist depending on whether the samples are from bone (B) or tissue (H).\u003c/p\u003e \u003cp\u003eHP and HM samples are less hydrolyzed than BM samples, have an average MW above 40 KDa (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and these properties appear to result in a well-defined denaturation peak although the low value of the denaturation enthalpy indicates a low content of collagen triple helix-like structures. In particular, the HP4 and HM4 samples appear to be those with a lower degree of structural order given their low enthalpy value (about 1 J/g). This is likely due to the conditions used during the drying process after extraction, the storage conditions and the possible presence of covalent cross-linking that hinders renaturation.\u003c/p\u003e \u003cp\u003eBM3, BM4, and BM5 exhibited a calorimetric curve significantly different from the other samples. Specifically, the signal consists of several overlapping signals, likely due to the higher degree of hydrolysis in the gelatine, resulting in molecules with broader molecular weight distribution. This suggests that a well-defined three-dimensional network is unlikely to form, but rather several aggregates with lower structural order. In contrast, BM1 and BM2 displayed a denaturation signal similar to that of HP and HM samples, indicating a higher degree of renaturation compared to the other BM samples.\u003c/p\u003e \u003cp\u003eFTIR results revealed a relative high content of β-structures in all the glue samples, gelatine and collagen. This highlights that is extremely difficult to obtain native collagen after the extraction process from tissue and it explains the low denaturation enthalpy obtained from collagen. In general the structural analysis of gelatine in the literature focuses on evaluating the triple helix content using X-ray diffraction, supported by DSC results [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Structure property studies link the mechanical properties of glues to the triple helix content, and vice versa [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, β-structures can also play a role in the mechanical and adhesive properties of glues and gelatine. In fact, a few studies have reported that in silk protein toughness and tensile strength are positively correlated with β-sheet content [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and in gelatin-electrospun materials stiffness increases with a higher β-sheet content [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, we believe that in gelatine-based adhesives, it is crucial to consider not only the amount of triple-helix-like structures but also the amount of β-sheet structures to explain the structure-dependent mechanical properties.\u003c/p\u003e \u003cp\u003eAnother observation in this study is the presence of physical ageing in all the glue samples analysed, regardless of animal origin or tissue type. As reported in the literature, the enthalpy relaxation value is influenced by storage conditions, including temperature and humidity. Since the glass transition temperature is higher than room temperature, physical aging will inevitably occur, and because it affects mechanical properties, it should be considered when using gelatin-based adhesives.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMuseo Nacional del Prado (Madrid) and restoration workshop of the University Suor Orsola Benincasa (Naples) are acknowledged for providing the animal glues samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElena Pulidori:\u0026nbsp;\u003c/strong\u003eConceptualization, Investigation, Data Curation, Writing - Original Draft.\u0026nbsp;\u003cstrong\u003eCelia Duce:\u0026nbsp;\u003c/strong\u003eConceptualization, Writing - Review \u0026amp; Editing, Supervision, Funding acquisition.\u0026nbsp;\u003cstrong\u003eEmilia Bramanti:\u003c/strong\u003e Conceptualization, Writing - Review \u0026amp; Editing, Supervision, Funding acquisition.\u0026nbsp;\u003cstrong\u003eLeila Birolo:\u0026nbsp;\u003c/strong\u003eResources, Writing - Review \u0026amp; Editing, Funding acquisition.\u0026nbsp;\u003cstrong\u003eBrunella Cipolletta\u003c/strong\u003e: Writing - Review \u0026amp; Editing.\u0026nbsp;\u003cstrong\u003eLaura Dello Ioio\u003c/strong\u003e: Resources, Writing - Review \u0026amp; Editing.\u0026nbsp;\u003cstrong\u003eIlaria Bonaduce:\u0026nbsp;\u003c/strong\u003eWriting - Review \u0026amp; Editing, Supervision, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by MUR project PRIN 2022 PNRR ArtDECOW (project code P2022HWL7L). The project is financed by European Union-Next Generation EU, Mission 2, Component 1 (M2C1) CUP: I53D23005990001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNtasi, G. et al. Proteomic Characterization of Collagen-Based Animal Glues for Restoration. \u003cem\u003eJ. 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Calorim.\u003c/em\u003e \u003cb\u003e93\u003c/b\u003e, 595\u0026ndash;598 (2008).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Gelatine, animal glue, gelatine adhesives, secondary structure, thermal analysis, physical ageing","lastPublishedDoi":"10.21203/rs.3.rs-5670377/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5670377/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnimal glues have been used for centuries, but their popularity decreased in the 20th century with the rise of synthetic adhesives, leading to their current primary use in restoration. Despite this decline, gelatine, derived from denatured and partially hydrolysed collagen, has gained popularity in various applications. This study focuses on gelatinous glue samples derived from animal bone and hide tissues, examining their secondary structure and thermal properties to identify structure-property correlations. Infrared spectroscopy analysis has revealed differences in the secondary structures, with hide glues exhibiting more β-structures than bone glues, indicating a higher degree of aggregation. Thermogravimetric analysis and differential scanning calorimetry also have highlighted differences between hide and bone glues, showing that the latter are more hydrolysed. Furthermore, the calorimetric curves have showed different values of denaturation enthalpy thus indicating a different degree of gelatine renaturation. Additionally, the calorimetric analysis has demonstrated the physical ageing of gelatinous glue samples, a key factor in maintaining adhesive properties for long-term use under specific storage conditions.\u003c/p\u003e \u003cp\u003eIn a context prioritizing the use of waste biomass over fossil fuels, understanding the properties of gelatine in glues is crucial for enhancing their performance and promoting their adoption as sustainable alternatives to non-renewable adhesives.\u003c/p\u003e","manuscriptTitle":"Characterization of the secondary structure, renaturation and physical ageing of gelatine adhesives","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-23 13:18:44","doi":"10.21203/rs.3.rs-5670377/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-10T07:11:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-04T07:52:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29827583058841327505773337182213030004","date":"2025-01-03T18:23:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199715868472460615716864785641255303784","date":"2024-12-25T06:47:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-22T08:08:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"244501187343747503684955085526425281873","date":"2024-12-20T02:47:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-19T19:28:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-19T19:13:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-12-19T18:06:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-19T11:24:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-12-18T14:35:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f3311ca1-8da6-4edb-8c8e-295467eb34d4","owner":[],"postedDate":"December 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41928131,"name":"Physical sciences/Chemistry"},{"id":41928132,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2025-07-07T16:03:49+00:00","versionOfRecord":{"articleIdentity":"rs-5670377","link":"https://doi.org/10.1038/s41598-025-04910-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-02 15:57:50","publishedOnDateReadable":"July 2nd, 2025"},"versionCreatedAt":"2024-12-23 13:18:44","video":"","vorDoi":"10.1038/s41598-025-04910-8","vorDoiUrl":"https://doi.org/10.1038/s41598-025-04910-8","workflowStages":[]},"version":"v1","identity":"rs-5670377","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5670377","identity":"rs-5670377","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}