Enhancement of the hygrothermal ageing properties of gelatine films by ethylene glycol diglycidyl ether

Owing to the instability of gelatine in hygrothermal environments, gelatine-based cultural heritage undergo various deterioration processes, such as cracking, peeling, warping, curling and fracture, posing significant threats to its long-term preservation. Building on previous research, this study investigates the stability of polyol glycidyl ether– gelatine composite films under high-humidity and high-temperature conditions using ethylene glycol diglycidyl ether (EGDE) as a model compound. The hygrothermal ageing properties of EGDE–gelatine composite films are evaluated in terms of macrosize, mesoscopic structure, surface properties and mechanical properties. Results indicate that EGDE enhances the dimensional stability and swelling ratios of the composite films, stabilizes the pore structure and distribution and maintains the hydrophilicity and molecular structural stability under hygrothermal ageing conditions. Furthermore, the incorporation of EGDE leads to superior stress–strain properties of the composite films in such challenging environments. This study provides valuable experimental data for the preparation and conservation applications of gelatine-based cultural heritage materials.


Introduction
Gelatine, a natural polymer derived from animal protein, has been historically utilised as a sizing material when producing cultural heritage items such as ancient calligraphy items and paintings [1,2].In contemporary settings, the emergence of the photographic technology has accelerated the development of photosensitive emulsions.As a primary film-forming substance used in photosensitive emulsions, gelatine is extensively employed in photographic archives, including photographs, films and glass negatives [3][4][5].Previous studies have indicated that unstable dry-wet cycling conditions can cause protein molecule aggregation in the gelatine emulsion film, leading to shrinkage and deformation, ultimately leading to embrittlement, shedding, curling and fracture of the emulsion layer in gelatine-based photographic archives [6,7].Our recent observations indicated that the combined effect of heat and humidity further exacerbates the shrinkage deformation of gelatine films.Therefore, developing a simple method to enhance the stability of gelatine films in high-humidity and high-temperature environments is crucial for preserving of such cultural heritage.
However, toxicity associated with aldehydes raises safety concerns when used in applications involving human contact.Recent in vitro studies have shown that the cytotoxicity levels of epoxy compounds are within acceptable limits [15], leading to their adoption in pharmaceutical and biomaterial applications [16][17][18].Among the multifunctional epoxy crosslinking agents, water-soluble polyol glycidyl ethers offer a promising yet underexplored alternative to crosslinking polymers containing hydroxyl, amine and carboxyl groups [19].These agents are also used in surface modifications to bind hydroxyl groups with proteins and in membrane technologies [20][21][22].For instance, 1,4-butanediol diglycidyl ether has been used to chemically crosslink gelatine with chondroitin and hyaluronic acid, considerably influencing the physical and biochemical properties of the resulting hydrogels based on the degree of chemical crosslinking and position of reaction substitution [23][24][25].Ethylene glycol diglycidyl ether (EGDE) has been incorporated into adhesive systems as a viscosity-reducing agent, plasticiser and crosslinking agent, enhancing the performance of soybean-based adhesives [26].Polyethylene glycol diglycidyl ether has been used to crosslink and modify silk fibroin proteins and alginates, improving their biological properties, hydrophobicity and solubility through chemical functional group modifications [27].Recently, our research group employed glycerol triglycidyl ether (GTE) as crosslinking agent and plasticiser in photographic gelatine films, examining variations in hygroscopicity, swelling and flexibility after the modification [28].The chemical structures of EGDE and GTE molecules have typical ethylene oxide end groups, presenting high reactivity, which can crosslink with the amino groups in gelatine molecules through a ring-opening reaction to form C-O-C bonds [29].Polyols are commonly used as plasticizers to regulate the flexibility and water-holding properties of gelatine films in food and biomedical applications [30,31], such as glycerol, propylene glycol, diethylene glycol, and ethylene glycol, which can be inserted into the gelatine films to form a hydrogen bonding network [32][33][34][35][36][37].Therefore, previous studies have primarily focused on the physical and chemical modifications of gelatine, polysaccharides and other biological materials using polyol glycidyl ether (for example, GTE, EGDE, etc.) as a crosslinking agent or plasticiser.
However, reports on the ageing resistance and longevity of modified films remain scarce.Notably, elucidating the anti-ageing properties of modified gelatine films under extreme conditions is crucial for guiding the stable preservation of gelatine-based cultural heritage.To address this gap, we used EGDE as a representative compound to investigate the stability of polyol glycidyl ethergelatine composite films under severe hygrothermal conditions (RH = 85% and T = 60 °C).The hygrothermal ageing performance of the films was extensively evaluated, considering aspects such as macrosize, mesoscopic structure, surface properties and mechanical properties.The research methodology is depicted in Fig. 1.This study explored the dimensional changes, swelling properties, surface morphologies and pore structures of the EGDEgelatine composite films before and after hygrothermal ageing.The effect of varying the EGDE concentrations on the macroscopic and mesoscopic stabilities of the EGDEgelatine composite films under these conditions was also assessed.In addition, the study analysed changes in surface hydrophobicity, crystallinity, molecular functional groups and stress-strain properties to understand the effects of hygrothermal ageing on the films at different EGDE concentrations.The findings offer valuable experimental data and theoretical insights for the restoration and protection of fragile gelatine-based cultural heritage.

Sample preparation
An appropriate amount of gelatine powder (photographic grade, Aladdin Chemical Reagents Ltd.) was weighed and immersed in distilled water.After it was completely dissolved, it was heated at 60 °C for 50 min with stirring to produce a transparent homogeneous solution with a 5 wt% concentration.Then, an appropriate amount of EGDE (biological grade, Aladdin Chemical Reagent Co., Ltd.) was added to the above transparent solution to make a mixture, and stirring was continued at 60 °C for 90 min to prepare EGDE-gelatine solutions with concentrations of 0 wt% (control), 0.5 wt%, 1 wt%, 1.5 wt% and 2 wt%, respectively.The above mixtures were transferred to petri dishes using the flow-through film-forming method and dried naturally at room temperature to form a film.

Characterisation methods
(1) Hygrothermal ageing conditions: the relative humidity (RH) of hygrothermal ageing was 85%, the temperature (T) was 60 °C, and the ageing time was 144 h.In order to investigate the physicochemical (2) Dissolvability test: all the composite films before and after ageing were dried in a blower oven at 60 °C for 48 h, and these samples were weighted to obtain W 1 .Afterwards, they were immersed into a container with deionized water and fully dissolved for 8 min, and then the films were taken out of the container, wiped with a filter paper to remove the excess water on the surface, and these films were weighed to obtain W 2 .The weight swelling ratio was calculated with Eq. ( 1).
(3) Scanning electron microscopy (SEM) test: to investigate the effect of hygrothermal ageing on the pore structure of the composite films, the dissolved composite films were freeze-dried, and then they were attached on an aluminium sample stage with conductive adhesive.The samples surface was sprayed with gold for 80 s using an ion sputtering device (SCD005, Baltek, Liechtenstein, Germany), and the surface morphologies and the sample structures were observed by scanning electron microscopy (SEM, TM3030, Hitachi, Japan).(4) Low-field nuclear magnetic resonance (NMR) test: to further investigate the effect of hygrothermal ageing on the pore sizes and distributions of these composite films, the prepared gelatine film samples with different shrinkage rates were swollen in distilled water, and then the T2 inversion spectra of gelatine films with different shrinkage rates were tested by a NMR analyzer (MicroMR12-025 V).Low-field NMR is used to infer porosity, pore size distribution, and other information related to pore structure by measuring the intensity and characteristic parameters of the NMR signal.(5) Contact angle test: the contact angle of gelatine film's surface was investigated.The OCA20 static video contact angle meter (Dataphysics, Stuttgart, Germany) was used to detect the contact angle changes of the composite films before and after hygrothermal ageing.(6) X-ray diffraction test: the crystal structures of the composite films were analysed using X-ray diffraction (XRD; Smart Lab, Rigaku Corporation, Japan) using a voltage of 45 kV, current of 200 mA, scan speed of 5°/min, and 2θ scan range of 2-50°.
(1) Weight swelling ratio (%) (7) Fourier transform infrared (FTIR) spectroscopy test: the gelatine films were tested at room temperature and ambient humidity using a FTIR PerkinElmer Spectrum 2 (Vertex 70, Bruker, Karlsruhe, Germany) with total reflection mode.The wavenumber range was between 500 and 4000 cm −1 with a resolution of 4 cm −1 , and the scan number was 32 times.
In order to reduce the influence of carbon dioxide and water vapor on the infrared spectrum, the infrared spectrum of the sample was deducted from the background contribution.(8) Stress-strain test: the mechanical properties of these films were tested with a speed of 5 mm/min at room temperature using a universal material testing machine with PT-1176PC computerized servosystem (Dongguan Baoda Instrument Co., Ltd., China), and then the stress-strain curves before and after hygrothermal ageing were recorded for the EGDE-gelatine composite films.

Macrosize and swelling properties
To study the anti-ageing properties of gelatine films as influenced by EGDE, we monitored changes in the size of the composite films with varying EGDE concentrations before and after ageing from a macro perspective.The photographs (Fig. 2) showed that the control sample (films untreated with EGDE) underwent considerable dimensional shrinkage, showing varied length and width, after hygrothermal ageing.In contrast, the composite films with different EGDE concentrations exhibited negligible dimensional changes after ageing, indicating that they possessed substantial resistance to hygrothermal ageing.The effect of EGDE on the swelling properties of the gelatine films was also investigated by recording changes in the mass of the films with different EGDE concentrations before and after ageing [28].The results (Fig. 3) indicated that the swelling ratio of the control sample sharply decreased following hygrothermal ageing.This reduction in weight swelling ratio may be attributed to hygrothermal ageing affecting the distribution of hydrophilic and hydrophobic functional groups on the gelatine film's surface, resulting in the aggregation of hydrophilic groups and exposure of hydrophobic groups.Figure 3a shows that the weight swelling ratio decreased following EGDE modification, and this decline continued with increasing EGDE concentration, albeit the overall change was minimal.Figure 3b shows that the weight swelling ratio of the control sample reached 672.1% after hygrothermal ageing.With increasing EGDE concentration, the weight swelling ratio gradually decreased to 2% before and after ageing.These results demonstrate that EGDE enhances the hygrothermal stability of the gelatine films.
Although photographic gelatin is a solid layer, gelatine also presents strong hygroscopicity and swelling, and EGDE and water could formulate an emulsion to be sprayed onto the surface of the gelatine film through the swelling effect into the gelatine layer, then play a role in crosslinking and plasticizing, and ultimately realize the restoration and protection of gelatine-based cultural heritage, the application research results will be embodied in the subsequent publications.

Mesoscopic structure
To further explore the effect of EGDE on the porosity of the gelatine films at a micro-mesoscale level, the control film and composite films with different EGDE concentrations were freeze-dried and SEM analysis was performed to assess changes in pore.The results (Fig. 4a-e) showed considerable porosity of the control film.After treatment with varying concentrations of EGDE, a notable reduction in the pore size was observed and the large pores progressively diminished.Comparison of Fig. 4a and a1 indicates that the pore size of the gelatine films was substantially reduced following hygrothermal ageing.The composite films with different concentrations of EGDE almost retained their surface morphologies and pore structures before and after hygrothermal ageing, corroborating the observed changes in macroscopic sizes and weight swelling ratios as influenced by the EGDE treatment; this result was also consistent with the conclusions derived from Fig. 3.
Low-field nuclear magnetic resonance (NMR) is a method with unique sensitivity to pore water, as it is based on the magnetization and relaxation behaviour of the spin magnetic moment of hydrogen atoms forming water molecules [38].As illustrated in Fig. 5, the control film and composite films treated with various EGDE concentrations exhibited three peaks in the T2 inversion spectrum, corresponding to bound water, capillary water and free water within the gelatine films [6,39], which are arranged according to increasing relaxation times in the T2 spectrum.The signal intensity in the spectrum indicates the water content, with free water predominantly located in the macropores [6]. Figure 5a shows that the highest signal intensity in the T2 spectrum of the control film appears near a relaxation time of 1000 ms, indicating that the control film primarily possessed a macroporous structure, wherein free water was distributed.With increasing EGDE concentration, the relaxation time in the T2 spectrum gradually shifted to 100 ms, indicating the disappearance of the large pore structure associated with free water and its transformation into a smaller pore structure representing capillary water.This relaxation time tended to stabilise with further increase in the EGDE Fig. 3 Weight swelling ratio (a) and its variation (b) for the gelatine films before and after hygrothermal ageing as a function of EGDE concentration in the films concentration.Figure 5b shows that the relaxation time of the control film after ageing was centred at ~ 100 ms in the T2 spectrum, indicating that hygrothermal ageing altered the pore structure of the control film.However, the pore structure of the composite films treated with varying EGDE concentrations showed minimal changes before and after ageing.Combining these results with the SEM results, it is speculated that EGDE facilitates the transition of the pore structure of the gelatine films from a large to small scale and the EGDE-gelatine composite films exhibit notable stability during hygrothermal ageing.

Surface properties
To examine changes in the surface hydrophilicity and hydrophobicity of the EGDE-gelatine composite films before and after hygrothermal ageing, static optical contact angle tests were conducted on the control film and composite films with varying EGDE concentrations before and after hygrothermal ageing [40][41][42].The results are depicted in Fig. 6.A lower contact angle suggests increased hydrophilicity.Figure 6a reveals that the control film and composite films with different EGDE concentrations initially exhibited hydrophilic properties.As the EGDE concentration increased, the contact angle progressively decreased, enhancing the hydrophilicity of the film surfaces.This enhancement is likely due to the hydrolysis of the epoxy end-groups of EGDE molecules or their ring-opening reactions with the amino groups of gelatine, leading to the formation of abundant hydroxyl groups, thereby considerably increasing the hydrophilicity of the films.Figure 6b shows that the contact angle of the control film after hygrothermal ageing was as high as 118.4°, indicating increased hydrophobicity.However, the contact angles of the composite films after ageing, while being generally higher than those before ageing, ranged between 60.2° and 68.1°, indicating retained hydrophilicity.This implies that EGDE can enhance the hydrophilicity of gelatine films even after hygrothermal ageing, improving their flexibility.Gelatine films exhibit a typical crystalline structure, with characteristic peaks at 2θ = 7.8° and 22°, representing helical structures in the gelatine molecule [43,44].The sharp peak at 2θ = 8° correlates with the diameter and number of gelatine triple helices, while the broad peak at 2θ = 20° pertains to the amorphous region related to the distance between amino acid residues along the helix.Figure 7a illustrates that the EGDE treatment considerably affected the crystallinity of the gelatine films: with increasing EGDE concentration, the peak intensity at 2θ = 7.8° gradually diminished until the peak disappeared and the broad peak at 2θ = 22° weakened until it stabilised.As shown in Fig. 7b, the crystallisation peak of the control sample vanished after hygrothermal ageing and the amorphous peak notably weakened.Conversely, the composite films with varying EGDE concentrations largely retained their pre-ageing structure.This persistence is likely due to EGDE molecules being integrated in the gelatine macromolecular network, obstructing the formation of a gelatine crystalline structure, thereby enhancing the mobility and flexibility of gelatine molecules and effectuating the plasticisation of the gelatine films.In addition, the composite films maintained the pre-ageing amorphous molecular structure after hygrothermal ageing, showing preserved stable configuration.
To elucidate the interactions between EGDE and gelatine molecules, the results from FTIR spectroscopic analyses of the control sample and composite films are presented in Fig. 8.The infrared spectra of the control sample [28,45] features the stretching vibration peak of the carbonyl group in the amide I band at 1650 cm −1 , N-H in-plane deformation vibration peak in the amide II band at 1550 cm −1 and C-N stretching vibration peak in the amide III band at 1236 cm −1 .Figure 8a shows that for different EGDE concentrations, the composite films preserved the characteristic peak of the gelatine protein amide bond, with a distinct C-O stretching vibration peak emerging at 1084 cm −1 .As the EGDE concentration increased, this peak also showed a slightly increased intensity, possibly due to the epoxy end-groups of the EGDE molecules progressively accumulating on the film surfaces.Figure 8b reveals that the typical characteristic peaks of the control sample diminished after hygrothermal ageing, likely due to the aggregation of gelatine proteins, which diminished the intensity of the peaks of side chain groups.The composite films maintained a very typical gelatine characteristic peak, indicating that EGDE molecules could sustain the structural stability of gelatine molecules under hygrothermal ageing.After hygrothermal ageing, there was a decrease in the intensity of the characteristic peak at 1084 cm −1 in the composite films, indicating partial consumption of the C-O bond in the epoxy group at the end of the EGDE molecule.This could be attributed to the migration of small EGDE molecules from the surface to the interior of gelatine under a hightemperature and high-humidity condition, preserving the structural integrity of gelatine molecules.

Mechanical property
The stress-strain curves [46] of the control film and composite films with different EGDE concentrations before and after hygrothermal ageing are presented in Fig. 9. Figure 9a depicts the curve before ageing; the control sample exhibited a maximum strain of 4.2% and stress of 64.2 MPa.As the EGDE concentration increased, the maximum stress and strain of the composite films decreased.At an EGDE concentration of 0.5%, the maximum strain and stress values were 26.0% and 70.2 MPa, respectively.At 1.0% concentration, the maximum stress remained unchanged and strain decreased to 15.1%.Higher EGDE concentrations of 1.5% and 2.0% led to a considerable reduction in the stress and strain values.This indicates that a low EGDE concentration enhances the synergistic effects of its crosslinking and plasticising functions, improving the film's mechanical properties.However, excessive EGDE may compromise the strength of gelatine films, leading to a considerable reduction in the maximum stress.Figure 9b illustrates the stressstrain curve after ageing.After hygrothermal ageing, the control sample exhibited severe shrinkage and deformation, making it impossible to obtain a stress-strain curve.As the EGDE concentration increased, the maximum stress of the composite films gradually decreased while the strain increased, likely due to EGDE molecules gradually migrating within the gelatine molecular system during ageing, acting as plasticisers.Therefore, the optimal EGDE concentration is recommended to be between 0.5 and 1.0%.

Conclusion
This study focused on the physical and chemical stabilities of EGDE-gelatine composite films under hygrothermal ageing, which is a critical aspect in the preservation of gelatine-based cultural heritage.The comprehensive evaluation undertaken herein addressed aspects such as macroscopic size, mesoscopic structure, surface properties and mechanical properties.Results indicated that EGDE molecules stabilize the macroscopic sizes, swelling ratios, pore structures and distribution of gelatine films; maintain their hydrophilicity and molecular structures; and improve the stress-strain properties of the films under hygrothermal conditions.The synergistic effects of EGDE's crosslinking and plasticising functions considerably improve the hygrothermal ageing

Fig. 1
Fig. 1 Research idea and experimental design of this study

Fig. 8
Fig. 8 FTIR spectra of the EDGE-gelatine composite films before (a) and after (b) hygrothermal ageing as a function of the EGDE concentration of the films

Fig. 9
Fig. 9 Stress-strain curves of the EDGE-gelatine composite films before (a) and after (b) hygrothermal ageing as a function of the EGDE concentration of the films