Functional characterization of biodegradable films obtained from whole Paecilomyces variotii biomass

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Martinez, Andres G. Salvay, Macarena R. Sanchez-Díaz, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3783428/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The indiscriminate use of petroleum-based polymers and plastics for single-use food packaging has led to serious environmental problems due the non-biodegradable characteristics. Thus, much attention has been focused on the research of new biobased and biodegradable materials. Yeast and fungal biomass are low-cost and abundant sources of biopolymers with highly promising properties for the development of biodegradable materials. This study aimed to select a preparation method to develop new biodegradable films using the whole biomass of Paecilomyces variotii subjected to successive physical treatments including ultrasonic homogenization (US) and heat treatment. Sterilization process had an important impact on the final filmogenic dispersion and mechanical properties of the films. Longer US treatments produced a reduction in the particle size and the application of an intermediate UT treatment contributed favorably to the breaking of agglomerates allowing the second US treatment to be more effective, achieving an ordered network with a more uniform distribution. Samples that were not filtrated after the sterilization process presented mechanical properties similar to plasticized materials. On the other hand, the filtration process after sterilization eliminated soluble and hydratable compounds, which produced a reduction in the hydration properties of the films. biobased films ultrasound homogenization sterilization cell rupture casting Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Food packaging is mainly used to protect food from the environment and to provide consumers with information about ingredients and nutritional values. Materials traditionally used for food packaging include glass, metal, paper and cardboard, and plastics. The use of plastics over glass and metals has continued to increase due to their good material properties and low cost [1,2]. For this reason, in recent years, much attention has been focused on research to replace petroleum-based commodity plastics, cost-effectively, with biodegradable materials offering competitive mechanical properties [3]. Biopolymers, such as polysaccharides and proteins, have been considered the most promising materials for this purpose [3-6]. Biodegradable films and coatings made from these materials may be used to cover food surfaces. They can serve as barriers to control the transfer of moisture, oxygen, carbon dioxide, lipids, and flavour components, thus preserving the quality of food and extending its shelf life [7]. Among other remarkable properties, they can be used as carriers of functional agents, as antimicrobials or antioxidant compounds, and to improve appearance and handling [8]. Film characteristics are dependent on the structure and chemical properties of the biopolymers that make up the film. Polysaccharides and proteins interact strongly with water; therefore, films made of these biopolymers are hydrophilic films. These films also include those obtained from the whole cell biomass of microorganisms such as yeast, which contain both polysaccharides and proteins [9-11]. Polysaccharides and proteins are generally used for their ability to establish polymer interactions and create a continuous network responsible for the functional properties of biodegradable films [12,13]. Yeast and fungal biomass are low-cost and abundant sources of biopolymers with highly promising properties for the development of biodegradable materials [14]. Their properties make them a remarkably interesting system for the development of biodegradable films with potential applications in food packaging. Also, they could be obtained as a residue from some industrial processes such as the brewing industry [15,16] or other biotechnological processes [17] where the biomass (cells or mycelia) is discarded after obtaining the final product. Several research works have investigated the use of yeasts and their constituents as film-forming materials [9-11,18], but in contrast, there is not much specific literature on the use of filamentous fungi as film-forming agents. Filamentous fungi biomass is mainly used for human consumption, and fungal enzymes and bioactive compounds production in the medical and food industry [19-21]. New investigations have demonstrated that the use of mycelial structure is promising for new application areas, such as, the fabrication of structures that contain a vegetal fibre filler, glued by fungal mycelium [14,22,23]. Additionally, the development of sustainable products by using fungi biomass was described for the textile, packaging, and automotive industries [24]. Recent studies by our research group have shown that it is possible to obtain films from mycelia of Penicillium nalgiovense, Mucor hiemalis, and Aspergillus niger by casting of filmogenic dispersions [25]. This study showed that the properties of the films, such as colour and mechanical properties, strongly depend on the fungal strain they are made of. Many authors have reported that the formation of films based on isolated and purified β-glucan from brewer's yeast and plant β-glucan from oats is possible [18,26,27]. Both, filamentous fungi, and yeasts are important sources of β-glucans. Kyanko et al. (2013) investigated and characterized the total dietary fibre, β-glucan, protein, and RNA content of 37 non-toxic filamentous fungal strains isolated from various sources [28]. Paecilomyces variotii presented the highest content of β-glucan (23.8 ± 2.4 % w/w) and total dietary fibre (51.7 ± 5.5% w/w) among all filamentous fungi studied. These results suggested the potential use of this fungus as a film-forming agent. The genus Paecilomyces was split from Penicillium by Bainier in 1907 based on their differences in phialide shape and conidial colour. Paecilomyces species are important as soil fungi and insect pathogens. Only two species are commonly isolated from foods: Paecilomyces variotii and Paecilomyces lilacinus [29]. P. variotii has not been reported as a mycotoxin producer and is used in the food industry as a producer of enzymes [29]. Traditionally, the most common approach to developing new biodegradable materials has been to purify biopolymers of interest from their original biomass by physical or chemical modification to improve their ability to form films. In this work, the whole biomass of Paecilomyces variotii is used, thus this novel approach contributes to a more efficient process with less waste. Films derived from biopolymers are usually fragile and brittle, so they require the addition of plasticizers to improve their functional properties and meet industry requirements [3,30]. Therefore, it is important to study the effect of plasticizers in polymer matrices. Glycerol is the most commonly used plasticizer in biopolymer-based films due to its suitable properties of miscibility and low cost [11]. The aim of this study was to select a preparation method to develop a new biodegradable film using the whole biomass of Paecilomyces variotii subjected to successive physical treatments including ultrasonic homogenization and heat treatment. Functional characterization of the material was carried out, examining the thermal, mechanical, hydration, and water vapour barrier properties of the resulting films. 2. Materials and Methods 2.1. Materials Paecilomyces variotii was obtained from the fungal culture collection of the Food Mycology Laboratory of National University of Quilmes. The strain was reactivated by growing it for 5 days at 25 °C in potato dextrose agar medium (PDA, Britania, Argentina). The reagents and salts used in this work were of analytical grade. 2.2. Production of fungal biomass The conidia production was carried out in Malt Extract Agar (MEA) plates with incubation of 5 days at 37°C. The conidia formed were collected and a suspension in sterile distilled water was performed to inoculate 250 ml of Yeast Extract Sucrose (YES) broth at a concentration of 10 5 conidia/ml. The cultures were incubated in a SONTEC OS22 orbital shaker (Sontec Científica, Buenos Aires, Argentina) at 37 ºC, with shaking at 135 rpm, for 5 days. The formed biomass was collected by vacuum filtration using Whatman Nº1 filter paper, to eliminate the growth medium Then, two washes were performed with sterile distilled water, and dry weight was determined in triplicate in an oven at 105 °C for 2 hours. The filtered biomass was stored at -18°C. 2.3. Preparation of fungal dispersions and film development For the preparation of fungal dispersion, two methodologies were performed. In the first one, a 3% dry weight/volume dispersion was prepared using the filtered biomass and coded as “Treatment 1” (T1). Then, the second one was performed using 100 g of filtered biomass in distilled water, these dispersions were coded as “Treatment 2” (T2). T2 was assayed for the sterilization of wet biomass with a non-specific relation of dry biomass to water. For both methodologies, a standard sterilization heat treatment (121 °C, 15 min) was performed. The T1 dispersion was transferred to a plastic vessel for further treatment by ultrasound (US) and high-speed homogenization (UT). For the dispersion T2, after the heat treatment, the biomass was filtered again, and the dry weight was determined for the preparation of a 3% dry weight/volume (%wt) dispersion, then it was transferred to a plastic vessel for further treatment by UT and US [25]. The cell disruption procedure was developed by different conditions, using a VCX-750 ultrasonic processor (Sonics and Materials, Inc., Newtown, CT U.S.A), at a power of 100%. The working temperature was controlled with a thermocouple placed inside the vessel. A limit of 40°C was established, since above this temperature the collapse of the bubbles that occur during cavitation is weaker, reducing the effect of sonication [31]. The high-speed homogenization was carried out using an Ultraturrax IKA T25 device (IKA ® Works, Inc. Wilmington, USA) at 12000 rpm for 1 minute. Then, the different conditions for cell rupture were: US 15 minutes (1US15') US 30 minutes (1US30') US 15 minutes + UT + US 15 minutes (2US15') Then, 25% wt (concerning dry matter of fungal biomass) of glycerol (Gly) was added to the treated dispersions as a plasticizer, based on the dry weight of the biomass present in the dispersion. The dispersions were homogenized by magnetic stirring for 20 minutes at room temperature. A control dispersion without plasticizer was also prepared and subjected to the same magnetic stirring treatment. The films were prepared by placing 20 g of dispersion in 90 mm diameter plastic Petri dishes and finally obtained in an oven at 35 °C for 24 hours. 2.4. Characterization of filmogenic dispersion To evaluate the degree of cell rupture in the dispersion a qualitative evaluation of the degree of fragmentation of the cellular structures due to the US treatments was performed by using a light microscope ECLIPSE E200 (Nikon, Japan). The samples were stained with lactophenol cotton blue and observed at 40X magnification. In addition, the particle size distribution (PSD) was determined by using a Mastersizer 2000E light scattering kit (Malvern Instruments, Worcestershire, UK) in the range of 0.1 to 1000 μm was used. The added volume of the dispersions was 2 ml in 600 ml of water, with stirring at 2000 rpm (Hydro Pump 2000MU Unit, Malvern Instruments, Worcestershire, UK) at room temperature. Experiments were performed in triplicates. Results were reported as Volume and Number frequency (x100). The optical parameters applied were the relative refractive index of the dispersed particle of 1.52 and the adsorption coefficient of 0.1. 2.5. Characterization of developed films 2.5.1. Mechanical Test The uniaxial tensile tests were carried out by a universal mechanical test machine Megatest ® TC-500 series II (Micrometric SRL, Buenos Aires, Argentina) at 24 °C, equipped with a 30 N cell load and experiments were performed at 5 mm min −1 . The tests were carried out on rectangular specimens of 50 mm x 10 mm, the initial grip separation was 25 mm, and 10 replicates were made per formulation and the mean values were reported with their respective standard deviations. The parameters studied were tensile strength (TS, MPa), Young's modulus (YM, MPa), and elongation at break (ɛ%). Before testing, samples were conditioned at 53% relative humidity (rh). Thicknesses of films were measured with a digital micrometer (Insize Co. Ltd., Japan) at 10 random positions on the film. 2.5.2. Thermogravimetric analysis (TGA) The mass loss as a function of temperature was registered by TGA in a Q-500 equipment (TA Instruments, Delaware, USA) in the temperature range of 30 to 550 °C. Between 6 to 10 mg of sample were weighted and analyzed at 10°C/min under a nitrogen atmosphere, with a flow of 40 ml/min (purge gas) and 60 ml/min (reactive gas). Initial degradation temperature (T 0 ) was determined at 15% of mass loss, while temperatures at the maximum degradation rate (T max ) for each degradation stage were determined from peaks of derivative curves. All samples were analyzed in duplicate. 2.5.3 Hydration and water vapour transport 2.5.3.1 Water sorption isotherms The hydration properties of films were analysed through the water sorption isotherms and were determined gravimetrically at 22°C according to the standard procedure previously described [32]. Dried samples of films of a superficial area of 58 cm 2 and thicknesses of 0.10 ± 0.01 mm were placed in 4 L desiccators and equilibrated at different water activities a w ( a w =% r.h./100). For this, saturated solutions of LiCl, MgCl 2 , NaBr, NaCl, and BaCl 2 were used to generate conditions of a w of 0.11, 0.33, 0.57, 0.75, and 0.90, respectively. Dried atmospheres were obtained using silica gel. Samples were periodically weighed using an analytical balance (±10 −4 g) and the evolution to equilibrium at each moisture condition was checked until constant weight. The hydration or water content h , given in units of g of water per g of dry matter (d.m.), was obtained by taking the difference between the mass of the hydrated film and that of the dried film and was evaluated as a function of a w . Experiments were performed in triplicates. Isotherms were fitted using the Guggenheim-Anderson-De Boer (GAB) model [33] through Eq. 1: where N is the monolayer water content (g of water per g of d.m.) related to primary binding sites of water molecules, c is a parameter linked to the sorption heat monolayer that represents the force of the water-binding to primary binding sites, and k is related to sorption heat multilayer that represents the capability of water to bind to the multilayer [34]. 2.5.3.2 Experimental water vapor permeability measurements Water vapour transport was assayed by measuring water vapor permeability following the standard ASTM-E96 (2016) [35]. For this, films were sealed on the top of cups containing a saturated salt solution of BaCl 2 that provides the highest r.h. of 90%. Test cups were placed in desiccators maintained at a constant temperature of 22 °C and containing a saturated solution of NaOH that provides the lowest r.h. of 10%. Therefore, water vapor flux was determined from the weight loss of the cup. A fan was used to maintain uniform conditions inside the desiccators over the films [36]. Weight loss measurements were taken by weighing the test cup using an analytical balance (± 10 −3 g). Weight loss m versus time t was plotted and when the steady-state (straight line) was reached 36 hours further were registered. The experimental water vapor permeability P w exp was calculated according to Eq. 2 [37]: where P w exp is given in units of g s −1 m −1 Pa −1 , A is the effective area of exposed film (2.2×10 -3 m 2 ), (Δ m /Δ t ) is the slope of a linear regression of weight loss versus time, L is the film thickness (0.10 ± 0.01 mm), and Δ p w =( p w1 - p w2 ) is the differential water vapour partial pressure across the film, p w1 and p w2 are the partial pressures (Pa) of water vapour at the film surface inside and outside the cup, respectively. p w1 was corrected by the mean air gap distance (5 ×10 -3 m in the present study) between the saturated solution level in the cup and the film position [38]. Experiments were performed in triplicate. 2.5.4 Statistical Analysis An analysis of variance (ANOVA) was carried out and it was verified if there were significant differences in the variables measured for each sample in mechanical tests of the films, through a Tukey multiple comparison analysis using PSPP 0.8.5 (Free Software Foundation, Boston, USA), with a confidence level of a p <0.05. 3. Results & Discussion 3.1 Characterization of filmogenic dispersion Figure 1 shows the images of dispersions T1 and T2 submitted to different US treatments. Figures 1a and 1d show dispersions T1 and T2 submitted to 1US15´.In both cases it was possible to observe the presence of longer hyphae than the other treatments. Regarding 30-minute ultrasound treatments, those dispersions submitted to 2US15’ (Figures 1c and 1f for T1 and T2, respectively) presented the most severe rupture, since is possible to see shorter hyphae. Therefore, the ultraturrax treatment after the first US treatments led to a greater breakage of the fungal cells. This corresponds to what was reported for Aspergillus niger and Penicillium nalgiovense biomass dispersions (3% wt, on a dry basis) treated with 2US15' [25]. Add Figure 1 Regarding the optical microscopy images, the severity of the US treatments to break cellular structures of Paecylomyces variotii, focusing on the rupture grade for both T1 and T2, could be ordered as follows: 2US15’>1US30’>1US15’. Dispersions and the effect of US treatments were also characterized through the particle size distribution (PSD) of dispersion T1 and T2. Figure 2 shows the PSD of the tested samples. Figures 2a and b are showed %volume and %number of T1 dispersions, respectively. The particle size distributions (% volume) of T1 dispersions 1US15' and 1US30' were similar, with populations centred on 5, 100, and 700 µm. This variety of peaks is probably related to the fact that these dispersions were not filtered after heating treatment with the autoclave, and therefore there was a greater amount of particles of different sizes. Bzducha-Wrobel et al. (2014) studied the effect of sterilization as a method of cell disruption in yeasts and they found that those compounds released during the heating treatment, were part of the cell wall of microorganisms and they were solubilized at high temperatures [39]. Dispersion T1-2US15´ presented a bimodal PSD, with maxima at 0.7 and 5 µm. In this case, the order of the treatments applied to the dispersion played an important role, since the first 15 minutes of US treatment, may produce cells fracture or breakage but also might break possible aggregates of particles produced during autoclaving, which with high-speed homogenization with ultraturrax (UT) they were broken or leave them more exposed, and through the second US treatment of 15 minutes, smaller particles were generated. Figure 2b shows the PSD in % number of the T1 dispersions. Samples 1US15' and 1US30' showed similar populations of less than 10 µm with a maximum at 2.5 µm. In the case of sample 2US15', a different result was obtained compared to samples 1US15' and 1US30' (in agreement with what was seen in the size distributions in % volume), where two populations with maxima at 0.7 µm and 2.8 μm were observed. Dispersions T2 showed different profiles in particle distribution with respect T1. Figure 2c demonstrated that %volume distribution presented a bimodal profile for all US treatments (1US15’, 1US30’ y 2US15’), with two populations well defined, a minor population centred at 0.7 µm and a major one centred at 6 µm. No differences were found between US treatments in smaller populations. However, differences were observed in the amplitude of the larger population, where 1US15´ dispersions presented a span value that doubled approximately the span value of 1US30 and 2US15 (Table 2). This result indicates that the 2US15' dispersion was the one with the most homogeneous particle size distribution (% volume). Sanchez Díaz et al. [25] also reported more homogeneous particle distributions for Aspergillus niger and Penicillium nalgiovense biomass dispersions (3% wt, on a dry basis) treated with 2US15', but in these cases monomodal distributions were achieved by this treatment. The PSD of the T2 dispersions in % number (Figure 2d) also presented two populations, where the largest number of particles had a maximum of around 0.7 µm and the smallest around 3 µm. It is interesting to note that in these distributions no peaks in sizes greater than 10 µm were observed, indicating that the fraction of the total number of particles (% number) in that area is very small. No differences were observed in the distributions for the three treatments in sample T2. Add Figure 2 Table 1 shows the parameters obtained from the PSD in %volume. In both dispersions T1 and T2, the span value and D 4.3 values indicated that in those longer US treatments, there was a reduction in the particle size. The values obtained for T2 1US15’ are comparable with those reported for Aspergillus niger and Penicillium nalgiovense biomass dispersions (3% wt, on a dry basis) treated with 2US15' [25]. Comparing those US treatments with the same time of US (1US30´and 2US15´), the intermediate homogenization at high speed contributed positively to the breaking of agglomerates, generating a more uniform distribution, being the span value lower for these dispersions. These corresponds to what was observed for Mucor hiemalis and Aspergillus niger dispersions treated by 15-minute ultrasound with and without an intermediate UT homogenization [25]. Add Table 1 3.2. Characterization of films based on Paecilomyces variotii biomass 3.2.1. Mechanical Test To evaluate the effect of the US treatments on the obtained films from dispersions T1 and T2 tensile tests were carried out and Table 2 shows the results of plasticized and non-plasticized samples. The films obtained from T2 dispersions without plasticizer showed a typical pattern of a brittle material, since the maximum tensile strength values (TS) and Young's modulus values (YM) of these samples were very high and the elongation at break (ɛ%) values was low [40]. It was observed that there were no significant differences between the ɛ% values for the three rupture treatments studied, but there were differences in the TS values. The highest values were obtained in those films formed with 30 minutes of US, either with or without the intermediate ultraturrax treatment. While for the YM values, significant differences were observed between 1US15' and 2US15'. The T1 films 1US15' and 1US30' without glycerol also showed a typical pattern of a brittle material, although in these cases it is highlighted that the YM values were lower compared to those of the T2 films. In particular, for T1-2US15' sample without the addition of plasticizer, it was interesting to note that the mechanical parameters were very similar to those of the plasticized samples, with low values of YM and TS and the value of ɛ% was increased, in comparison to the films 1US15' and 1US30'. These may be due to the presence of low molecular weight compounds formed during heating treatment that remained in the media since these samples were not filtered after the sterilization process. Add Table 2 The mechanical properties of the fungal films obtained in this work could be compared with those reported by other authors in the literature [25,41,42]. The values of the parameters TS, YM, and ɛ% depend on the biopolymer that conforms the film matrix. Generally, films based on polysaccharides are more rigid compared to those based on proteins, which are more extensible than the former [30,43]. Then, the mechanical properties of films based on mixtures of polysaccharides and proteins depend on the ratio of these biomolecules in the dispersions. Pranoto et al. found that the strength and flexibility of films composed of a mixture of gelatine and gellan gum, could be modified by varying the proportion of each biopolymer, and related the best mechanical properties with an optimal level of interaction between biomolecules [44]. In films formed from fungal biomass composed of polysaccharides and proteins, only glycerol was added as an extra component, so the interaction between biomolecules might be modified through the US treatments, and in this study, the optimal level of interaction was found in the sample T2 2US15´ with 25% Gly. The values of the parameters TS, YM, and ɛ% obtained in the sample T2 2US15´ with 25% Gly were comparable with those reported for Aspergillus niger and Penicillium nalgiovense films [25] obtained from 2US15´ treated dispersions with 25% Gly. Nevertheless, no significant differences were found in TS, YM, and ɛ% among the different cell rupture treatments except for P. nalgiovense films. On the other hand, films obtained from dispersions T1 2US15' without glycerol were less rigid and more extensible than those reported by other authors. This result was interesting since those films presented characteristics of a plasticized material. This phenomenon was probably related to the content of solubilized compounds during the sterilization process already described in the particle size determinations. 3.2.2 Thermogravimetric analysis (TGA) Figure 3 shows the derivative curve of weight versus temperature (DTGA, %/°C) of the films T1 and T2. Thermograms showed that all the studied films presented a degradation profile with several stages as expected for biobased films [11,16,25]. The first stage is from 30 to 100 °C (information not shown in the Figures), corresponding to the loss of moisture and possible low molecular weight compounds present in the formulations [45,46]. The second stage was extended from 100 to 240 ºC, where the loss of the plasticizer and initial decomposition of proteins and other compounds were mostly observed [11]. Regarding this zone, Figures 3b and 3d show the thermograms of the plasticized samples where a pronounced peak appeared in the degradation zone of glycerol [16]. Ramos et al. studied the thermal degradation of films based on whey proteins with glycerol and concluded that in the degradation zone of the plasticizer, other compounds were lost such as structurally bound water, volatilization of compounds associated with glycerol, and incipient protein degradation [47]. However, in the plasticized samples T2 2US15', only a shoulder was observed in this area, corresponding to the degradation of the plasticizer, while in the other two samples 1US15' and 1US30' a notable peak was observed. Figure 3a, which showed the thermogram of sample T1 without plasticizer, presented a shoulder in this second zone of degradation, indicating that small compounds from the fungal matrix were present and were degraded at these temperatures. Finally, the stage that extends between 240 to 550 °C corresponds to the pyrolysis of β-glucan and other carbohydrates and massive protein degradation [11,25,48,49]. Add Figure 3 The parameters determined by TGA were the initial degradation temperature considered when the films registered a weight loss of 15% (T 0 ), the temperature of the maximum degradation rate of the material in each stage (T max ), corresponding to the peak temperature in the DTGA curves. All these parameters are shown in Table 3. Add Table 3 From the table, it could be seen that the sample T2 2US15' 25%G showed the highest thermal resistance compared. This could be explained by the UT treatment carried out after the first US treatment, which breaks agglomerates that allowed the second US treatment to be more effective, achieving a more ordered network more resistant to thermal degradation. This corresponds to the reported for A. niger and M. hiemalis films obtained with an intermediate UT treatment with 15 min of US [25]. Table 3 also shows that the addition of plasticizer produced a decrease in the T 0 , both in the T1 and T2 films. The presence of the plasticizer reduces the interaction between the polymer chains of the film matrix, exposing them to degradation and thus reducing their thermal resistance [16]. All films presented the greatest percentage of weight loss at 260 °C. Without glycerol, T2 samples presented higher thermal stability than T1, maybe due to the presence of small molecules that remained in T1 samples after sterilization that in samples T2 were eliminated with the filtration process. 3.3.3. Hydration and water vapour transport Water sorption isotherms of Paecilomyces variotii biomass biomass-based films are shown in Figure 4. Experimental points were fitted with the GAB model (Eq. 1) and fitted parameters are displayed in Table 4. Table 4 also shows the measured values of P w exp . Add Figure 4 It could be observed from Figure 4a that the shape of isotherms of all unplasticized T1 films showed a slight increase in the hydration water content at low values of a w , and a sharp increase for a w >0.6. This shape of isotherms suggested the existence of a small amount of water directly bound to the polymeric matrix, forming the hydration monolayer. Then, most of the hydration water was forming multilayers and was indirectly bound to the polymeric matrix. In contrast, the isotherms of unplasticized T2 films displayed a larger increase at low values of a w , and a soft increase for a w >0.6, as compared with T1 films. Add Table 4 The convexity or concavity of isotherms in the region at a w <0.6 is linked to the value of parameter c of Eq. 1 [34,50], which is related to the force of the water-binding to the monolayer. As could be observed in Figure 4A and Table 4, the values of parameter c for T2 films are larger compared to T1 films, and the isotherms of T2 films at a w <0.6 becomes more concave. Parameter N related to the number of primary binding sites of hydration did not present drastic differences between unplasticized films T1 and T2. However, parameter k related to the capability of water to be bounded to the multilayer was higher in the T1 films, resulting in higher hydration for a w >0.6 and consequently in greater values of h 90%r.h. (see Table 4). In this way, in unplasticized T1 films, most of the hydration water was forming multilayers and was indirectly bound to the polymeric matrix. Therefore, as compared with unplasticized T2 films, the hydration water in T1 films is more susceptible to being moved by the diffusion mechanism [51]. This is reflected in the higher P w exp values obtained for the unplasticized sample T1, as compared to T2 (see Table 4). This difference could be due to the presence of low molecular weight compounds in the T1 films, which would promote the separation of the polymeric chains in the film matrix, increasing the free volume to be occupied by mobile water [2,33]. On the other hand, it could be observed that the ultrasonic homogenisation treatment affected the hydration and the water vapour permeability of the unplasticized films T1 and T2 in a different way. In particular, regarding values of h 90%rh and P w exp in Table 4, the difference produced by the treatments was more noticeable in the T1 films, where the unplasticized samples T1 1US30’ and T1 2US15’ presented higher values of h 90%rh and P w exp as compared to T1 1US15. This could be due to the increased aggregate breakdown and size reduction of the polymer chains produced by a longer ultrasound application time, which increases hydration and water mobility. Concerning the effect of glycerol, it could be observed in Figure 4 and Table 4 that the addition of the plasticizer in all samples increases the global hydration and P w exp values. It was observed in thermogravimetric studies (section 3.2.2) that glycerol incorporated into the film matrix decreased the attractive forces between polymer chains, favouring thermal degradation. Consequently, the addition of glycerol increases free volume and segmental motions, producing a global increase in hydration water content and allowing greater mobility to water molecules that increase P w exp [2]. Furthermore, in a similar way to what was observed for the unplasticized films, T1 samples with 25% glycerol showed higher values of h 90%rh and P w exp as compared with T2 films with the plasticizer. 4. Conclusion This research contributes to reinforcing the novel idea that it is feasible to obtain materials from whole biomass of filamentous fungi by casting from filmogenic dispersions. This approach provides an innovative alternative source of biopolymers that do not require further purification and is not limited by crop failure or climate conditions. In this work it was feasible to obtain films from all treated dispersions of P. variotti regardless of the rupture condition applied.The three combined conditions of ultrasound (US) and ultraturrax (UT) were effective for cell rupture, verified by optical microscopy of dispersions and PDS. Longer US treatments produced a reduction in the particle size and the application of the intermediate UT treatment contributed favorably to the breaking of agglomerates allowing the second US treatment to be more effective, achieving an ordered network with a more uniform distribution. This arrangement gave a greater thermal resistance to the fungal biomass films. It was possible to determine that the filtration process after sterilization when obtaining the filmogenic dispersions, eliminated soluble and hydratable compounds, which produced a reduction in the hydration properties of the films produced with T2 dispersions. Through the characterization of mechanical tests, it is highlighted that in the T1 2US15' 0%G films, the values obtained were very similar to those of the plasticized samples, with low values of YM and TS. Declarations Authors Contributions All authors contributed to the study's conception and design. Material preparation, data collection and analysis were performed by Ezequiel A. Martinez, Macarena R. Sanchez-Díaz, Andres G. Salvay, Vanesa Ludemann, and Mercedes A. Peltzer. The funds were managed by Vanesa Ludemann. The manuscript was written and approved by all authors. Acknowledgment The authors of this article acknowledge the financial support from National University of Quilmes (UNQ, Argentina) through R&D program (Expediente 1300/19). References Tang XZ, Kumar P, Alavi S, Sandeep KP (2012) Recent advances in biopolymers and biopolymer-based nanocomposites for food packaging materials. Crit Rev Food Sci Nutr.52 (5):426-442. https://doi.org/10.1080/10408398.2010.500508. 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Journal of Thermal Analysis and Calorimetry 93(2):489-494. Ramírez Tapias YA, Di Monte MV, Peltzer MA, Salvay AG (2021) Bacterial cellulose films production by Kombucha symbiotic community cultured on different herbal infusions. Food Chemistry 372:131346. https://doi.org/10.1016/j.foodchem.2021.131346. Delgado JF, Peltzer MA, Salvay AG (2022) Water Vapour Transport in Biopolymeric Materials: Effects of Thickness and Water Vapour Pressure Gradient on Yeast Biomass-Based Films. J Polym Environ 30:2976–2989. https://doi.org/10.1007/s10924-022-02412-6 Tables Table 1. Parameters obtained from % Volume distribution: Span and D 4.3 values Sample SPAN D 4,3 T1 1US15' 33 105 ± 41 1US30' 10 50 ± 18 2US15' 2.6 9 ± 1 T2 1US15' 4.6 14.5 ± 0.5 1US30' 2.7 8.7 ± 0.4 2US15' 2.1 8.7 ± 0.3 Table 2. Tensile parameters from mechanical assay Sample 0% Glycerol 25% Glycerol TS (MPa) YM (MPa) ɛ% (%) TS (MPa) YM (MPa) ɛ% T1 1US15' 11±2 a,c 499±63 a 3±1 a 1,0±0,1 c 9±2 c 13±3 a 1US30' 8±1 a,e 529±91 a 2±1 a 1,0±0,2 c 11±3 c 12±2 a 2US15' 4±1 e 77±9 c 12±2 b 2±1 c 9±1 c 20±3 b T2 1US15' 9±1 a 712±107 a 2±1 a 4±1 a 63±10 a 10±1 a 1US30' 17±3 b 1006±333 a,b 2±1 a 4±1 a 84±13 b 10±2 a 2US15' 14±2 b,c 1312±149 b 2±1 a 6±1 b 99±11 b 13±2 a *Different letters in the same column indicate significant differences (p < 0.05) between treatments by Tukey’s test Table 3. Parameters from thermal analysis Samples 0% Glycerol 25% Glycerol T 0 (°C) T max (°C) T 0 (°C) T max1 (°C) T max2 (°C) T1 1US15´ 157±17 256±3 140±2 169±4 265±2 1US30´ 204±3 255±1 154±10 178±18 264±2 2US15´ 182±1 254±6 145±2 183±3 265±2 T2 1US15´ 228±1 261±1 138±1 145±4 266±1 1US30´ 204±10 258±1 140±5 171±8 265±3 2US15´ 236±1 266±3 180±8 207±8 270±1 Table 4. Values of the GAB parameters fitted for the water sorption isotherms displayed in Figure X and experimental water vapour permeability of films. The reported values of the statistical parameter R 2 indicate a very good acceptance of the fit model. Errors in GAB parameters were estimated from the fit analysis. h 90%r.h. refers to the hydration equilibrium value at 90% r.h. Different letters in the same column corresponding to h 90%r.h. and P w exp indicate statistically significant differences ( p <0.05). Units of N and h 90%r.h. are g H 2 O per g d.m and units of P w exp are 10 -10 g s -1 m -1 Pa -1 . Sample Water sorption isotherms P w exp R 2 N c K h 90%rh 0 % Glycerol T1 1US15’ 0.999 0.058±0.002 3.23±0.57 0.953±0.006 0.39±0.02 a 2.4±0.1 a 1US30’ 0.999 0.077±0.008 1.80±0.54 0.969±0.008 0.56±0.03 b 2.7±0.1 b 2US15’ 0.999 0.080±0.004 1.13±0.12 0.937±0.006 0.44±0.02 c 3.0±0.1 c T2 1US15’ 0.997 0.064±0.008 4.38±0.94 0.852±0.008 0.26±0.02 d 2.3±0.1 a 1US30’ 0.995 0.049±0.009 3.93±0.51 0.877±0.009 0.22±0.02 e 2.1±0.1 d 2US15’ 0.996 0.086±0.011 4.51±0.98 0.796±0.009 0.28±0.02 d 2.0±0.1 d 25 % Glycerol T1 1US15’ 0.999 0.121±0.011 1.38±0.27 0.942±0.007 0,70±0,03 g 5.7±0.1 e 1US30’ 0.999 0.115±0.08 1.74±0.42 0.959±0.006 0,77±0,03 h 6.0±0.1 f 2US15’ 0.999 0.128±0.005 1.55±0.17 0.967±0.004 0,90±0,03 i 6.1±0.1 f T2 1US15’ 0.999 0.088±0.003 1.92±0.19 0.927±0.005 0,48±0,02 j 4.5±0.1 g 1US30’ 0.998 0.094±0.008 2.24±0.93 0.969±0.007 0,69±0,03 g 4.2±0.1 h 2US15’ 0.999 0.108±0.005 1.68±0.18 0.938±0.005 0,62±0,03 k 4.3±0.1 h Additional Declarations No competing interests reported. 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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-3783428","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":262082251,"identity":"7f8950c5-9ad6-45e2-89cf-b27369293cce","order_by":0,"name":"Ezequiel A. Martinez","email":"","orcid":"","institution":"University of Quilmes","correspondingAuthor":false,"prefix":"","firstName":"Ezequiel","middleName":"A.","lastName":"Martinez","suffix":""},{"id":262082252,"identity":"864739da-a298-413d-b9bb-4e05470b757c","order_by":1,"name":"Andres G. 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(a) Unplasticized films. (b) Plasticized films with 25 % glycerol. Experimental data were fitted with the GAB model using Eq. 1. The fitting parameters are shown in Table 4\u003c/p\u003e","description":"","filename":"FIG4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3783428/v1/2bc1ecd45d436a77f7b384c3.jpg"},{"id":49146843,"identity":"f0fefe03-4d57-4451-a1b9-4cf1e7474388","added_by":"auto","created_at":"2024-01-03 20:33:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":803066,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3783428/v1/cd2ae990-3d0e-4d4b-b9b5-779b35d94d87.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional characterization of biodegradable films obtained from whole Paecilomyces variotii biomass","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFood packaging is mainly used to protect food from the environment and to provide consumers with information about ingredients and nutritional values. Materials traditionally used for food packaging include glass, metal, paper and cardboard, and plastics. The use of plastics over glass and metals has continued to increase due to their good material properties and low cost [1,2]. For this reason, in recent years, much attention has been focused on research to replace petroleum-based commodity plastics, cost-effectively, with biodegradable materials offering competitive mechanical properties [3]. Biopolymers, such as polysaccharides and proteins, have been considered the most promising materials for this purpose [3-6]. Biodegradable films and coatings made from these materials may be used to cover food surfaces. They can serve as barriers to control the transfer of moisture, oxygen, carbon dioxide, lipids, and flavour components, thus preserving the quality of food and extending its shelf life [7]. Among other remarkable properties, they can be used as carriers of functional agents, as antimicrobials or antioxidant compounds, and to improve appearance and handling [8].\u003c/p\u003e\n\u003cp\u003eFilm characteristics are dependent on the structure and chemical properties of the biopolymers that make up the film. Polysaccharides and proteins interact strongly with water; therefore, films made of these biopolymers are hydrophilic films. These films also include those obtained from the whole cell biomass of microorganisms such as yeast, which contain both polysaccharides and proteins [9-11]. Polysaccharides and proteins are generally used for their ability to establish polymer interactions and create a continuous network responsible for the functional properties of biodegradable films [12,13].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYeast and fungal biomass are low-cost and abundant sources of biopolymers with highly promising properties for the development of biodegradable materials [14]. Their properties make them a remarkably interesting system for the development of biodegradable films with potential applications in food packaging. Also, they could be obtained as a residue from some industrial processes such as the brewing industry [15,16]\u0026nbsp;or other biotechnological processes [17] where the biomass (cells or mycelia) is discarded after obtaining the final product. Several research works have investigated the use of yeasts and their constituents as film-forming materials [9-11,18], but in contrast, there is not much specific literature on the use of filamentous fungi as film-forming agents.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFilamentous fungi biomass is mainly used for human consumption, and fungal enzymes and bioactive compounds production in the medical and food industry [19-21]. New investigations have demonstrated that the use of mycelial structure is promising for new application areas, such as, the fabrication of structures that contain a vegetal fibre filler, glued by fungal mycelium [14,22,23].\u0026nbsp;Additionally, the development of sustainable products by using fungi biomass was described for the textile, packaging, and automotive industries [24]. Recent studies by our research group have shown that it is possible to obtain films from mycelia of \u003cem\u003ePenicillium nalgiovense, Mucor hiemalis,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAspergillus niger\u003c/em\u003e by casting of filmogenic dispersions [25]. This study showed that the properties of the films, such as colour and mechanical properties, strongly depend on the fungal strain they are made of. Many authors have reported that the formation of films based on isolated and purified \u0026beta;-glucan from brewer\u0026apos;s yeast and plant \u0026beta;-glucan from oats is possible [18,26,27]. Both, filamentous fungi, and yeasts are important sources of \u0026beta;-glucans. Kyanko et al. (2013) investigated and characterized the total dietary fibre, \u0026beta;-glucan, protein, and RNA content of 37 non-toxic filamentous fungal strains isolated from various sources [28]. \u003cem\u003ePaecilomyces variotii\u003c/em\u003e presented the highest content of \u0026beta;-glucan (23.8 \u0026plusmn; 2.4 % w/w) and total dietary fibre (51.7 \u0026plusmn; 5.5% w/w) among all filamentous fungi studied. These results suggested the potential use of this fungus as a film-forming agent.\u003c/p\u003e\n\u003cp\u003eThe genus \u003cem\u003ePaecilomyces\u003c/em\u003e was split from \u003cem\u003ePenicillium\u003c/em\u003e by Bainier in 1907 based on their differences in phialide shape and conidial colour. \u003cem\u003ePaecilomyces\u0026nbsp;\u003c/em\u003especies are important as soil fungi and insect pathogens. Only two species are commonly isolated from foods: \u003cem\u003ePaecilomyces variotii\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePaecilomyces lilacinus\u003c/em\u003e [29]. \u003cem\u003eP. variotii\u0026nbsp;\u003c/em\u003ehas not been reported as a mycotoxin producer and is used in the food industry as a producer of enzymes [29].\u003c/p\u003e\n\u003cp\u003eTraditionally, the most common approach to developing new biodegradable materials has been to purify biopolymers of interest from their original biomass by physical or chemical modification to improve their ability to form films. In this work, the whole biomass of \u003cem\u003ePaecilomyces variotii\u003c/em\u003e is used, thus this novel approach contributes to a more efficient process with less waste.\u003c/p\u003e\n\u003cp\u003eFilms derived from biopolymers are usually fragile and brittle, so they require the addition of plasticizers to improve their functional properties and meet industry requirements [3,30]. Therefore, it is important to study the effect of plasticizers in polymer matrices. Glycerol is the most commonly used plasticizer in biopolymer-based films due to its suitable properties of miscibility and low cost [11].\u003c/p\u003e\n\u003cp\u003eThe aim of this study was to select a preparation method to develop a new biodegradable film using the whole biomass of \u003cem\u003ePaecilomyces variotii\u003c/em\u003e subjected to successive physical treatments including ultrasonic homogenization and heat treatment. Functional characterization of the material was carried out, examining the thermal, mechanical, hydration, and water vapour barrier properties of the resulting films.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePaecilomyces variotii\u003c/em\u003e was obtained from the fungal culture collection of the Food Mycology Laboratory of National University of Quilmes. The strain was reactivated by growing it for 5 days at 25 \u0026deg;C in potato dextrose agar medium (PDA, Britania, Argentina). The reagents and salts used in this work were of analytical grade.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Production of fungal biomass\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe conidia production was carried out in Malt Extract Agar (MEA) plates with incubation of 5 days at 37\u0026deg;C. The conidia formed were collected and a suspension in sterile distilled water was performed to inoculate 250 ml of Yeast Extract Sucrose (YES) broth at a concentration of 10\u003csup\u003e5\u003c/sup\u003e conidia/ml. The cultures were incubated in a SONTEC OS22 orbital shaker (Sontec Cient\u0026iacute;fica, Buenos Aires, Argentina) at 37 \u0026ordm;C, with shaking at 135 rpm, for 5 days. The formed biomass was collected by vacuum filtration using Whatman N\u0026ordm;1 filter paper, to eliminate the growth medium Then, two washes were performed with sterile distilled water, and dry weight was determined in triplicate in an oven at 105 \u0026deg;C for 2 hours. The filtered biomass was stored at -18\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Preparation of fungal dispersions and film development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the preparation of fungal dispersion, two methodologies were performed. In the first one, a 3% dry weight/volume dispersion was prepared using the filtered biomass and coded as \u0026ldquo;Treatment 1\u0026rdquo; (T1). Then, the second one was performed using 100 g of filtered biomass in distilled water, these dispersions were coded as \u0026ldquo;Treatment 2\u0026rdquo; (T2). T2 was assayed for the sterilization of wet biomass with a non-specific relation of dry biomass to water. For both methodologies, a standard sterilization heat treatment (121 \u0026deg;C, 15 min) was performed. The T1 dispersion was transferred to a plastic vessel for further treatment by ultrasound (US) and high-speed homogenization (UT). For the dispersion T2, after the heat treatment, the biomass was filtered again, and the dry weight was determined for the preparation of a 3% dry weight/volume (%wt) dispersion, then it was transferred to a plastic vessel for further treatment by UT and US [25]. The cell disruption procedure was developed by different conditions, using a VCX-750 ultrasonic processor (Sonics and Materials, Inc., Newtown, CT U.S.A), at a power of 100%. The working temperature was controlled with a thermocouple placed inside the vessel. A limit of 40\u0026deg;C was established, since above this temperature the collapse of the bubbles that occur during cavitation is weaker, reducing the effect of sonication [31]. The high-speed homogenization was carried out using an Ultraturrax IKA T25 device (IKA\u003csup\u003e\u0026reg;\u003c/sup\u003e Works, Inc. Wilmington, USA) at 12000 rpm for 1 minute. Then, the different conditions for cell rupture were:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eUS 15 minutes (1US15\u0026apos;)\u003c/li\u003e\n \u003cli\u003eUS 30 minutes (1US30\u0026apos;)\u003c/li\u003e\n \u003cli\u003eUS 15 minutes + UT + US 15 minutes (2US15\u0026apos;)\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThen, 25% wt (concerning dry matter of fungal biomass) of glycerol (Gly) was added to the treated dispersions as a plasticizer, based on the dry weight of the biomass present in the dispersion. The dispersions were homogenized by magnetic stirring for 20 minutes at room temperature. A control dispersion without plasticizer was also prepared and subjected to the same magnetic stirring treatment.\u003c/p\u003e\n\u003cp\u003eThe films were prepared by placing 20 g of dispersion in 90 mm diameter plastic Petri dishes and finally obtained in an oven at 35 \u0026deg;C for 24 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Characterization of filmogenic dispersion\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the degree of cell rupture in the dispersion a qualitative evaluation of the degree of fragmentation of the cellular structures due to the US treatments was performed by using a light microscope ECLIPSE E200 (Nikon, Japan). The samples were stained with lactophenol cotton blue and observed at 40X magnification. In addition, the particle size distribution (PSD) was determined by using a Mastersizer 2000E light scattering kit (Malvern Instruments, Worcestershire, UK) in the range of 0.1 to 1000 \u0026mu;m was used. The added volume of the dispersions was 2 ml in 600 ml of water, with stirring at 2000 rpm (Hydro Pump 2000MU Unit, Malvern Instruments, Worcestershire, UK) at room temperature. Experiments were performed in triplicates. Results were reported as Volume and Number frequency (x100). The optical parameters applied were the relative refractive index of the dispersed particle of 1.52 and the adsorption coefficient of 0.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Characterization of developed films\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.1. Mechanical Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe uniaxial tensile tests were carried out by a universal mechanical test machine Megatest\u003csup\u003e\u0026reg;\u003c/sup\u003e TC-500 series II (Micrometric SRL, Buenos Aires, Argentina) at 24 \u0026deg;C, equipped with a 30 N cell load and experiments were performed at 5 mm min\u003csup\u003e\u0026minus;1\u003c/sup\u003e. The tests were carried out on rectangular specimens of 50 mm x 10 mm, the initial grip separation was 25 mm, and 10 replicates were made per formulation and the mean values were reported with their respective standard deviations. The parameters studied were tensile strength (TS, MPa), Young\u0026apos;s modulus (YM, MPa), and elongation at break (ɛ%). Before testing, samples were conditioned at 53% relative humidity (rh). Thicknesses of films were measured with a digital micrometer (Insize Co. Ltd., Japan) at 10 random positions on the film.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.2. Thermogravimetric analysis (TGA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mass loss as a function of temperature was registered by TGA in a Q-500 equipment (TA Instruments, Delaware, USA) in the temperature range of 30 to 550 \u0026deg;C. Between 6 to 10 mg of sample were weighted and analyzed at 10\u0026deg;C/min under a nitrogen atmosphere, with a flow of 40 ml/min (purge gas) and 60 ml/min (reactive gas). Initial degradation temperature (T\u003csub\u003e0\u003c/sub\u003e) was determined at 15% of mass loss, while temperatures at the maximum degradation rate (T\u003csub\u003emax\u003c/sub\u003e) for each degradation stage were determined from peaks of derivative curves. All samples were analyzed in duplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHydration and water vapour transport\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.3.1 Water sorption isotherms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe hydration properties of films were analysed through the water sorption isotherms and were determined gravimetrically at 22\u0026deg;C according to the standard procedure previously described [32]. Dried samples of films of a superficial area of 58 cm\u003csup\u003e2\u003c/sup\u003e and thicknesses of 0.10 \u0026plusmn; 0.01 mm were placed in 4 L desiccators and equilibrated at different water activities \u003cem\u003ea\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e (\u003cem\u003ea\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e=% r.h./100). For this, saturated solutions of LiCl, MgCl\u003csub\u003e2\u003c/sub\u003e, NaBr, NaCl, and BaCl\u003csub\u003e2\u003c/sub\u003e were used to generate conditions of \u003cem\u003ea\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e of 0.11, 0.33, 0.57, 0.75, and 0.90, respectively. Dried atmospheres were obtained using silica gel. Samples were periodically weighed using an analytical balance (\u0026plusmn;10\u003csup\u003e\u0026minus;4\u003c/sup\u003e g) and the evolution to equilibrium at each moisture condition was checked until constant weight. The hydration or water content \u003cem\u003eh\u003c/em\u003e, given in units of g of water per g of dry matter (d.m.), was obtained by taking the difference between the mass of the hydrated film and that of the dried film and was evaluated as a function of \u003cem\u003ea\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e. Experiments were performed in triplicates. Isotherms were fitted using the Guggenheim-Anderson-De Boer (GAB) model [33] through Eq. 1:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" style=\"width: 671px; height: 73.5342px;\" width=\"671\" height=\"73.5342\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere \u003cem\u003eN\u003c/em\u003e is the monolayer water content (g of water per g of d.m.) related to primary binding sites of water molecules,\u003cem\u003e\u0026nbsp;c\u003c/em\u003e is a parameter linked to the sorption heat monolayer that represents the force of the water-binding to primary binding sites, and \u003cem\u003ek\u003c/em\u003e is related to sorption heat multilayer that represents the capability of water to bind to the multilayer [34].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.3.2 Experimental water vapor permeability measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWater vapour transport was assayed by measuring water vapor permeability following the standard\u0026nbsp;ASTM-E96 (2016) [35]. For this,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efilms were sealed on the top of cups containing a saturated salt solution of BaCl\u003csub\u003e2\u003c/sub\u003e that provides\u003csub\u003e\u0026nbsp;\u003c/sub\u003ethe highest r.h. of 90%. Test cups were placed in desiccators maintained at a constant temperature of 22 \u0026deg;C and containing a saturated solution of NaOH that provides the lowest r.h. of 10%. Therefore, water vapor flux was determined from the weight loss of the cup. A fan was used to maintain uniform conditions inside the desiccators over the films [36]. Weight loss measurements were taken by weighing the test cup using an analytical balance (\u0026plusmn; 10\u003csup\u003e\u0026minus;3\u003c/sup\u003e g). Weight loss \u003cem\u003em\u003c/em\u003e versus time \u003cem\u003et\u003c/em\u003e was plotted and when the steady-state (straight line) was reached 36 hours further were registered. The experimental water vapor permeability \u003cem\u003eP\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eexp\u003c/sup\u003e was calculated according to Eq. 2 [37]:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" style=\"width: 783px; height: 76.2371px;\" width=\"783\" height=\"76.2371\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere \u003cem\u003eP\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eexp\u003c/sup\u003e is given in units of g s\u003csup\u003e\u0026minus;1\u003c/sup\u003em\u003csup\u003e\u0026minus;1\u003c/sup\u003ePa\u003csup\u003e\u0026minus;1\u003c/sup\u003e, \u003cem\u003eA\u003c/em\u003e is the effective area of exposed film (2.2\u0026times;10\u003csup\u003e-3\u003c/sup\u003e m\u003csup\u003e2\u003c/sup\u003e), (\u0026Delta;\u003cem\u003em\u003c/em\u003e/\u0026Delta;\u003cem\u003et\u003c/em\u003e) is the slope of a linear regression of weight loss versus time, \u003cem\u003eL\u003c/em\u003e is the film thickness (0.10 \u0026plusmn; 0.01 mm), and \u0026Delta;\u003cem\u003ep\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e=(\u003cem\u003ep\u003c/em\u003e\u003csub\u003ew1\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003e\u003csub\u003ew2\u003c/sub\u003e) is the differential water vapour partial pressure across the film, \u003cem\u003ep\u003c/em\u003e\u003csub\u003ew1\u003c/sub\u003e and \u003cem\u003ep\u003c/em\u003e\u003csub\u003ew2\u0026nbsp;\u003c/sub\u003eare the partial pressures (Pa) of water vapour at the film surface inside and outside the cup, respectively. \u003cem\u003ep\u003c/em\u003e\u003csub\u003ew1\u003c/sub\u003e was corrected by the mean air gap distance (5 \u0026times;10\u003csup\u003e-3\u0026nbsp;\u003c/sup\u003em in the present study) between the saturated solution level in the cup and the film position [38]. Experiments were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.4 Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn analysis of variance (ANOVA) was carried out and it was verified if there were significant differences in the variables measured for each sample in mechanical tests of the films, through a Tukey multiple comparison analysis using PSPP 0.8.5 (Free Software Foundation, Boston, USA), with a confidence level of a p \u0026lt;0.05.\u003c/p\u003e"},{"header":"3.\tResults \u0026 Discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1 Characterization of filmogenic dispersion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 1 shows the images of dispersions T1 and T2 submitted to different US treatments. Figures 1a and 1d show dispersions T1 and T2 submitted to 1US15\u0026acute;.In both cases it was possible to observe the presence of longer hyphae than the other treatments. Regarding 30-minute ultrasound treatments, those dispersions submitted to 2US15\u0026rsquo; (Figures 1c and 1f for T1 and T2, respectively) presented the most severe rupture, since is possible to see shorter hyphae. Therefore, the ultraturrax treatment after the first US treatments led to a greater breakage of the fungal cells. This corresponds to what was reported for \u003cem\u003eAspergillus niger\u003c/em\u003e and \u003cem\u003ePenicillium nalgiovense\u003c/em\u003e biomass dispersions (3% wt, on a dry basis) treated with 2US15\u0026apos; [25].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdd Figure 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRegarding the optical microscopy images, the severity of the US treatments to break cellular structures of \u003cem\u003ePaecylomyces variotii,\u003c/em\u003e focusing on the rupture grade for both T1 and T2, could be ordered as follows: 2US15\u0026rsquo;\u0026gt;1US30\u0026rsquo;\u0026gt;1US15\u0026rsquo;.\u003c/p\u003e\n\u003cp\u003eDispersions and the effect of US treatments were also characterized through the particle size distribution (PSD) of dispersion T1 and T2. Figure 2 shows the PSD of the tested samples. \u0026nbsp;Figures 2a and b are showed %volume and %number of T1 dispersions, respectively. The particle size distributions (% volume) of T1 dispersions 1US15\u0026apos; and 1US30\u0026apos; were similar, with populations centred on 5, 100, and 700 \u0026micro;m. This variety of peaks is probably related to the fact that these dispersions were not filtered after heating treatment with the autoclave, and therefore there was a greater amount of particles of different sizes. Bzducha-Wrobel et al. (2014) studied the effect of sterilization as a method of cell disruption in yeasts and they found that those compounds released during the heating treatment, were part of the cell wall of microorganisms and they were solubilized at high temperatures [39]. Dispersion T1-2US15\u0026acute; presented a bimodal PSD, with maxima at 0.7 and 5 \u0026micro;m. In this case, the order of the treatments applied to the dispersion played an important role, since the first 15 minutes of US treatment, may produce cells fracture or breakage but also might break possible aggregates of particles produced during autoclaving, which with high-speed homogenization with ultraturrax (UT) they were broken or leave them more exposed, and through the second US treatment of 15 minutes, smaller particles were generated. Figure 2b shows the PSD in % number of the T1 dispersions. Samples 1US15\u0026apos; and 1US30\u0026apos; showed similar populations of less than 10 \u0026micro;m with a maximum at 2.5 \u0026micro;m. In the case of sample 2US15\u0026apos;, a different result was obtained compared to samples 1US15\u0026apos; and 1US30\u0026apos; (in agreement with what was seen in the size distributions in % volume), where two populations with maxima at 0.7 \u0026micro;m and 2.8 \u0026mu;m were observed.\u003c/p\u003e\n\u003cp\u003eDispersions T2 showed different profiles in particle distribution with respect T1. Figure 2c demonstrated that %volume distribution presented a bimodal profile for all US treatments (1US15\u0026rsquo;, 1US30\u0026rsquo; y 2US15\u0026rsquo;), with two populations well defined, a minor population centred at 0.7 \u0026micro;m and a major one centred at 6 \u0026micro;m. No differences were found between US treatments in smaller populations. However, differences were observed in the amplitude of the larger population, where 1US15\u0026acute; dispersions presented a span value that doubled approximately the span value of 1US30 and 2US15 (Table 2). This result indicates that the 2US15\u0026apos; dispersion was the one with the most homogeneous particle size distribution (% volume). Sanchez D\u0026iacute;az et al. [25] also reported more homogeneous particle distributions for \u003cem\u003eAspergillus niger\u003c/em\u003e and \u003cem\u003ePenicillium nalgiovense\u003c/em\u003e biomass dispersions (3% wt, on a dry basis) treated with 2US15\u0026apos;, but in these cases monomodal distributions were achieved by this treatment. The PSD of the T2 dispersions in % number (Figure 2d) also presented two populations, where the largest number of particles had a maximum of around 0.7 \u0026micro;m and the smallest around 3 \u0026micro;m. It is interesting to note that in these distributions no peaks in sizes greater than 10 \u0026micro;m were observed, indicating that the fraction of the total number of particles (% number) in that area is very small. No differences were observed in the distributions for the three treatments in sample T2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdd Figure 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 1 shows the parameters obtained from the PSD in %volume. In both dispersions T1 and T2, the \u003cem\u003espan\u003c/em\u003e value and D\u003csub\u003e4.3\u0026nbsp;\u003c/sub\u003evalues indicated that in those longer US treatments, there was a reduction in the particle size. The values obtained for T2 1US15\u0026rsquo; are comparable with those reported for \u003cem\u003eAspergillus niger\u003c/em\u003e and \u003cem\u003ePenicillium nalgiovense\u003c/em\u003e biomass dispersions (3% wt, on a dry basis) treated with 2US15\u0026apos; [25]. Comparing those US treatments with the same time of US (1US30\u0026acute;and 2US15\u0026acute;), the intermediate homogenization at high speed contributed positively to the breaking of agglomerates, generating a more uniform distribution, being the \u003cem\u003espan\u003c/em\u003e value lower for these dispersions. These corresponds to what was observed for \u003cem\u003eMucor hiemalis\u003c/em\u003e and \u003cem\u003eAspergillus niger\u003c/em\u003e dispersions treated by 15-minute ultrasound with and without an intermediate UT homogenization [25].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdd Table 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Characterization of films based on \u003cem\u003ePaecilomyces variotii\u003c/em\u003e biomass\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.1. Mechanical Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the effect of the US treatments on the obtained films from dispersions T1 and T2 tensile tests were carried out and Table 2 shows the results of plasticized and non-plasticized samples. The films obtained from T2 dispersions without plasticizer showed a typical pattern of a brittle material, since the maximum tensile strength values (TS) and Young\u0026apos;s modulus values (YM) of these samples were very high and the elongation at break (ɛ%) values was low [40]. It was observed that there were no significant differences between the ɛ% values for the three rupture treatments studied, but there were differences in the TS values. The highest values were obtained in those films formed with 30 minutes of US, either with or without the intermediate ultraturrax treatment. While for the YM values, significant differences were observed between 1US15\u0026apos; and 2US15\u0026apos;. The T1 films 1US15\u0026apos; and 1US30\u0026apos; without glycerol also showed a typical pattern of a brittle material, although in these cases it is highlighted that the YM values were lower compared to those of the T2 films. In particular, for T1-2US15\u0026apos; sample without the addition of plasticizer, it was interesting to note that the mechanical parameters were very similar to those of the plasticized samples, with low values of YM and TS and the value of ɛ% was increased, in comparison to the films 1US15\u0026apos; and 1US30\u0026apos;. These may be due to the presence of low molecular weight compounds formed during heating treatment that remained in the media since these samples were not filtered after the sterilization process.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdd Table 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mechanical properties of the fungal films obtained in this work could be compared with those reported by other authors in the literature [25,41,42]. The values of the parameters TS, YM, and ɛ% depend on the biopolymer that conforms the film matrix. Generally, films based on polysaccharides are more rigid compared to those based on proteins, which are more extensible than the former [30,43]. Then, the mechanical properties of films based on mixtures of polysaccharides and proteins depend on the ratio of these biomolecules in the dispersions. Pranoto \u003cem\u003eet al.\u003c/em\u003e found that the strength and flexibility of films composed of a mixture of gelatine and gellan gum, could be modified by varying the proportion of each biopolymer, and related the best mechanical properties with an optimal level of interaction between biomolecules [44]. In films formed from fungal biomass composed of polysaccharides and proteins, only glycerol was added as an extra component, so the interaction between biomolecules might be modified through the US treatments, and in this study, the optimal level of interaction was found in the sample T2 2US15\u0026acute; with 25% Gly. The values of the parameters TS, YM, and ɛ% obtained in the sample T2 2US15\u0026acute; with 25% Gly were comparable with those reported for \u003cem\u003eAspergillus niger\u003c/em\u003e and \u003cem\u003ePenicillium nalgiovense\u0026nbsp;\u003c/em\u003efilms [25] obtained from 2US15\u0026acute; treated dispersions with 25% Gly. Nevertheless, no significant differences were found in TS, YM, and ɛ% among the different cell rupture treatments except for \u003cem\u003eP. nalgiovense\u003c/em\u003e films. On the other hand, films obtained from dispersions T1 2US15\u0026apos; without glycerol were less rigid and more extensible than those reported by other authors. This result was interesting since those films presented characteristics of a plasticized material. This phenomenon was probably related to the content of solubilized compounds during the sterilization process already described in the particle size determinations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.2 Thermogravimetric analysis (TGA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 3 shows the derivative curve of weight versus temperature (DTGA, %/\u0026deg;C) of the films T1 and T2. Thermograms showed that all the studied films presented a degradation profile with several stages as expected for biobased films [11,16,25]. The first stage is from 30 to 100 \u0026deg;C (information not shown in the Figures), corresponding to the loss of moisture and possible low molecular weight compounds present in the formulations [45,46]. The second stage was extended from 100 to 240 \u0026ordm;C, where the loss of the plasticizer and initial decomposition of proteins and other compounds were mostly observed [11]. Regarding this zone, Figures 3b and 3d show the thermograms of the plasticized samples where a pronounced peak appeared in the degradation zone of glycerol [16]. Ramos et al. studied the thermal degradation of films based on whey proteins with glycerol and concluded that in the degradation zone of the plasticizer, other compounds were lost such as structurally bound water, volatilization of compounds associated with glycerol, and incipient protein degradation [47]. However, in the plasticized samples T2 2US15\u0026apos;, only a shoulder was observed in this area, corresponding to the degradation of the plasticizer, while in the other two samples 1US15\u0026apos; and 1US30\u0026apos; a notable peak was observed. Figure 3a, which showed the thermogram of sample T1 without plasticizer, presented a shoulder in this second zone of degradation, indicating that small compounds from the fungal matrix were present and were degraded at these temperatures. Finally, the stage that extends between 240 to 550 \u0026deg;C corresponds to the pyrolysis of \u0026beta;-glucan and other carbohydrates and massive protein degradation [11,25,48,49].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdd Figure 3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe parameters determined by TGA were the initial degradation temperature considered when the films registered a weight loss of 15% (T\u003csub\u003e0\u003c/sub\u003e), the temperature of the maximum degradation rate of the material in each stage (T\u003csub\u003emax\u003c/sub\u003e), corresponding to the peak temperature in the DTGA curves. All these parameters are shown in Table 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdd Table 3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom the table, it could be seen that the sample T2 2US15\u0026apos; 25%G showed the highest thermal resistance compared. This could be explained by the UT treatment carried out after the first US treatment, which breaks agglomerates that allowed the second US treatment to be more effective, achieving a more ordered network more resistant to thermal degradation. This corresponds to the reported for \u003cem\u003eA. niger\u003c/em\u003e and \u003cem\u003eM. hiemalis\u003c/em\u003e films obtained with an intermediate UT treatment with 15 min of US [25]. Table 3 also shows that the addition of plasticizer produced a decrease in the T\u003csub\u003e0\u003c/sub\u003e, both in the T1 and T2 films. The presence of the plasticizer reduces the interaction between the polymer chains of the film matrix, exposing them to degradation and thus reducing their thermal resistance [16]. All films presented the greatest percentage of weight loss at 260 \u0026deg;C. Without glycerol, T2 samples presented higher thermal stability than T1, maybe due to the presence of small molecules that remained in T1 samples after sterilization that in samples T2 were eliminated with the filtration process.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.3. Hydration and water vapour transport\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWater sorption isotherms of \u003cem\u003ePaecilomyces variotii\u0026nbsp;\u003c/em\u003ebiomass biomass-based films are shown in Figure 4. Experimental points were fitted with the GAB model (Eq. 1) and fitted parameters are displayed in Table 4. Table 4 also shows the measured values of \u003cem\u003eP\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eexp\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdd Figure 4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt could be observed from Figure 4a that the shape of isotherms of all unplasticized T1 films showed a slight increase in the hydration water content at low values of \u003cem\u003ea\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e, and a sharp increase for \u003cem\u003ea\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u0026gt;0.6. This shape of isotherms suggested the existence of a small amount of water directly bound to the polymeric matrix, forming the hydration monolayer. Then, most of the hydration water was forming multilayers and was indirectly bound to the polymeric matrix. In contrast, the isotherms of unplasticized T2 films displayed a larger increase at low values of \u003cem\u003ea\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e, and a soft increase for \u003cem\u003ea\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u0026gt;0.6, as compared with T1 films.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdd Table 4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe convexity or concavity of isotherms in the region at \u003cem\u003ea\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u0026lt;0.6 is linked to the value of parameter \u003cem\u003ec\u003c/em\u003e of Eq. 1 [34,50], which is related to the force of the water-binding to the monolayer. As could be observed in Figure 4A and Table 4, the values of parameter \u003cem\u003ec\u003c/em\u003e for T2 films are larger compared to T1 films, and the isotherms of T2 films at \u003cem\u003ea\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u0026lt;0.6 becomes more concave. \u0026nbsp;Parameter \u003cem\u003eN\u003c/em\u003e related to the number of primary binding sites of hydration did not present drastic differences between unplasticized films T1 and T2. However, parameter \u003cem\u003ek\u003c/em\u003e related to the capability of water to be bounded to the multilayer was higher in the T1 films, resulting in higher hydration for \u003cem\u003ea\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u0026gt;0.6 and consequently in greater values of \u003cem\u003eh\u003c/em\u003e\u003csub\u003e90%r.h.\u003c/sub\u003e (see Table 4). In this way, in unplasticized T1 films, most of the hydration water was forming multilayers and was indirectly bound to the polymeric matrix. Therefore, as compared with unplasticized T2 films, the hydration water in T1 films is more susceptible to being moved by the diffusion mechanism [51]. This is reflected in the higher \u003cem\u003eP\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eexp\u003c/sup\u003e values obtained for the unplasticized sample T1, as compared to T2 (see Table 4). This difference could be due to the presence of low molecular weight compounds in the T1 films, which would promote the separation of the polymeric chains in the film matrix, increasing the free volume to be occupied by mobile water [2,33].\u003c/p\u003e\n\u003cp\u003eOn the other hand, it could be observed that the ultrasonic homogenisation treatment affected the hydration and the water vapour permeability of the unplasticized films T1 and T2 in a different way. In particular, regarding values of\u0026nbsp;\u003cem\u003eh\u003c/em\u003e\u003csub\u003e90%rh\u003c/sub\u003e and\u0026nbsp;\u003cem\u003eP\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eexp\u003c/sup\u003e in Table 4, the difference produced by the treatments was more noticeable in the T1 films, where the unplasticized samples T1 1US30\u0026rsquo; and T1 2US15\u0026rsquo; presented higher values of\u0026nbsp;\u003cem\u003eh\u003c/em\u003e\u003csub\u003e90%rh\u003c/sub\u003e and\u0026nbsp;\u003cem\u003eP\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eexp\u003c/sup\u003e as compared to T1 1US15. This could be due to the increased aggregate breakdown and size reduction of the polymer chains produced by a longer ultrasound application time, which increases hydration and water mobility.\u003c/p\u003e\n\u003cp\u003eConcerning the effect of glycerol, it could be observed in Figure 4 and Table 4 that the addition of the plasticizer in all samples increases the global hydration and\u0026nbsp;\u003cem\u003eP\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eexp\u003c/sup\u003e values.\u0026nbsp;It was observed in thermogravimetric studies (section 3.2.2) that glycerol incorporated into the film matrix decreased the attractive forces between polymer chains, favouring thermal degradation. \u0026nbsp;Consequently, the addition of glycerol increases free volume and segmental motions, producing a global increase in hydration water content and allowing greater mobility to water molecules that increase \u003cem\u003eP\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eexp\u003c/sup\u003e [2]. \u0026nbsp;Furthermore, in a similar way to what was observed for the unplasticized films, T1 samples with 25% glycerol showed higher values of \u003cem\u003eh\u003c/em\u003e\u003csub\u003e90%rh\u003c/sub\u003e and\u0026nbsp;\u003cem\u003eP\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eexp\u003c/sup\u003e as\u0026nbsp;compared with T2 films with the plasticizer.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"4.\tConclusion","content":"\u003cp\u003eThis research contributes to reinforcing the novel idea that it is feasible to obtain materials from whole biomass of filamentous fungi by casting from filmogenic dispersions.\u0026nbsp;This approach provides an innovative alternative source of biopolymers that do not require further purification and is not limited by crop failure or climate conditions. In this work it was feasible to obtain films from all treated dispersions of \u003cem\u003eP. variotti\u003c/em\u003e regardless of the rupture condition applied.The three combined conditions of ultrasound (US) and ultraturrax (UT) were effective for cell rupture, verified by optical microscopy of dispersions and PDS. Longer US treatments produced a reduction in the particle size and the application of the intermediate UT treatment contributed favorably to the breaking of agglomerates allowing the second US treatment to be more effective, achieving an ordered network with a more uniform distribution. This arrangement gave a greater thermal resistance to the fungal biomass films. It was possible to determine that the filtration process after sterilization when obtaining the filmogenic dispersions, eliminated soluble and hydratable compounds, which produced a reduction in the hydration properties of the films produced with T2 dispersions. Through the characterization of mechanical tests, it is highlighted that in the T1 2US15\u0026apos; 0%G films, the values obtained were very similar to those of the plasticized samples, with low values of YM and TS.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study\u0026apos;s conception and design. Material preparation, data collection and analysis were performed by Ezequiel A. Martinez, Macarena R. Sanchez-D\u0026iacute;az, Andres G. Salvay, Vanesa Ludemann, and Mercedes A. Peltzer. The funds were managed by Vanesa Ludemann. The manuscript was written and approved by all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors of this article acknowledge the financial support from National University of Quilmes (UNQ, Argentina) through R\u0026amp;D program (Expediente 1300/19).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTang XZ, Kumar P, Alavi S, Sandeep KP (2012) Recent advances in biopolymers and biopolymer-based nanocomposites for food packaging materials. Crit Rev Food Sci Nutr.52 (5):426-442. https://doi.org/10.1080/10408398.2010.500508. \u003c/li\u003e\n\u003cli\u003eComa ME, Peltzer MA, Delgado JF, Salvay AG (2019) Water k\u0026eacute;fir grains as an innovative source of films: Study of plasticizer content on film properties. 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Biotechnol 29:1412\u0026ndash;1423. https://doi.org/10.4014/jmb.1905.05015\u003c/li\u003e\n\u003cli\u003eSanthosh BS, Bhavana DR, Rakesh MG (2018) Mycelium composites: An emerging green building material. Int. Res. J. Eng. Technol 5:3066\u0026ndash;3068.\u003c/li\u003e\n\u003cli\u003eSun W, Tajvidi M, Hunt CG, McIntyre G, Gardner DJ (2019) Fully Bio-Based Hybrid Composites Made of Wood, Fungal Mycelium and Cellulose Nanofibrils. Sci. Rep. 9: 3766. https://doi.org/10.1038/s41598-019-40442-8\u003c/li\u003e\n\u003cli\u003eCerimi K, Akkaya KC, Pohl C, Schmidt B, Neubauer P (2019) Fungi as source for new bio-based materials: A patent review. Fungal Biol. Biotechnol. 6:17. https://doi.org/10.1186/s40694-019-0080-y\u003c/li\u003e\n\u003cli\u003eSanchez-D\u0026iacute;az MR, Lazarte MS, Moavro A, Mercedes A Peltzer, Vanesa Ludemann. (2023) Naturally Multicomponent Materials Obtained from Filamentous Fungi: Impact of Different Cell Rupture Treatment on Film Properties. 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Applied Biochemistry and Microbiology 49 (1): 41\u0026ndash;45. https://doi.org/10.1134/s0003683813010080\u003c/li\u003e\n\u003cli\u003ePitt JI, Hocking AD (2009) Fungi and Food Spoilage. 3rd. ed. Springer Dordrecht Heidelberg London New York. https://doi.org/10.1007/978-0-387-92207-2\u003c/li\u003e\n\u003cli\u003eFarahnaky A, Saberi B, Majzoobi M (2013) Effect of glycerol on physical and mechanical properties of wheat starch edible films. J. Texture Stud. 44:176\u0026ndash;186. https://doi.org/10.1111/jtxs.12007 \u003c/li\u003e\n\u003cli\u003eZhang L, Ye X Xue SJ, Zhang X, Liu D, et al. (2013) Effect of high-intensity ultrasound on the physicochemical properties and nanostructure of citrus pectin. Journal of the Science of Food and Agriculture 93:2028-2036. https://doi.org/10.1002/jsfa.6011\u003c/li\u003e\n\u003cli\u003eRam\u0026iacute;rez Tapias YA, Peltzer MA, Delgado JF, Salvay AG (2020) Kombucha tea by-product as source of novel materials: Formulation and characterization of films. Food Bioproc Tech. 13:1166\u0026ndash;1180. https://doi.org/10.1007/s11947-020-02471-4\u003c/li\u003e\n\u003cli\u003eGuggenheim EA (1966) Applications of statistical mechanics. 1st. ed. Oxford: Claredon Press. ISBN 10:0198553315. https://doi.org/10.1002/ange.19670791621\u003c/li\u003e\n\u003cli\u003eSalvay AG, Colombo MF, Grigera JR (2003) Hydration effects on the structural properties and haem-haem interactions in haemoglobin. Phys Chem Chem Phys 5:192-197. https://doi.org/10.1039/B209560B\u003c/li\u003e\n\u003cli\u003eASTM-E96 (2016) Standard test methods for water vapor transmission of materials. 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LWT - Food Science and Technology 40(5):766\u0026ndash;774. https://doi.org/10.1016/j.lwt.2006.04.005\u003c/li\u003e\n\u003cli\u003eZohuriaan M,Shokrolahi F. Thermal studies on natural and modified gums. Polymer Testing 23(5): 575\u0026ndash;579. https://doi.org/10.1016/j.polymertesting.2003.11.001\u003c/li\u003e\n\u003cli\u003eHoque S, Benjakul S, Prodpran T (2011) Effects of partial hydrolysis and plasticizer content on the properties of film from cuttlefish (Sepia pharaonis) skin gelatin. Food Hydrocolloids 25:82-90. https://doi.org/10.1016/j.foodhyd.2010.05.008\u003c/li\u003e\n\u003cli\u003eRamos \u0026Oacute;L, Reinas I, Silva SI, Fernandes JC, Cerqueira, MA, Pereira RN, et al. (2013) Effect of whey protein purity and glycerol content upon physical properties of edible films manufactured therefrom. Food Hydrocolloids 30(1):110\u0026ndash;122. https://doi.org/10.1016/j.foodhyd.2012.05.001\u003c/li\u003e\n\u003cli\u003eKagimura FY, da Cunha MAA, Barbosa A, Dekker RFH, Malfatti CRM (2015) Biological activities of derivatized d-glucans: A review. International Journal of Biological Macromolecules 72: 588\u0026ndash;598. https://doi.org/10.1016/j.ijbiomac.2014.09.008 \u003c/li\u003e\n\u003cli\u003eStawski D, Rabiej, S, Herczyńska L, Draczyński Z (2008) Thermogravimetric analysis of chitins of different origin. Journal of Thermal Analysis and Calorimetry 93(2):489-494. \u003c/li\u003e\n\u003cli\u003eRam\u0026iacute;rez Tapias YA, Di Monte MV, Peltzer MA, Salvay AG (2021) Bacterial cellulose films production by Kombucha symbiotic community cultured on different herbal infusions. Food Chemistry 372:131346. https://doi.org/10.1016/j.foodchem.2021.131346.\u003c/li\u003e\n\u003cli\u003eDelgado JF, Peltzer MA, Salvay AG (2022) Water Vapour Transport in Biopolymeric Materials: Effects of Thickness and Water Vapour Pressure Gradient on Yeast Biomass-Based Films. J Polym Environ 30:2976\u0026ndash;2989. https://doi.org/10.1007/s10924-022-02412-6\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eParameters obtained from % Volume distribution: Span and D\u003csub\u003e4.3\u003c/sub\u003e values\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"257\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.96498054474708%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eSample\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.957198443579767%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eSPAN\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.07782101167315%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eD\u003csub\u003e4,3\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.84046692607004%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.124513618677042%\" valign=\"top\"\u003e\n \u003cp\u003e1US15\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.957198443579767%\" valign=\"top\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.07782101167315%\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; 105 \u0026plusmn; 41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.678571428571427%\" valign=\"top\"\u003e\n \u003cp\u003e1US30\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.339285714285715%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.982142857142854%\"\u003e\n \u003cp\u003e50 \u0026plusmn; 18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.678571428571427%\" valign=\"top\"\u003e\n \u003cp\u003e2US15\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.339285714285715%\" valign=\"top\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.982142857142854%\"\u003e\n \u003cp\u003e9 \u0026plusmn; 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.84046692607004%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.124513618677042%\" valign=\"top\"\u003e\n \u003cp\u003e1US15\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.957198443579767%\" valign=\"top\"\u003e\n \u003cp\u003e4.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.07782101167315%\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;14.5 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.678571428571427%\" valign=\"top\"\u003e\n \u003cp\u003e1US30\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.339285714285715%\" valign=\"top\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.982142857142854%\"\u003e\n \u003cp\u003e8.7 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.678571428571427%\" valign=\"top\"\u003e\n \u003cp\u003e2US15\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.339285714285715%\" valign=\"top\"\u003e\n \u003cp\u003e2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.982142857142854%\"\u003e\n \u003cp\u003e8.7 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eTensile parameters from mechanical assay\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"604\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.543046357615893%\" colspan=\"2\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eSample\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.72185430463576%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e0% Glycerol\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.735099337748345%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e25% Glycerol\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.10794297352342%\" valign=\"top\"\u003e\n \u003cp\u003eTS (MPa)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.95926680244399%\" valign=\"top\"\u003e\n \u003cp\u003eYM (MPa)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052953156822811%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;ɛ% (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.10794297352342%\" valign=\"top\"\u003e\n \u003cp\u003eTS (MPa)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.718940936863543%\" valign=\"top\"\u003e\n \u003cp\u003eYM (MPa)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052953156822811%\" valign=\"top\"\u003e\n \u003cp\u003eɛ%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.291873963515755%\" rowspan=\"3\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.281923714759536%\" valign=\"top\"\u003e\n \u003cp\u003e1US15\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.930348258706468%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; 11\u0026plusmn;2\u003csup\u003ea,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.25207296849088%\" valign=\"top\"\u003e\n \u003cp\u003e499\u0026plusmn;63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\" valign=\"top\"\u003e\n \u003cp\u003e3\u0026plusmn;1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.930348258706468%\" valign=\"top\"\u003e\n \u003cp\u003e1,0\u0026plusmn;0,1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.427860696517413%\" valign=\"top\"\u003e\n \u003cp\u003e9\u0026plusmn;2\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\" valign=\"top\"\u003e\n \u003cp\u003e13\u0026plusmn;3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.211573236889693%\" valign=\"top\"\u003e\n \u003cp\u003e1US30\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; 8\u0026plusmn;1\u003csup\u003ea,e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.72151898734177%\" valign=\"top\"\u003e\n \u003cp\u003e529\u0026plusmn;91\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.47739602169982%\" valign=\"top\"\u003e\n \u003cp\u003e2\u0026plusmn;1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\" valign=\"top\"\u003e\n \u003cp\u003e1,0\u0026plusmn;0,2\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.732368896925859%\" valign=\"top\"\u003e\n \u003cp\u003e11\u0026plusmn;3\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.47739602169982%\" valign=\"top\"\u003e\n \u003cp\u003e12\u0026plusmn;2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.211573236889693%\" valign=\"top\"\u003e\n \u003cp\u003e2US15\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\" valign=\"top\"\u003e\n \u003cp\u003e4\u0026plusmn;1\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.72151898734177%\" valign=\"top\"\u003e\n \u003cp\u003e77\u0026plusmn;9\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.47739602169982%\" valign=\"top\"\u003e\n \u003cp\u003e12\u0026plusmn;2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\" valign=\"top\"\u003e\n \u003cp\u003e2\u0026plusmn;1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.732368896925859%\" valign=\"top\"\u003e\n \u003cp\u003e9\u0026plusmn;1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.47739602169982%\" valign=\"top\"\u003e\n \u003cp\u003e20\u0026plusmn;3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.291873963515755%\" rowspan=\"3\"\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.281923714759536%\" valign=\"top\"\u003e\n \u003cp\u003e1US15\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.930348258706468%\" valign=\"top\"\u003e\n \u003cp\u003e9\u0026plusmn;1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.25207296849088%\" valign=\"top\"\u003e\n \u003cp\u003e712\u0026plusmn;107\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\" valign=\"top\"\u003e\n \u003cp\u003e2\u0026plusmn;1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.930348258706468%\" valign=\"top\"\u003e\n \u003cp\u003e4\u0026plusmn;1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.427860696517413%\" valign=\"top\"\u003e\n \u003cp\u003e63\u0026plusmn;10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\" valign=\"top\"\u003e\n \u003cp\u003e10\u0026plusmn;1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.211573236889693%\" valign=\"top\"\u003e\n \u003cp\u003e1US30\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\" valign=\"top\"\u003e\n \u003cp\u003e17\u0026plusmn;3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.72151898734177%\" valign=\"top\"\u003e\n \u003cp\u003e1006\u0026plusmn;333\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.47739602169982%\" valign=\"top\"\u003e\n \u003cp\u003e2\u0026plusmn;1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\" valign=\"top\"\u003e\n \u003cp\u003e4\u0026plusmn;1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.732368896925859%\" valign=\"top\"\u003e\n \u003cp\u003e84\u0026plusmn;13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.47739602169982%\" valign=\"top\"\u003e\n \u003cp\u003e10\u0026plusmn;2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.211573236889693%\" valign=\"top\"\u003e\n \u003cp\u003e2US15\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\" valign=\"top\"\u003e\n \u003cp\u003e14\u0026plusmn;2\u003csup\u003eb,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.72151898734177%\" valign=\"top\"\u003e\n \u003cp\u003e1312\u0026plusmn;149\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.47739602169982%\" valign=\"top\"\u003e\n \u003cp\u003e2\u0026plusmn;1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\" valign=\"top\"\u003e\n \u003cp\u003e6\u0026plusmn;1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.732368896925859%\" valign=\"top\"\u003e\n \u003cp\u003e99\u0026plusmn;11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.47739602169982%\" valign=\"top\"\u003e\n \u003cp\u003e13\u0026plusmn;2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Different letters in the same column indicate significant differences (p \u0026lt; 0.05) between treatments by Tukey\u0026rsquo;s test\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. \u0026nbsp;\u003c/strong\u003eParameters from thermal analysis\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.548123980424144%\" colspan=\"2\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eSamples\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.548123980424144%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e0% Glycerol\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.90375203915171%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e25% Glycerol\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003eT\u003csub\u003e0\u003c/sub\u003e (\u0026deg;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e (\u0026deg;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003eT\u003csub\u003e0\u003c/sub\u003e (\u0026deg;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003eT\u003csub\u003emax1\u003c/sub\u003e (\u0026deg;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003eT\u003csub\u003emax2\u0026nbsp;\u003c/sub\u003e(\u0026deg;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.317073170731708%\" rowspan=\"3\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.13821138211382%\" valign=\"top\"\u003e\n \u003cp\u003e1US15\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.308943089430894%\" valign=\"top\"\u003e\n \u003cp\u003e157\u0026plusmn;17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.308943089430894%\" valign=\"top\"\u003e\n \u003cp\u003e256\u0026plusmn;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.308943089430894%\" valign=\"top\"\u003e\n \u003cp\u003e140\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.308943089430894%\" valign=\"top\"\u003e\n \u003cp\u003e169\u0026plusmn;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.308943089430894%\" valign=\"top\"\u003e\n \u003cp\u003e265\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.80701754385965%\" valign=\"top\"\u003e\n \u003cp\u003e1US30\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e204\u0026plusmn;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e255\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e154\u0026plusmn;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e178\u0026plusmn;18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e264\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.80701754385965%\" valign=\"top\"\u003e\n \u003cp\u003e2US15\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e182\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e254\u0026plusmn;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e145\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e183\u0026plusmn;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e265\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.317073170731708%\" rowspan=\"3\"\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.13821138211382%\" valign=\"top\"\u003e\n \u003cp\u003e1US15\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.308943089430894%\" valign=\"top\"\u003e\n \u003cp\u003e228\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.308943089430894%\" valign=\"top\"\u003e\n \u003cp\u003e261\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.308943089430894%\" valign=\"top\"\u003e\n \u003cp\u003e138\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.308943089430894%\" valign=\"top\"\u003e\n \u003cp\u003e145\u0026plusmn;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.308943089430894%\" valign=\"top\"\u003e\n \u003cp\u003e266\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.80701754385965%\" valign=\"top\"\u003e\n \u003cp\u003e1US30\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e204\u0026plusmn;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e258\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e140\u0026plusmn;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e171\u0026plusmn;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e265\u0026plusmn;3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.80701754385965%\" valign=\"top\"\u003e\n \u003cp\u003e2US15\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e236\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e266\u0026plusmn;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e180\u0026plusmn;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e207\u0026plusmn;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.43859649122807%\" valign=\"top\"\u003e\n \u003cp\u003e270\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e Values of the GAB parameters fitted for the water sorption isotherms displayed in Figure X and experimental water\u0026nbsp;vapour\u0026nbsp;permeability of films. The reported values of the statistical parameter \u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e indicate a very good acceptance of the fit model. Errors in GAB parameters were estimated from the fit analysis. \u003cem\u003eh\u003c/em\u003e\u003csub\u003e90%r.h.\u003c/sub\u003e refers to the hydration equilibrium value at 90% r.h. Different letters in the same column corresponding to \u003cem\u003eh\u003c/em\u003e\u003csub\u003e90%r.h.\u003c/sub\u003e and \u003cem\u003eP\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eexp\u003c/sup\u003e indicate statistically significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). Units of \u003cem\u003eN\u003c/em\u003e and \u003cem\u003eh\u003c/em\u003e\u003csub\u003e90%r.h.\u003c/sub\u003e are g H\u003csub\u003e2\u003c/sub\u003eO per g d.m and units of \u003cem\u003eP\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eexp\u003c/sup\u003e are 10\u003csup\u003e-10\u003c/sup\u003eg s\u003csup\u003e-1\u003c/sup\u003em\u003csup\u003e-1\u003c/sup\u003ePa\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.61855670103093%\" colspan=\"3\" rowspan=\"2\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"1.0309278350515463%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.97938144329896%\" colspan=\"5\" valign=\"bottom\"\u003e\n \u003cp\u003eWater sorption isotherms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.0618556701030926%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eexp\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.5384615384615385%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.307692307692308%\"\u003e\n \u003cp\u003e\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e\u003cem\u003eN\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.46153846153846%\"\u003e\n \u003cp\u003e\u003cem\u003ec\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.46153846153846%\"\u003e\n \u003cp\u003e\u003cem\u003eh\u003c/em\u003e\u003csub\u003e90%rh\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.076923076923077%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.25531914893617%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.638297872340425%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"1.0638297872340425%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.51063829787234%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.957446808510639%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.76595744680851%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.957446808510639%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.76595744680851%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.127659574468085%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.638297872340425%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" rowspan=\"7\"\u003e\n \u003cp\u003e0 % Glycerol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.25531914893617%\" rowspan=\"3\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.638297872340425%\"\u003e\n \u003cp\u003e1US15\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"1.0638297872340425%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.51063829787234%\"\u003e\n \u003cp\u003e0.999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.957446808510639%\"\u003e\n \u003cp\u003e0.058\u0026plusmn;0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.76595744680851%\"\u003e\n \u003cp\u003e3.23\u0026plusmn;0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.957446808510639%\"\u003e\n \u003cp\u003e0.953\u0026plusmn;0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.76595744680851%\"\u003e\n \u003cp\u003e0.39\u0026plusmn;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.127659574468085%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.638297872340425%\"\u003e\n \u003cp\u003e2.4\u0026plusmn;0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.764705882352942%\"\u003e\n \u003cp\u003e1US30\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"1.1764705882352942%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.411764705882353%\"\u003e\n \u003cp\u003e0.999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.647058823529413%\"\u003e\n \u003cp\u003e0.077\u0026plusmn;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.117647058823529%\"\u003e\n \u003cp\u003e1.80\u0026plusmn;0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.647058823529413%\"\u003e\n \u003cp\u003e0.969\u0026plusmn;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.117647058823529%\"\u003e\n \u003cp\u003e0.56\u0026plusmn;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.3529411764705883%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.764705882352942%\"\u003e\n \u003cp\u003e2.7\u0026plusmn;0.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.764705882352942%\"\u003e\n \u003cp\u003e2US15\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"1.1764705882352942%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.411764705882353%\"\u003e\n \u003cp\u003e0.999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.647058823529413%\"\u003e\n \u003cp\u003e0.080\u0026plusmn;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.117647058823529%\"\u003e\n \u003cp\u003e1.13\u0026plusmn;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.647058823529413%\"\u003e\n \u003cp\u003e0.937\u0026plusmn;0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.117647058823529%\"\u003e\n \u003cp\u003e0.44\u0026plusmn;0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.3529411764705883%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.764705882352942%\"\u003e\n \u003cp\u003e3.0\u0026plusmn;0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.49438202247191%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"1.1235955056179776%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.98876404494382%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.853932584269664%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.48314606741573%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.853932584269664%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.48314606741573%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.247191011235955%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.49438202247191%\" rowspan=\"3\"\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\"\u003e\n \u003cp\u003e1US15\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"1.1235955056179776%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.98876404494382%\"\u003e\n \u003cp\u003e0.997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.853932584269664%\"\u003e\n \u003cp\u003e0.064\u0026plusmn;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.48314606741573%\"\u003e\n \u003cp\u003e4.38\u0026plusmn;0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.853932584269664%\"\u003e\n \u003cp\u003e0.852\u0026plusmn;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.48314606741573%\"\u003e\n \u003cp\u003e0.26\u0026plusmn;0.02\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.247191011235955%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\"\u003e\n \u003cp\u003e2.3\u0026plusmn;0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.764705882352942%\"\u003e\n \u003cp\u003e1US30\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"1.1764705882352942%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.411764705882353%\"\u003e\n \u003cp\u003e0.995\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.647058823529413%\"\u003e\n \u003cp\u003e0.049\u0026plusmn;0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.117647058823529%\"\u003e\n \u003cp\u003e3.93\u0026plusmn;0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.647058823529413%\"\u003e\n \u003cp\u003e0.877\u0026plusmn;0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.117647058823529%\"\u003e\n \u003cp\u003e0.22\u0026plusmn;0.02\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.3529411764705883%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.764705882352942%\"\u003e\n \u003cp\u003e2.1\u0026plusmn;0.1\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.764705882352942%\"\u003e\n \u003cp\u003e2US15\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"1.1764705882352942%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.411764705882353%\"\u003e\n \u003cp\u003e0.996\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.647058823529413%\"\u003e\n \u003cp\u003e0.086\u0026plusmn;0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.117647058823529%\"\u003e\n \u003cp\u003e4.51\u0026plusmn;0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.647058823529413%\"\u003e\n \u003cp\u003e0.796\u0026plusmn;0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.117647058823529%\"\u003e\n \u003cp\u003e0.28\u0026plusmn;0.02\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.3529411764705883%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.764705882352942%\"\u003e\n \u003cp\u003e2.0\u0026plusmn;0.1\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.25531914893617%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.638297872340425%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"1.0638297872340425%\"\u003e\n 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\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"biobased films, ultrasound homogenization, sterilization, cell rupture, casting","lastPublishedDoi":"10.21203/rs.3.rs-3783428/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3783428/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe indiscriminate use of petroleum-based polymers and plastics for single-use food packaging has led to serious environmental problems due the non-biodegradable characteristics. Thus, much attention has been focused on the research of new biobased and biodegradable materials. Yeast and fungal biomass are low-cost and abundant sources of biopolymers with highly promising properties for the development of biodegradable materials. This study aimed to select a preparation method to develop new biodegradable films using the whole biomass of \u003cem\u003ePaecilomyces variotii\u003c/em\u003e subjected to successive physical treatments including ultrasonic homogenization (US) and heat treatment. Sterilization process had an important impact on the final filmogenic dispersion and mechanical properties of the films. Longer US treatments produced a reduction in the particle size and the application of an intermediate UT treatment contributed favorably to the breaking of agglomerates allowing the second US treatment to be more effective, achieving an ordered network with a more uniform distribution. Samples that were not filtrated after the sterilization process presented mechanical properties similar to plasticized materials. On the other hand, the filtration process after sterilization eliminated soluble and hydratable compounds, which produced a reduction in the hydration properties of the films.\u003c/p\u003e","manuscriptTitle":"Functional characterization of biodegradable films obtained from whole Paecilomyces variotii biomass","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-03 20:17:17","doi":"10.21203/rs.3.rs-3783428/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d028bb05-77ec-4b60-9ed1-9921bc728a72","owner":[],"postedDate":"January 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-29T08:46:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-03 20:17:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3783428","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3783428","identity":"rs-3783428","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}