Characterization of bacterial cellulose films combined with chitosan and polyvinyl alcohol: Evaluation of mechanical and barrier properties

1 Characterization of bacterial cellulose films combined with chitosan and polyvinyl 1 alcohol: Evaluation of mechanical and barrier properties 2 3 Patricia Cazón1,2, Gonzalo Velázquez1*, Manuel Vázquez2* 4 1Instituto Politécnico Nacional. CICATA unidad Querétaro. Cerro Blanco No. 141. 5 Colinas del Cimatario, Querétaro, 76090, México. 6 2Department of Analytical Chemistry, Faculty of Veterinary, University of Santiago de 7 Compostela, 27002-Lugo, Spain 8 *Corresponding author: [email protected] 9 gvel[email protected]; phone +52 (442)-229-0804 Ext 81058. 10 11 Abstract 12 Bacterial cellulose (BC) produced by Komagataeibacter xylinus is a biomaterial with a 13 unique three-dimensional structure. To improve the mechanical properties and reinforce 14 the BC films, they were immersed in polyvinyl alcohol (0-4%) and chitosan (0-1%) 15 bathes. Moisture content, mechanical properties and water vapour permeability were 16 measured to assess the effect of polyvinyl alcohol and chitosan. The morphology, 17 optical, structural and thermal properties were evaluated by scanning electron 18 microscopy, spectral analysis, thermogravimetry and differential scanning calorimetry. 19 Results showed that moisture content was significantly affected by the chitosan 20 presence. Tensile strength values in the 20.76 - 41.65 MPa range were similar to those 21 of synthetic polymer films. Percentage of elongation ranged from 2.28 to 21.82% and 22 Young's modulus ranged from 1043.88 to 2247.82 MPa. The water vapour permeability 23 *Manuscript Click here to view linked References 2 (1.47·10-11 - 3.40·10-11 g/m·s·Pa) decreased with the addition of polyvinyl alcohol. The 24 developed films own UV light barrier properties and optimal visual appearance. 25 26 Keywords: Films; bacterial cellulose; water vapor permeability; chitosan; polyvinyl 27 alcohol; UV protection. 28 29 1. Introduction 30 In the last decade, research in biopolymers has been widely carried out to design 31 and develop renewable, biodegradable and biocompatible products with applications in 32 medicine, pharmacy, cosmetics, food industry and biotechnology (Hu, Chen, Yang, Li, 33 & Wang, 2014). Among biopolymers, cellulose is the most abundant natural compound 34 on the earth. This low-cost biopolymer has renewability, non-toxicity, biocompatibility, 35 biodegradability and chemical stability (Cazón, Velazquez, Ramírez, & Vázquez, 36 2017). Due to the properties of cellulose, it has become a very interesting material to 37 develop new biodegradable polymers, like biofilms aimed to food packaging 38 applications. 39 Cellulose is a highly crystalline polymer formed by a linear chain with two 40 anhydroglucose rings ((C6H10O5)n), covalently linked through an oxygen in a β(1-4) 41 glycosidic bond. Cellulose possesses abundant hydroxyl groups forming plenty inter42 and intra-molecular hydrogen bonds (Cazón et al., 2017). It is found in plant cell wall 43 and accordingly, it can be obtained from wood, cotton, hemp and plant-based materials. 44 Furthermore, cellulose is also produced by tunicates, several species of algae, and by 45 some species of bacteria including Acetobacter, Agrobacterium, Pseudomonas, 46 3 Rhizobium, Sarcina and Komagataeibacter xylinus. The last one was renamed as 47 Acetobacter xylinum and more widespread as Gluconoacetobacter xylinus (Szymańska-48 Chargot et al., 2017; Yamada et al., 2012). It produces high amounts of cellulose, being 49 one of the most commonly studied sources of bacterial cellulose (BC) 50 (Mohammadkazemi, Azin, & Ashori, 2015; Ruka, Simon, & Dean, 2012). 51 In a suitable culture medium and static conditions, G. xylinus synthesizes BC in 52 the form of pellicle on the surface of the liquid medium. This microorganism produces 53 glucose chains from the carbon source contained in the medium. The glucose chains are 54 extruded out through tiny pores present on their cell wall. Combining the glucose 55 chains, the microorganism forms microfibrils that aggregate to form cellulose ribbons. 56 These ribbons form a three-dimensional structure consisting of an ultrafine network of 57 cellulose nanofibers with an expanded surface area and high porosity. The three-58 dimensional structure determines its physical and mechanical properties (Jozala et al., 59 2016; Shah, Ul-Islam, Khattak, & Park, 2013; Shao et al., 2016). Unlike vegetable 60 cellulose, BC is obtained with high purity, free of vegetable remains such as lignin and 61 hemicellulose (Jozala et al., 2016). 62 Several studies have been focused on developing new films based on BC. 63 Usually, BC films are combined with other polymers or plasticizers to improve or 64 modify the physicochemical properties and expand its potential applications. There are 65 two main strategies to combine BC with other polymers or plasticizers, keeping the film 66 structure obtained from the fermentation process. One option is by supplementation the 67 culture medium with the desirable reinforcing agents, such as Aloe vera (Saibuatong & 68 Phisalaphong, 2010a), chitosan (Phisalaphong & Jatupaiboon, 2008), polyvinyl alcohol 69 4 (PVOH) (Gea, Bilotti, Reynolds, Soykeabkeaw, & Peijs, 2010) or polyethylene oxide 70 (Brown & Laborie, 2007). Other strategy is by immersion of the BC film in a bath with 71 a solution of the desirable polymer or plasticizer, e.g. polyvinyl alcohol (PVOH) (Gea et 72 al., 2010), polyethylene glycol or diacrylate (Cai & Kim, 2010; Numata, Sakata, 73 Furukawa, & Tajima, 2015). 74 A widespread practice is to use nanofibers or nanowhiskers of BC as reinforcing 75 agent. BC pellicles are usually subjected to a hydrolysis process using strong acids, 76 breaking down the structure of the material into nanofibers or nanocrystals (Martínez-77 Sanz, Lopez-Rubio, & Lagaron, 2013). These BC nanocomponents are incorporated 78 into the matrix of a polymer to modify the properties of the composite film such as 79 starch (Martins et al., 2009), PVOH (Jipa et al., 2012), arabinogalactan and xyloglucan 80 (Lucyszyn et al., 2016), chitosan (Salari, Sowti Khiabani, Rezaei Mokarram, 81 Ghanbarzadeh, & Samadi Kafil, 2018; Velásquez-Cock et al., 2014; Wang, Xie, et al., 82 2018), polylactic acid, polyethylene glycol (Martínez-Sanz et al., 2013), fish proteins 83 (Shabanpour, Kazemi, Ojagh, & Pourashouri, 2018), gelatin (George & Siddaramaiah, 84 2012) or agar (Wang, Guo, et al., 2018) among other biopolymers. 85 From the strategies mentioned to obtain BC-polymers composite films, it is 86 interesting to pay special attention to the immersion method. It is a simple technique 87 that allows using the films formed from the culture medium. This method allows 88 avoiding previous steps of dissolution, regeneration or homogeneous dispersion of the 89 cellulose, simplifying the process. In addition, this technique allows taking advantage of 90 the unique structure of the BC as the main component of the final matrix. On the other 91 hand, unlike the supplementation culture media method, by immersion allows 92 5 expanding the range and concentration of the possible combinations. Some 93 supplementation components in the culture media can interfere with the cellulose 94 production yield. Hence, the concentration of these components in the formulation film 95 are limited (Phisalaphong & Jatupaiboon, 2008; Saibuatong & Phisalaphong, 2010a). 96 Following the strategy of combining polymers to improve the properties of the 97 final material, PVOH and chitosan can improve the potential applications of BC-based 98 films. PVOH is a hydrophilic semi-crystalline polymer produced by polymerization of 99 vinyl acetate to polyvinyl acetate, followed by a hydrolysis process. It is a synthetic 100 polymer, water soluble, non-toxic, biodegradable, with film forming properties, optimal 101 transparency and good elasticity properties (Carvalho et al., 2009; Cazón, Vázquez, & 102 Velazquez, 2018a). These properties make PVOH an ideal polymer to combine with BC 103 by immersion. 104 Chitosan is one of the most studied polysaccharides with potential applications 105 in biomedical, food, and chemical industries. It is the second most abundant 106 polysaccharide in nature. It can be obtained mainly from residues of the shellfish 107 industry. This polymer is non-toxic, biodegradable, with film forming properties and 108 soluble in dilute organic acids such as acetic acid. One of the most interesting properties 109 of the chitosan is its antimicrobial activity against a wide range of foodborne 110 filamentous fungi, yeast, and bacteria, being more active against yeasts (Helander, 111 Nurmiaho-Lassila, Ahvenainen, Rhoades, & Roller, 2001; No, Meyers, Prinyawiwatkul, 112 & Xu, 2007). Among these properties, its solubility and antimicrobial activity make it 113 an ideal polymer to combine with BC by immersion. Incorporating antimicrobial agents 114 from natural source into BC composite films addresses the current consumer demand of 115 6 a natural alternative to chemically synthesized antimicrobial polymers. In food 116 packaging, a strategy to increase the shelf life is to develop active films with 117 antimicrobial properties. The direct contact with the active films inhibit the growth of 118 microorganisms on the surface of the food (Broek Van Den, Knoop, Kappen, & Boeriu, 119 2015; Moreira, Roura, & Ponce, 2011). 120 In previous studies of our group, films based on regenerated vegetable cellulose-121 PVOH-chitosan were characterized. The results obtained suggested an adequate 122 interaction of cellulose-chitosan-PVOH to combine both polymers by immersion, 123 improving the film properties (Cazón, Vázquez, & Velazquez, 2018b; Cazón et al., 124 2018a). Nevertheless, as mentioned, there are important structural differences between 125 vegetable cellulose and BC. These differences could affect the interactions of cellulose-126 PVOH-chitosan, improving the properties of the cellulose-based films and expanding its 127 potential applications. Besides, it was observed the low transmittance of regenerated 128 cellulose and chitosan films developed, manifested UV protect properties of cellulose-129 based films (Cazón et al., 2018a). Packaging materials against UV light has received a 130 great deal of attention since UV-light is one of those responsible factors of the oxidative 131 process in vitamins, lipids and proteins. These oxidative process produce undesirable 132 off-flavours that decrease the shelf-life of the products (Olarte, Sanz, Federico 133 Echávarri, & Ayala, 2009). Hence, the UV-barrier properties observed increase the 134 interest in the development of BC-based films for food applications. 135 On the other hand, the application of BC-PVOH-chitosan components in the 136 field of controlled drug administration has been also studied. The results suggested that 137 BC-PVOH-chitosan composites could be used as a biopolymeric carrier for drug 138 7 delivery applications (Pavaloiu, Dobre, & Hlevca, 2013). Despite the good interaction 139 among these biodegradable polymers, the characterization of these films has not been 140 carried out to aim applications like biopolymers for active food packaging. 141 The aim of this work was to developed biodegradable films based on BC with 142 chitosan and PVOH to enhance the mechanical and optical properties to obtain cellulose 143 films more manageable and transparent with antimicrobial and UV-barrier properties. 144 The effect of the ratio of these polymers incorporated to the BC matrix by immersion, 145 maintaining intact the characteristic BC structure, was studied and compared to 146 regenerated cellulose, previously analysed. Polynomial models were used to evaluate 147 the effect of the composition of the blend on the moisture content, mechanical 148 properties (tensile strength, percentage of elongation to break and Young’s Modulus) 149 and water vapour permeability. Besides, microstructure, optical properties, structure and 150 thermal analyses of the BC-PVOH-chitosan films were also evaluated. 151 152 2. Materials and methods 153 Komagateibacter xylinus was obtained from the “Colección Española de 154 Cultivos Tipo” (CECT, Valencia, Spain). Extra pure anhydrous sodium bromide (99 %), 155 sodium hydroxide (98 %) and D(+)-glucose monohydrate (99% extra pure) were 156 purchased from Acros organics (Geel, Belgium). Yeast extract was provided by 157 Scharlau Microbiology (Barcelona, Spain). Full-hydrolyzed (>98%) polyvinyl alcohol 158 with average molecular weight (Mw) of 30,000 g/mol and ester value of 12-25 were 159 supplied by Merck (Billerica, MA, US). Chitosan (Mw 100000-300000) was purchased 160 from Acros organics (Geel, Belgium). 161 8 162 2.1. Preparation of bacterial cellulose films 163 The preparation process is shown in Figure 1. Initial culture medium was 164 prepared from 10% of glucose and 1% of yeast extract and sterilized at 121ºC for 15 165 min. A pre-culture was prepared by transferring 5 mL of a stock culture to 300 mL of 166 medium in a Petri dish and incubated statically at 30 ºC for 2 days. Then, Petri dishes 167 were prepared with 75 ml of culture medium and inoculated with 5 ml of the pre-168 culture. Petri dish was stored at 30 ºC for 8 days. After that time, the film was 169 suspended in the air-liquid medium. The films were treated with 1% (w/v) of NaOH at 170 90 ºC for 1 h to remove bacterial cells and washed with running distillate water until pH 171 7. Finally, BC films were dried at room temperature for 48 h. 172 173 9 174 Fig. 1. Overall process proposed for the production of bacterial cellulose-based films. 175 176 2.2. Preparation of bacterial cellulose-chitosan-PVOH composite films 177 The resulting wet bacterial films were placed between two sheets of filter paper 178 to remove excessive water. Then, films were immersed in a bath of the mixture 179 chitosan-PVOH. The solution of chitosan was prepared from an aqueous solution of 1% 180 (v/v) of acetic acid at room temperature with vigorous agitation. The PVOH solution 181 was prepared from an aqueous solution heated at 80 ºC with vigorous agitation for 2 h. 182 Finally, the BC-PVOH-chitosan films were dried in a Petri dish at room temperature for 183 H2O (2.5 L) Glucose (10 % w/w) Yeast extract (1% w/w) Sterilize in an autoclave 121ºC, 15 min Incubate without agitation at 30ºC for 2 days Culture Medium Mixing and shaking until room temperature 300 ml of solution in Petri dish Gluconacetobacter xylinus 5 ml of a preinoculum 75 ml of solution in Petri dish Incubate without agitation at 30ºC for 8 days Gluconacetobacter xylinus 5 ml of the preinoculum Treat with 1% NaOH 90ºC, 1 h Treat with destilled water until pH 7 Cellulose films Dry at room temperature for 48 h 16 Table 1. Formulations assayed for the study of bacterial cellulose based films combined with chitosan and polyvinyl alcohol and experimental results for the mechanical and permeability properties of the films achieved. MC is the equilibrium moisture content of each sample after being stored in desiccators with saturated salt of sodium bromide for 5 days, TS is tensile strength, %E is percentage of elongation at break, YM is Young’s modulus and WVP is water vapour permeability. Exp. Chitosan PVOH Thickness MC TS %E YM WVP % (w/w) % (w/w) mm % MPa % MPa g/m s Pa 1 0 0 2.08·10-2 1.82 20.76 2.28 1043.88 2.38·10-11 2 0 2 5.07·10-2 8.58 30.59 4.60 1595.16 1.47·10-11 3 0 4 6.62·10-2 8.06 27.50 9.99 1055.57 1.85·10-11 4 0.5 0 2.36·10-2 6.41 33.37 4.60 1443.45 2.46·10-11 5 0.5 2 3.98·10-2 8.73 38.89 4.71 2247.82 2.06·10-11 6 0.5 4 5.04·10-2 8.46 38.29 5.57 1825.22 2.21·10-11 7 1 0 2.99·10-2 12.55 39.28 7.08 1579.59 2.79·10-11 8 1 2 5.17·10-2 10.68 41.65 13.20 1363.63 2.68·10-11 9 1 4 5.71·10-2 9.89 36.81 21.82 1174.37 3.40·10-11 295 296 3. Results and discussion 297 Biodegradable films were obtained using bacterial cellulose, PVOH and 298 chitosan at several concentrations following the experimental design showed in Table 1. 299 The concentration of PVOH in the bath ranged from 0 to 4% (w/w) and chitosan ranged 300 17 from 0 to 1% (w/w). At higher concentrations of chitosan and PVOH, the bath was very 301 viscous resulting in too thick films. The average thickness of the films and the results 302 obtained for dependent variables (moisture content, TS, %E, YM and WVP) are listed 303 in Table 1. The effect of the PVOH and chitosan on the dependent variables (moisture 304 content, TS, %E, YM and WVP) were modelled using a second-order polynomial 305 equation. The ANOVA results of the dependent variables are shown in Table 2. Table 2 306 also shows the fit statistics values of r2, predicted r2, adjusted r2 and adequate precision 307 for each dependent variable analyzed. 308 309 Table 2. Analysis of variance (ANOVA) for each of the study dependent variables. TS is tensile strength, %E is percentage of elongation at break, YM is Young’s modulus, WVP is water vapour permeability, MC is the equilibrium moisture content of each sample after being stored in desiccators with saturated salt of sodium bromide for 5 days. MC TS %E YM WVP Source F-value p-value F-value p-value F-value p-value F-value p-value F-value p-value Model 9.04 0.0184 30.63 0.0089 9.80 0.0129 302.83 0.0033 20.11 0.0163 A-Chitosan 15.96 0.0104 107.55 0.0019 10.39 0.0181 63.87 0.0153 68.39 0.0037 B-PVOH 2.35 0.1862 5.98 0.0920 9.21 0.0230 153.05 0.0065 0.19 0.6905 AB 8.81 0.0312 9.05 0.0573 79.11 0.0124 13.08 0.0363 A2 14.23 0.0326 1054.5 0.0009 2.78 0.1942 B2 16.35 0.0272 655.51 0.0015 16.13 0.0277 18 r2 0.98 0.98 0.77 0.99 0.99 Adjusted r2 0.97 0.95 0.69 0.99 0.99 Predicted r2 0.95 0.79 0.45 0.98 0.95 Adequate precision 25.55 16.94 8.85 48.82 39.39 310 3.1. Scanning electron microscopy (SEM) 311 The top and bottom surfaces of both sides and cross section of the films obtained 312 in experiments 1, 3 and 7 were observed by scanning electron microscopy (SEM). 313 Figure 2 shows the top and bottom surface of the films as well as the cross section. The 314 selected experiments allowed to analyse the effect of the PVOH and chitosan on the 315 microstructure of the bacterial cellulose films. As shown Figure 2a, bacterial cellulose 316 films presented a continuous matrix with a porous surface. Bottom side showed a denser 317 surface and a rough layer on the opposite side. The difference between both sides 318 depended on the fermentation process. Probably, the less dense and porous side was the 319 most recently formed. The cross section of the bacterial cellulose films (Figure 2a) 320 showed a laminated structure. The acetic bacteria form the pellicle layer by layer. In 321 general, the addition of PVOH and chitosan resulted in a softening of the surface, 322 decreasing the roughness and porosity (Figure 2b and 2c). The bottom sides were 323 smoother than the top because they were in contact with the Petri dish during the 324 drying. According to the cross-section micrographs of the experiment 3 (Figure 2b) and 325 7 (Figure 2c), PVOH and chitosan increased the thickness and the density of the film 326 promoting a more compact structure. The thickness increase was more important in 327 formulations containing PVOH than those with chitosan. Chitosan and PVOH had a 328 19 similar effect on the surface and on the microstructure than that on vegetable 329 regenerated cellulose films studied in previous work (Cazón et al., 2018b, 2018a). 330 331 332 Fig. 2. Scanning electron microscopy of the samples as well of the section of the films 333 of experiments 1, 3 and 7. A) Pure bacterial cellulose sample. B) Bacterial cellulose-334 poly(vinyl alcohol) 4% (w/w). C) Bacterial cellulose-chitosan 1% (w/w). 335 336 Top Bottom A B C Section 20 3.2. Moisture content at equilibrium 337 The content of the moisture of the samples were analysed. Bacterial cellulose, 338 chitosan and PVOH are hydrophilic components, but with different affinity and 339 interaction capacity with water. The moisture content can modify the properties of the 340 films, mainly the mechanical properties. Hence, determining the moisture content of 341 each formulation can help to interpret the other tests. The moisture content of the 342 developed films conditioned in desiccators with saturated sodium bromide solution for 5 343 days ranged from 1.82 to 12.55%. Data fitted well to a two-factor interaction 344 mathematical model. The trial 2 was ignored for ANOVA analysis as it was detected as 345 an outlier in the Cook's distance test. The F-value of the model was 68.85 indicating 346 that the model was significant. The p-values of the model terms were significant (p 347 <0.05). The r2 value was 0.98. Adjusted r2 compares the goodness-of-fit for regression 348 models that contain differing numbers of independent variables. Predicted r2 is a 349 measure of how well the model predicts a response value. The adjusted r2 and predicted 350 r2 should be within approximately 0.20 of each other to be in reasonable agreement. In 351 this case, the difference between the predicted r2 (0.98) and the adjusted r2 (0.95) was 352 less than 0.2, which is reasonable. Adequate precision is a signal/noise ratio. It 353 compares the range of the predicted values at the design points to average prediction 354 error. Ratios greater than 4 indicate adequate model discrimination. The adequate 355 precision obtained was 25.55, implying an adequate signal. 356 The F-values of the terms allow determining which component has greater effect 357 on the response. The F-values of the model terms indicated that the moisture content of 358 the samples was mainly affected by the chitosan concentration, followed by the 359 21 interaction between chitosan-PVOH. Equation 7 predicts the moisture content of the 360 bacterial cellulose films as a function of chitosan and PVOH concentrations. 361 362 363 (Eq. 7) 364 365 According to the response surface (Figure 3a), the presence of chitosan or 366 PVOH in bacterial cellulose films increased the moisture content. Mainly the chitosan is 367 responsible for the increase of moisture content. This increase was due to the 368 hydrophilic nature of the chitosan and PVOH. The chitosan effect was more significant 369 due to its large amount of hydrophilic amino and hydroxyl groups. The moisture 370 adsorption behaviour of chitosan-PVOH blends in different humidity environments 371 were investigated in previous studies (Liu et al., 2018). Results in this study 372 demonstrated a higher moisture content of the composites when increasing the chitosan 373 concentration. 374 375 376 22 377 378 Fig. 3. Prediction of the model for the effect on A) moisture content in equilibrium and 379 B) water vapour permeability (WVP) of chitosan and poly(vinyl alcohol) on bacterial 380 cellulose films. 381 0 1 2 3 4 00.2 0.4 0.6 0.8 1 0 2 4 6 8 10 12 14 Moisture content (%) Chitosan(%) PVOH (%) 0 1 2 3 4 00.2 0.4 0.6 0.8 1 1·10-11 1.5·10-11 2·10-11 2.5·10-11 3·10-11 3.5·10-11 WVP (g/m s Pa) Chitosan(%) PVOH (%) A B 23 3.3. Water vapour permeability (WVP) 382 The WVP values ranged from 1.47·10-11 to 3.40·10-11 g/m·s·Pa. Data were well 383 fitted to a quadratic model. Experiment 3 was ignored because it was identified as an 384 outlier by the Cook's distance test. The F-value of the model was 143.37 with a p-value 385 of 0.0069, indicating that the mathematical model was significant. The value of r⁠2 was 386 0.99. The predicted r2 (0.95) was in reasonable agreement with the adjusted r2 (0.99). 387 The adequate precision was higher than 4 (39.39), implying an adequate signal. The F-388 values indicate that the chitosan had the most important effect on the WVP response, 389 followed by the interaction chitosan-PVOH. 390 Equation 8 predicts the response on WVP as a function of chitosan and PVOH. 391 392 393 394 (Eq. 8) 395 As shown in the response surface (Figure 3b), the WVP pattern depended on the 396 components blended with the BC. BC-chitosan blends increased the WVP when the 397 chitosan content increased. The hydrophilic property of the chitosan could explain the 398 increase of WVP. Conversely, BC-PVOH films had lower permeability than pure 399 cellulose films. Pure BC films showed a WVP of 2.38·10-11 (g/m·s·Pa) and pure PVOH 400 films reported a WVP of 1.70·10-11 g/m·s·Pa at 30 ºC (Jipa et al., 2012). Pure BC films 401 have an open porous structure, which facilitate the diffusion of the water molecules 402 through cellulose fibers. Some voids between the cellulose fibers could have been filled 403 by the PVOH decreasing the porosity and hindering the diffusion of the water 404 24 molecules. PVOH is a hydrophilic polymer, but its interaction capacity with water is 405 lower than that of chitosan (Liu et al., 2018). For this reason, bacterial cellulose-PVOH 406 films showed lower WVP values than that of bacterial cellulose-chitosan films. 407 However, this behaviour changed in presence chitosan-PVOH, producing a higher 408 increase of the permeability when the concentration of both polymers increased. This 409 effect has been reported in previous studies as chitosan-PVOH films manifested higher 410 WVP values than pure chitosan or PVOH films. The authors justified this effect as a 411 result of the formation of a more open matrix. The hydration layers of the chitosan 412 resulted in a lower density of the polymeric matrix. This structure facilitated the transfer 413 of water molecules through the network (Bonilla, Fortunati, Atarés, Chiralt, & Kenny, 414 2014). Regenerated cellulose-chitosan-PVOH films showed the same effect on WVP 415 (Cazón et al., 2018b). 416 417 3.4. Mechanical properties 418 The TS values ranged from 20.76 to 41.65 MPa. Data fitted well to a quadratic 419 equation. The F-value of the model was 30.63, indicating that the model was significant. 420 All terms in the quadratic model were significant (p < 0.05). The value of r⁠2 was 0.98 421 and the predicted r2 was in reasonable agreement with the adjusted r2. The adequate 422 precision obtained was 16.94, implying an adequate signal. The F-values of the model 423 terms indicated that TS was highly affected by the chitosan. Equation 9 forecasts the 424 values of TS as a function of chitosan and PVOH concentrations. 425 426 25 427 (Eq. 428 9) 429 430 Pure regenerated cellulose film reported a TS value of 38.30 MPa from a 431 dissolution of 4% of cellulose microcrystalline (Cazón et al., 2018b). Pure BC films 432 showed a TS values of 20.76 MPa. BC films showed lower TS values than regenerated 433 cellulose films, despite BC has higher crystallinity, because in its structure there are not 434 remains of amorphous structures. There are two reasons responsible of this TS 435 difference. One reason is the variation of the thickness. Pure regenerated cellulose 436 studied was three folds thicker than pure BC films. Lower thickness could imply lower 437 interaction among the cellulose fibers. It was observed that the TS values of the 438 regenerated cellulose films depended on the cellulose concentration (Cazón et al., 439 2018b). In addition, bacterial cellulose had a less packed structure than regenerated 440 cellulose, as shown the SEM images. These differences in the microstructure could 441 affect the resistance to rupture, but also could promote the interaction with other 442 polymers in the blend. Probably, longer fermentation time of the acetic bacteria to 443 obtain thicker films, could help to increase the hydrogen bond interactions and increase 444 the TS values. In literature, TS values have been reported between 5 and 96 MPa for 445 pure BC films (Cai & Kim, 2010; Phisalaphong & Jatupaiboon, 2008; Saibuatong & 446 Phisalaphong, 2010b; Szymańska-Chargot et al., 2017). The wide range of TS values 447 could be explained because the properties of cellulose depend on the specific 448 assembling and supramolecular order controlled by the culture medium, fermentation 449 32 Fig. 5. Prediction of the model for the effect on the Young´s Modulus of chitosan and 549 poly(vinyl alcohol) on bacterial cellulose films. 550 551 3.5. Light barrier properties, transparency and opacity 552 One of the main objectives of active packaging is maintaining the quality of 553 food to increase the shelf life. Hence, to develop biofilms with good optical barrier in 554 the region of UV-radiation is a feasible strategy. UV-light is the main responsible for 555 the appearance of the singlet oxygen, the most common cause of lipid oxidation. This 556 oxidative reaction alters the odour, taste and colour of food, diminishing its organoleptic 557 qualities, that affect the acceptability of food to consumers (Goudarzi, Shahabi-558 Ghahfarrokhi, & Babaei-Ghazvini, 2017; Vilela et al., 2017). On the other hand, 559 biodegradable films with UV-light barriers properties should have an adequate 560 transparency. Biopolymers with good values of transparency applied directly on food 561 provide a good visual appearance, increasing their consumer acceptancy (Cazón et al., 562 2018a). 563 The lipids are more susceptible to oxidative reactions in the region from 200 to 564 280 nm wavelength. Figure 6a shows the transmittance of the experiments 1, 3 and 7 in 565 the 200-280 nm UV range. The select samples allow to analyse the optical properties of 566 pure bacterial cellulose, and the effect of the PVOH and chitosan on the composite 567 films. In this region, pure bacterial cellulose showed transmittance values of 0% up to a 568 wavelength of 230 nm, then increasing up to 2%. Pure bacterial cellulose films showed 569 lower transmittance values than pure regenerated cellulose which ranged from 0.4 to 570 5.3% (Cazón et al., 2018a). This difference is probably due to the purity of the BC and 571 33 the structural differences between the films. The addition of PVOH increased the 572 transmittance at 210 nm up to 11.65%. BC-chitosan spectra showed that the 573 transmittance values remained low, without significant differences when compared to 574 pure bacterial cellulose. In previous studies, chitosan showed lower transmittance 575 values in the UV region between 200 and 280 nm (Kalaycıoğlu, Torlak, Akın-Evingür, 576 Özen, & Erim, 2017; Kanatt et al., 2012). That is the reason because BC-chitosan films 577 kept the low values. 578 In the UV-VIS region (280– 800 nm) (Figure 6b), pure BC films kept low values 579 of transmittance, with a maximum transmittance of 11%. Adding PVOH to BC resulted 580 in the greatest effect on transmittance, increasing the value up 46.6%. However, in 581 chitosan-BC samples, the transmittance increased constantly with the wavelength, up to 582 32%. This increase of the transmittance affected the visual properties modifying the 583 transparency and opacity of the films. 584 585 586 34 587 588 Fig. 6. Spectra profile A) and B) of UV-VIS and C) FT-IR of films from experiments 1, 589 3 and 7: Exp 1) Pure bacterial cellulose sample; Exp 3) Bacterial cellulose-poly(vinyl 590 alcohol) 4% (w/w); Exp 7) Bacterial cellulose-chitosan 1% (w/w). 591 592 Wavelength [nm] 280270260250240230220210200190 Transmittance [%] 12 10 8 6 4 2 0 Exp 1 Exp 3 Exp 7 Wavelength [nm] 800750700650600550500450400350300 Transmittance [%] 45 40 35 30 25 20 15 10 5 0 Wavenumbers [1/cm] 3500 3000 2500 2000 1500 1000 Absorbance 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 Exp 1 Exp 3 Exp 7 Exp 1 Exp 3 Exp 7 A) B) C) 35 Pure cellulose films had a smoked colour, but the formulations with PVOH 593 resulted in films with a translucent and shiny appearance. However, the transparency 594 values obtained for samples 1, 3 and 7 were 47.6, 24.5 and 48.0, respectively. 595 According to these results, pure BC film had the best transparency value, followed by 596 the chitosan film and then PVOH film. These values were strongly affected by the 597 thickness. Films with PVOH were three times thicker than pure BC samples. BC-598 chitosan films were twice as thick as pure BC. For this reason, it is necessary to 599 complete this information with the opacity. The opacity values calculated were 50.9, 6.2 600 and 20.9 for pure BC, BC-PVOH and BC-chitosan films, respectively. The transparency 601 and opacity values indicate that the PVOH, followed by the chitosan, improved the 602 optical properties of the films in the visible region, thus improving their appearance as 603 shown in Figure 7. In this region, the transmittance values were slightly lower than the 604 values obtained for regenerated cellulose-chitosan-PVOH films previously studied 605 (Cazón et al., 2018a). 606 607 36 608 Fig. 7. Visual appearance of the A) pure bacterial cellulose sample, B) bacterial 609 cellulose-poly(vinyl alcohol) 4% (w/w) and C) bacterial cellulose-chitosan 1% (w/w). 610 B) C) A) 37 611 3.6. Fourier transform infrared spectroscopy (FT-IR) 612 The interactions between BC-PVOH-chitosan at structural level were evaluated 613 by FT-IR measurements. Figure 6c shows the FT-IR spectra of the experiment 1, 3 and 614 7. The spectra show the characteristic absorption bands of BC, PVOH and chitosan. A 615 broad band in the 3000–3600 cm−1 region is attributed to the intermolecular hydrogen 616 bonding and -OH stretching vibrations in BC and PVOH. The addition of PVOH 617 resulted in a slight displacement of this peak at a higher wavenumber, probably due to 618 the increase of -OH groups and the formation of hydrogen bonding. The -NH stretching 619 vibration of chitosan is also located in this region. This increase of the absorbance could 620 also be associated to the interaction with -OH group (Cazón et al., 2018a; Pavaloiu, 621 Stoica-Guzun, Stroescu, Jinga, & Dobre, 2014). A weak signal at 2920 cm−1 appeared 622 in the three samples, due to the -CH stretching. 623 The absorbance peaks observed around 1550 cm⁠−1 were assigned to -NH2 624 deformation vibration of chitosan (Zhao, Teixeira, Gänzle, & Saldaña, 2018). However, 625 FT-IR spectra of pure BC and BC-PVOH films showed a peak of absorbance at 1550 626 cm⁠−1, with lower intensity. Probably, due to the presence of residues from the culture 627 medium that could not be completely removed during the washing process, since films 628 elaborated with regenerated cellulose did not show absorbance at this wavelength 629 (Cazón et al., 2018a). The absorption bands with a maximum at 1055 cm−1 and 1020 630 cm−1 are assigned to the C-O-C pyranose ring stretching vibrations and C-H ring 631 deformation, respectively. In this region, the BC-PVOH bands are shifted in comparison 632 38 to the pure BC spectra, indicating polymeric association through hydrogen bonding 633 (Pereira, de Arruda, & Stefani, 2015). 634 635 3.7. Thermal properties of films TGA-DSC 636 Thermogravimetry and differential scanning calorimetry of the samples from the 637 experiments 1, 3 and 7 were carried out simultaneously. These measurements allowed 638 assessing the stability of pure BC and the effect of adding PVOH and chitosan on the 639 thermal stability of the films. Figure 8 shows the thermograms obtained for each 640 sample. The first endothermic peak (60 - 110)°C corresponded to the volatilization of 641 water from the films samples (Martins et al., 2009). In agreement with the data obtained 642 in the measurement of moisture content in equilibrium, this first peak resulted in a 643 weight loss of 3.4, 3.9 and 5.3%, for the pure BC, BC-PVOH and BC-chitosan films, 644 respectively. 645 646 647 39 648 649 Exp. 1 Exp. 3 Exp. 7 40 Fig. 8. Thermogravimetry and differential scanning calorimetry of experiments 1, 3 and 650 7: 1) Pure bacterial cellulose sample; 3) Bacterial cellulose-poly(vinyl alcohol) 4% 651 (w/w); 7) Bacterial cellulose-chitosan 1% (w/w). 652 653 In Figure 8, the endothermic peak at 250°C corresponded to the onset 654 temperature of thermal degradation of cellulose, in accordance with the data observed in 655 previous works (Mohammadkazemi et al., 2015). At 400 °C the decomposition of the 656 BC corresponded to the 66.7% of the weight loss. The thermogram of the BC-PVOH 657 samples showed two endothermic peaks in the second region. The first endothermic 658 peak, at about 220 °C, corresponds to the thermal degradation of PVA (Bonilla et al., 659 2014). In the 200-240 ºC range, the percentage of the weight loss was 5.2-7.2% of the 660 total initial weight. The loss of weight in this interval was not significant, which 661 indicated that part of PVOH, degraded at higher temperatures probably due to the 662 interaction with cellulose. The second endothermic peak, at 260-340 ºC, corresponded 663 to the 11.1-74.4% weight loss of the initial weight. Data suggest that the BC-PVOH 664 interaction increased the thermal stability of the films, increasing the degradation 665 temperature of the PVOH and the BC. On the other hand, in presence of chitosan, the 666 degradation onset temperature of the sample was found in the 260-270 ºC region. The 667 presence of chitosan increased the degradation temperature of the sample compared to 668 that of pure BC. At the 260-340 ºC region, the weight loss was 12.7-45.0%, reaching a 669 weight loss of 55.1% up to a temperature of 400 ºC, indicating that the interaction BC-670 chitosan increased the thermal stability the sample. Regenerated cellulose-PVOH and 671 41 regenerated cellulose-chitosan also showed this increase in thermal stability due to the 672 interaction between the polymers (Cazón et al., 2018a). 673 674 4. Conclusions 675 Results showed that it is feasible to obtain BC-based films with potential 676 applications as active biopolymer for food packaging. Combining BC with PVOH and 677 chitosan allowed improving or modifying the mechanical, vapour permeability, thermal 678 and optical properties of the films. The equilibrium moisture content had an important 679 effect on the mechanical properties of the films. TS and %E values of the samples 680 increased with the presence of PVOH and chitosan. WVP decreased slightly when 681 PVOH was added into the formulation. Chitosan increased the permeability of the films. 682 PVOH and chitosan decreased the porosity of the BC films and increased the films 683 density. The UV-VIS spectra showed the optimal optical barrier properties of BC-based 684 films against UV-radiation. Adding PVOH increased the transmittance values in the 685 UV-VIS region and improved the transparency and visual appearance of the films. FT-686 IR and TGA-DSC indicated the interaction among the polymers. These properties could 687 be useful in food industry as an alternative to synthetic film preventing the lipid 688 oxidations in foods. 689 690 Acknowledgements 691 A grant from CONACYT (México) (#435948) to author Patricia Cazón is 692 gratefully acknowledged. 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