Journal Pre-proof

Regenerated cellulose films combined with glycerol and polyvinyl alcohol: Effect of
moisture content on the physical properties

Patricia Cazón, Gonzalo Velázquez, Manuel Vázquez

PII: S0268-005X(19)32639-6
DOI: https://doi.org/10.1016/j.foodhyd.2020.105657
Reference: FOOHYD 105657

To appear in: Food Hydrocolloids

Received Date: 8 November 2019

Revised Date: 7 January 2020
Accepted Date: 8 January 2020

Please cite this article as: Cazón, P., Velázquez, G., Vázquez, M., Regenerated cellulose films
combined with glycerol and polyvinyl alcohol: Effect of moisture content on the physical properties, Food
Hydrocolloids (2020), doi: https://doi.org/10.1016/j.foodhyd.2020.105657.

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CRediT author statement

Patricia Cazón: Investigation, Writing- Original draft preparation.

Gonzalo Velazquez: Methodology, Writing- Reviewing and Editing,

Manuel Vazquez: Conceptualization, Methodology, Writing- Reviewing and

Editing.
 1 Regenerated cellulose films combined with glycerol and polyvinyl alcohol: Effect of

2 moisture content on the physical properties

4 Patricia Cazón1,2, Gonzalo Velázquez1, Manuel Vázquez2*,

5
1
6 Instituto Politécnico Nacional. CICATA unidad Querétaro. Cerro Blanco No. 141.

7 Colinas del Cimatario, Querétaro, 76090, México.

2
8 Department of Analytical Chemistry, Faculty of Veterinary, University of Santiago de

9 Compostela, 27002-Lugo, Spain

10

11 *Corresponding author: manuel.vazquez@usc.es; phone +34 982 822 420. ORCID:

12 0000-0002-0392-1724

13

14

1
15 ABSTRACT

16 Regenerated cellulose-based films combined with glycerol and polyvinyl alcohol

17 (PVOH) show interesting UV-light barrier properties, with potential application in food

18 packaging to prevent oxidative deterioration. However, these materials are sensitive to

19 moisture, and their properties could be modified as a function of the relative humidity.

20 Hence, the objective of the present work was to evaluate the changes in the main

21 properties of regenerated cellulose-glycerol-PVOH films depending on the relative

22 humidity. Using the GAB adsorption isotherms, the moisture content was related with

23 the water activity of the films at several relative humidity conditions. According to the

24 obtained results, water molecules manifested a plasticizing effect modifying the

25 mechanical, water vapor permeability and optical properties of the developed films.

26 Tensile strength and Young’s modulus values ranged from 92.65 to 17.57 MPa and

27 from 3639.09 to 227.89 MPa, respectively. Both of them decreased when the moisture

28 content increased. The mechanical resistance to tensile and puncture of films enhanced

29 at high moisture content, changing from 5.88 to 15.97% and from 0.59 to 2.97 mm in

30 the tensile and puncture test, respectively. This effect was also observed for the burst

31 strength. Water vapor permeability increased from 5.15·10-10 to 5.44·10-9 g/ms Pa when

32 the moisture content increased, being more significative at higher values. No significant

33 variations were observed in the UV-VIS transmittance at different moisture contents.

34 The obtained results allow expanding the knowledge of the behavior of films based on

35 regenerated cellulose.

36

37 Keywords: adsorption isotherms; plasticization; regenerated cellulose; water vapour

38 permeability; moisture content

39

2
40 1. Introduction

41 Cellulose is the most abundant natural polymer on earth. Due to its properties

42 such as renewability, availability, non-toxicity, low cost, biocompatibility,

43 biodegradability and chemical stability is gaining attention. Recent studies have focused

44 on developing new biodegradable materials based on cellulose for several fields, such as

45 medicine (Stumpf, Yang, Zhang, & Cao, 2018), food (Patricia Cazon, Velazquez,

46 Ramírez, & Vázquez, 2017) or 3D printing (Dai et al., 2019), among others. The major

47 source of cellulose are plant fibers isolated from wood, cotton, hemp and other plant-

48 based materials. Plants produce over 1.5·1015 kg of cellulose per year, and

49 approximately 2 % of cellulose is used for production of regenerated cellulose fibers,

50 films and cellulose derivate, such as cellulose esters and ethers. In addition, cellulose

51 can also be synthesized by several fungi, bacteria and algae (Xu, Chen, Rosswurm, Yao,

52 & Janaswamy, 2016)

53 Cellulose is an unbranched homopolysaccharide, formed for two

54 anhydroglucose rings ((C6H10O5)n) of β-D-glucopyranoside, linked by (1 4) glucosidic

55 bonds. The pyranose rings are in the chair conformation 4C1, with the hydroxyl groups

56 in an equatorial position (Nisar et al., 2019).

57 Cellulose has a partially crystalline structure, formed by a strong network of

58 intermolecular and intramolecular hydrogen bonds, resulting in an insoluble material in

59 water or other common solvents (Geng et al., 2014). Important property that makes

60 cellulose a very interesting raw material to develop new biomaterials and that differs

61 from the rest of the polysaccharides most extensively studied for this purpose.

62 The insolubility of cellulose limits its utility to develop new materials (Xu et al.,

63 2016). However, the use of natural fibers as reinforcement in polymer composite

64 materials is constantly growing (López-Palestina et al., 2019; Pang, Cao, Cao, Sheb, &

3
65 Wang, 2019). Thus, the most widespread studies with cellulose are based on analyzing

66 its properties as a reinforcing agent, for example to develop new nanomaterials. Various

67 forms of cellulose, mainly cellulose nanocrystals and cellulose nanofibrils, exhibit

68 attractive properties and are potentially useful for a large number of industrial

69 applications such as nano or microfibers, whiskers or other structures (Dufresne, 2017).

70 Cellulose was evaluated as a reinforcing agent to modify the functional properties of

71 other polymer-based films such as polyvinyl alcohol (PVOH) (Cazón, Velazquez, &

72 Vázquez, 2020; Patricia Cazon, Velazquez, & Vázquez, 2019; Ibrahim, El-Zawawy, &

73 Nassar, 2010), tara gum (Ma, Hu, & Wang, 2016), kefiran (Shahabi-Ghahfarrokhi,

74 Khodaiyan, Mousavi, & Yousefi, 2015), chitosan (Rahmi, Lelifajri, Julinawati, &

75 Shabrina, 2017), amylopectin (Anna J., Samir, & Berglund, 2007), starch (Merci,

76 Marim, Urbano, & Mali, 2019), agar (Atef, Rezaei, & Behrooz, 2015), alginate (Huq et

77 al., 2012), κ-carrageenan (Popescu, Dogaru, Sun, Stoleru, & Simionescu, 2019), starch -

78 PVOH (Frone, Nicolae, Gabor, & Panaitescu, 2015) or chitosan-guar gum (Tang,

79 Zhang, Zhao, Guo, & Zhang, 2018), among others.

80 The growing interest and potential of cellulose to develop biodegradable

81 materials for medical applications (Tovar-Carrillo, Tagaya, & Kobayashi, 2013) or food

82 packaging, focused the attention of researchers on developing new methods to dissolve

83 cellulose (Tang et al., 2018). Some solvent systems for cellulose dissolution have been

84 developed, which allow using pure cellulose to obtain films without the need of

85 derivatives (Chen et al., 2015). One of these solvent is the NaOH/Urea aqueous system

86 that allow developing pure regenerated cellulose by a simple and eco-friendly process

87 (Cheng et al., 2019).

88 Pure regenerated cellulose films obtained by alkali/urea method possess some

89 drawbacks, mainly lower elasticity and opacity. Previous works showed a feasible

4
 90 strategy to enhance the film functional properties by the combination of regenerated

91 cellulose with glycerol and PVOH (P Cazon, Vazquez, & Velazquez, 2018; Patricia

92 Cazon et al., 2019). In this way, it was possible to maintain the biodegradability while

93 improving the film properties. Results showed regenerated cellulose films with better

94 mechanical properties by the addition of glycerol-PVOH that eased the handling of the

95 samples. Films with lower water vapour permeability than pure PVOH films and better

96 transparency values than pure cellulose. Cellulose-glycerol showed a very interesting

97 UV protective properties, with prospects application in food packaging to prevent

98 oxidative deterioration of food. In addition, the composite blends showed high adsorbed

99 water capacity, desirable to develop adsorbent pads in contact with food.

100 These materials are sensitive to moisture, and their properties could change with

101 the variations of the relative humidity (RH). Water molecules interacts with the

102 components of the composite blends by hydrogen bonds, resulting in an important

103 plasticizing effect (Ludwiczak & Mucha, 2010). It is possible to analyze the interactions

104 of the composite blends with the environmental moisture in a wide range of RH through

105 sorption isotherms studies. The water sorption isotherm represents the relationship

106 between equilibrium moisture content and water activity (aw) at a given temperature.

107 This phenomenon and the use of mathematical model such as GAB model, are the

108 major tool to describe and predict the water mobility in the films at different

109 environments (Tantala, Rachtanapun, Tongdeesoontorn, Jantanasakulwong, &

110 Rachtanapun, 2019). Previous works have analyzed the sorption isotherms of

111 biodegradable films, such as cellulose whiskers and microfibrils film (Meriçer, Minelli,

112 Giacinti Baschetti, & Lindström, 2017), chitosan-glycerol films (Monte, Moreno,

113 Senna, Arrieche, & Pinto, 2018), carboxymethyl chitosan films from different chitosan

114 sources (shrimp, crab, and squid) and molecular sizes (Tantala et al., 2019), fish gelatin

5
115 with ribose and fructose sugars (Hazaveh, Mohammadi Nafchi, & Abbaspour, 2015) or

116 starch-glycerol (Javier I. Enrione, Sandra E. Hill, & Mitchell, 2007), among others.

117 Most studies are limited to analyze the relationship between aw of the material and the

118 RH. The modification of the mechanical, permeability and optical properties that

119 depend on the moisture conditions have not been extensively studied in this kind of

120 materials (Sothornvit & Krochta, 2005). These properties are normally analyzed at a

121 unique humidity condition (standard conditions).

122 Mechanical properties of the novel developed biomaterials allows evaluating

123 and predicting their behaviour and integrity during processing, transport, handling and

124 storage of packaged foods (Rouhi, Razavi, & Mousavi, 2017). The barrier properties

125 such as water vapor (WVP) or ultraviolet (UV) radiation of a polymeric film are crucial

126 to predict the behaviour of the matrix and the shelf-life of the product when used as a

127 food packaging (Siracusa, Rocculi, Romani, & Rosa, 2008). The UV-radiations are

128 responsible for the activation of chemical reactions, such as lipid oxidation, vitamins

129 oxidations or loss of colour. These reactions result in a loss of the quality of packaged

130 foods (Guo, Ge, Li, Mu, & Li, 2014).

131 In previous reports (P Cazon et al., 2018; Patricia Cazon et al., 2019), it was

132 studied the effect of the composition of regenerated cellulose-glycerol-PVOH films on

133 their properties. Considering the changes of the properties of the materials at different

134 moisture contents, and the importance of predict the behaviour of the material, it is

135 important to extend the studies of the properties of the films under different moisture

136 content or a wide range of aw. Accordingly, the present study continues the investigation

137 initiated in previous reports (P Cazon et al., 2018; Patricia Cazon et al., 2019), in order

138 to extend the evaluation and knowledge of regenerated cellulose-glycerol-PVOH at

139 several environmental moisture contents. Therefore, the objective of this work was to

6
140 analyze the moisture adsorption isotherms and the effect of the moisture content on the

141 mechanical, water vapour permeability and optical properties of regenerated cellulose

142 films combined with glycerol and polyvinyl alcohol.

143

144 2. Materials and Methods

145

146 Microcrystalline extra pure cellulose, Urea (99.5 %), sodium hydroxide (98%),

147 extra pure anhydrous sodium bromide and acetic acid (99.5 %) were purchased from

148 Acros organics (Geel, Belgium). Full-hydrolyzed (>98%) PVOH with average

149 molecular weight of 30,000 g/mol and ester value of 12-25 were supplied by Merck

150 (Billerica, MA, US). Glycerol was purchased from Fisher Scientific (Pittsburgh, PA,

151 US). Silica gel 2.5-6 mm, extra pure lithium chloride, extra pure potassium carbonate,

152 extra pure potassium sulfate, extra pure barium chloride dihydrate, potassium acetate,

153 sodium chloride reagent grade were supplied by Scharlau Microbiology (Barcelona,

154 Spain).

155

156 2.1. Preparation of pure regenerated cellulose films

157 Pure regenerated cellulose films were obtained following the method described

158 in previous studies (Patricia Cazon et al., 2019). The modified NaOH/urea method was

159 used for dissolving cellulose microcrystalline (Zhang, Mao, Zhou, & Cai, 2005). An

160 aqueous solution containing NaOH/urea/H2O (7:14:79, w/w) was used to obtain a

161 solution of cellulose 4% (w/w). The microcrystalline cellulose in the NaOH/urea

162 solution was soaked for 1 h at room temperature under vigorously stirring. Then, a

163 freezing step of the cellulose-solvent solution at -20 °C for 24 h was necessary. After 24

164 h, the frozen mixture was thawed at room temperature with vigorous stirring during 1 h

7
165 to obtain a homogenous and translucent solution of cellulose 4% (w/w). In this step,

166 while maintaining the constant stirring of the mixture, a certain amount of glycerol was

167 added, drop by drop, until a final total glycerol concentration of 5% (w/w) was obtained

168 in the previous cellulose solution. Stirring was kept constant for 1 h to obtain a

169 homogeneous solution of glycerol and cellulose. The insolubilization and films

170 formation was obtained by immersion in an acid acetic bath. Cellulose films with

171 glycerol from the mixture solution was obtained by the pH decrease. The regenerated

172 cellulose films with glycerol were washed with distilled water until pH = 7. The

173 excessive water of the films was removed using filter paper. The resulting films were

174 dipped in a bath with a solution of PVOH (4% w/w) for 1 h. Finally, the wet

175 regenerated cellulose films with glycerol and PVOH films were formed in a Petri dish

176 and dried at room temperature for 48 h.

177 The dried films were peeled and cut to specific sizes for each test. The thickness of

178 each sample was measured at 5 random points of the sample, using a thickness meter

179 ET115S (Etari GmbH, Stuttgart, Germany). Finally, the samples were stored in

180 desiccators with the corresponding saturated salt or silica gel for 10 days. Time enough

181 to ensure that the samples have reached the moisture equilibrium before each test.

182

183 2.2. Moisture adsorption isotherms

184 The standard static gravimetric method developed by the European Cooperation

185 Project COST 90 (Wolf, Spiess, & Jung, 1985) was established to obtain the moisture

186 adsorption isotherms of regenerated cellulose-glycerol-PVOH films. The first step was

187 to calculate the moisture content of the samples conditioned in a microclimate at a

188 certain equilibrium relative humidity (RHeq). To recreate these microclimates, 6 airtight

189 containers were used. Each airtight container had a specific saturated saline solution of

8
190 LiCl, MgCl2, K2CO3, NaBr, NaCl and BaCl2 to obtain RHeq of 11.5, 32.7, 43.8, 57.7,

191 75.4 and 90.6%, respectively. In each container, 3 film samples of 3 ×3 cm2 were placed

192 to reach an equilibrium moisture content. The samples in the containers were stored in a

193 chamber at 30 °C for 10 day to reach the equilibrium.

194 Once the moisture equilibrium was reached, the samples were weighed (wet

195 weight) using a precision balance to the nearest 1·10-4 g. Then, the samples were dried

196 at 105 ˚C in a vacuum oven for 24 h until constant weight (dry weight). From these

197 data, the equilibrium moisture content (X) was calculated using the Equation 1:

198

199 Equilibrium moisture content X =
× 100 (Eq. 1)

200

201 The second step was to calculate the adsorption isotherm from the obtained

202 moisture content data using the Guggenheim-Anderson-De Boer (GAB) model.

203 Equation 2 of the GAB model was used to interpolate and plot the isothermal adsorption

204 data (Wolf et al., 1985).

205
#$·&·'·()
206 "= * '·() · * '·()+&·'·()
(Eq.2)

207

208 where Xm is the moisture content in the monolayer, C is a constant related to the

209 strength of water bound to primary adsorption sites, and K is related to the heat of

210 multilayer sorption (Bedane, Eić, Farmahini-Farahani, & Xiao, 2015). GAB-constants

211 (Xm, C and K) were determined by non-linear regression.

9
212

213 2.3. Water vapour permeability

214 The permeability properties of the films as a function of the moisture content

215 was evaluated following the method reported elsewhere (Aguirre-Loredo, Rodríguez-

216 Hernández, Morales-Sánchez, Gómez-Aldapa, & Velazquez, 2016). To determine the

217 WVP at different moisture contents a permeability chamber and cell were designed

218 allowing recreating microclimates with different RHeq inside and outside of the film.

219 The first microclimate (RHeq inside) was recreated using a saturated saline solution or

220 distilled water (11.5 - 100% RHeq) inside a wide-mouth cup. The cup with an area of

221 2.20610-3 m2 was sealed with a film sample. The second microclimate (RHeq outside)

222 was recreated using saline solutions or silica gel in a double wall chamber specially

223 designed for this test (Aguirre-Loredo et al., 2016). The aw of the samples for each test

224 was calculated following the equation 3.

225

-./01 234256∓./01 89:4256-

226 ,) = (Eq. 3)
; · *<<

227

228 Therefore, by combining different saline solutions into the chamber and the

229 wide mouth cup it was possible to study the WVP over a wide range of aw. The standard

230 gravimetric test method E96 (“Standard Test Methods for Water Vapor Transmission of

231 Materials,” 16AD) was followed to calculated the WVP in each region. The chamber

232 was placed on a precision scale, leaving the balance plate free in the center. The cup,

233 sealed with the sample, was placed on the plate. The partial pressure gradient between

234 both sides of the sample produced a driving force for the flow of water vapour through

235 the film. Water vapour flow resulted in a decrease or increase in cup weight depending

236 on the configuration tested. The temperature was controlled at 30 °C and the weight was

10
237 followed for at least 8 h and recorded every minute at the nearest 1·10-4 g. The WVP

238 values were calculated as indicated in previous works (Gennadios, Weller, & Gooding,

239 1994). Each test was performed by triplicate.

240

241 2.4. Mechanical properties

242 The mechanical properties were analyzed at different conditions of RHeq, and

243 therefore, according to the moisture content of the samples. Samples were cut at 15 ×

244 100 mm for tensile test and 30 × 30 mm for puncture test. The samples were

245 conditioned in airtight containers with a certain RHeq between 0 and 90.6% for 10 days

246 at 30 °C. A texturometer (TA-XTplus, Stable Micro System, United Kingdom) was

247 used to perform the mechanical tests. The tensile test was carried out following the

248 standard method ASTM D-882 (“Standard Test Method for Tensile Properties of Thin

249 Plastic Sheeting,” 18AD) at a constant crosshead speed of 0.08 mm/s, with an initial

250 separation of the grips of 40 mm. Load-distance data were recorded during the

251 deformation of the sample and used to calculate the tensile strength (TS), the percentage

252 of elongation at break (%E). The Young modulus (YM) was calculated from the stress-

253 strain data as indicated in previous studies (P Cazon et al., 2018; Patricia Cazon et al.,

254 2019).

255 The puncture test was carried out fixing the samples on a film support platform

256 (HDP/FSR). The samples were perforated by a cylindrical probe (d = 3 mm) at a

257 constant crosshead speed of 1 mm/s until rupture. The burst strength and distance to

258 burst were calculated automatically from the force (g) – deformation (mm) curves.

11
259

260 2.5. Light barrier properties, color, transparency and opacity

261 The optical properties of the samples were analyzed at different moisture content

262 using a spectrophotometer V-670 (Jasco Inc, Japan). The UV-VIS spectra (190 nm–800

263 nm) was used to analyze the samples in each region in transmittance mode at 2 nm. The

264 test was carried out in duplicate for each sample. The color was determined using the

265 software Spectra Manager (Jasco Inc, Japan) set at 2º standard observer with light

266 source of D65. The color matching function selected was JIS Z8701-1999 with data

267 pitch of 5 nm to obtain the CIE L* a* b* coordinates. The transparency and opacity of

268 the films were calculated from the data of VIS spectra following the Equation 4 and 5

269 (Han & Floros, 1997; Kanatt, Rao, Chawla, & Sharma, 2012).

270

EF % HIJJ
271 =>,?@A,>B?CD = K
(Eq. 4)

OP4QJJ
272 LA,CMND = K
(Eq. 5)

273

274 Where %T600 is the percent transmittance at 600 nm, x is the film thickness

275 (mm) and Abs500 is the absorbance at 500 nm.

276

277 2.6. Statistical analysis

278 The analysis of variance (ANOVA) was used to assess the effect of the relative

279 humidity condition on the properties of the films. Non-linear regression (Microsoft

280 Excel® 2010) was used to fit the GAB model to the adsorption isotherm. The mean

281 comparison was performed by Tukey’s test at p < 0.05. Analyses were performed using

282 Statistica 7.0 (StatSoft Inc.).

12
283

284 3. Results and Discussion

285 A film previously studied (P Cazon et al., 2018; Patricia Cazon et al., 2019) with

286 good elasticity and resistance to rupture, good UV-barrier properties, water adsorption

287 and appearance properties was selected to evaluate the plasticizing effect of the

288 adsorbed water molecules on its properties. Therefore, the present work evaluated the

289 results obtained from films based on a solution of glycerol 5% (w/w) and PVOH 5%

290 (w/w).

291

292 3.1. Moisture adsorption isotherms

293 The moisture adsorption isotherms of the films based on regenerated cellulose-

294 glycerol-PVOH were analyzed. The developed composites can interact with the

295 surrounding moisture due to their hydrophilic nature. The hydrophilic material gain or

296 lose moisture to reach the equilibrium with the environment. When the material reaches

297 the equilibrium moisture, the aw of the material sample becomes equivalent to RH of the

298 surrounding environment divided by 100 (Figura & Teixeira, 2007). The adsorption

299 isotherm of the developed material was obtained by the difference in weight between

300 the dry sample and the sample in equilibrium. Hence, it was possible to relate the

301 moisture content of each sample according to the aw by the adsorption isotherm (Figura

302 & Teixeira, 2007; Lewicki, 1997), as shown in Figure 1. Results showed that the

303 maximum moisture content of the samples was 20.80% at aw = 0.90. It was slightly

304 higher than the 14% of moisture content reported for pure regenerated cellulose from

305 microcrystalline cellulose measured at 25 ºC (Bedane et al., 2015). The difference could

306 be due to the sum of several factors. Mainly, due to the presence of glycerol and PVOH

307 that produced an increase of hydroxyl groups with great affinity for the water molecules

13
308 (Liu et al., 2018; Rachtanapun & Tongdeesoontorn, 2009). Previous study of adsorption

309 isotherms of chitosan-glycerol (Monte et al., 2018), starch-glycerol mixtures (Javier I.

310 Enrione et al., 2007) or microfibritalled cellulose-glycerol (Minelli et al., 2010)

311 observed that the presence of glycerol resulted in a significant increase of the moisture

312 content of the films at high aw values. Other reason could be difference in the

313 temperature at which the adsorption isotherms were obtained. The adsorption capacities

314 of cellulose films increase when the temperature increases, especially at low RH ranges

315 (Bedane et al., 2015). Finally, the method to obtain the regenerated cellulose films was

316 different, which could have an important effect on the structure of the film modifying

317 the adsorption properties.

318 The water molecules in the matrix of the film are in different states or are

319 interacting in a different way with the polymer chains. In the polymer matrix, there is

320 water that is strongly bound to active groups. This water nor freeze neither forms a

321 monolayer. Other water molecules appear in layers, in which the particles are not

322 completely free as they form the so-called partially bound water. Finally, there is

323 completely free water molecules, filling the pores of the capillary and voids in the

324 structure (Ludwiczak & Mucha, 2010). The shape of the isotherm obtained by the GAB

325 model and the GAB parameters could indicate the state of water molecules and the

326 interactions in the films (Srinivasa, Ramesh, Kumar, & Tharanathan, 2003).

327 Experimental adsorption data were well adjusted using the GAB equation (r2>

328 0.99). According to the Brunauer classification, the sigmoidal moisture adsorption

329 isotherm of regenerated cellulose-glycerol-PVOH films could be classified as type II

330 (Hazaveh et al., 2015). Type II is the most widespread sorption isotherm type in

331 materials of this nature, such as carboxymethyl chitosan (Tantala et al., 2019),

332 microfibrillated cellulose (Meriçer et al., 2017), carboxymethyl cellulose (Rachtanapun

14
333 & Tongdeesoontorn, 2009), chitosan (Endres & Weichold, 2019), chitosan-glycerol

334 (Monte et al., 2018), gelatin (Hazaveh et al., 2015), starch (Y. Zhang & Han, 2008). The

335 values of the GAB parameter were Xm=4.16, C=19.91 and K=0.89. Xm indicates the

336 maximum amount of water molecules that could be adsorbed in a single layer per gram

337 of dry film expressed as percentage. It is a parameter related to the number of

338 adsorption sites (Tantala et al., 2019). Previous studies reported slightly higher Xm

339 values for regenerated cellulose films from different resources (6.31-7.16 %) (Bedane et

340 al., 2015). These differences may be due to several factors. First, the interactions

341 between cellulose-glycerol-PVOH could decrease the number of available sites on the

342 surface of the film to interact with water molecules, compared to pure cellulose films.

343 Then, the presence of PVOH decreases the roughness and porosity of the surface of the

344 film, resulting in a decrease in the surface area and limiting the amount of water on

345 monolayer (Cazón et al., 2018). Finally, the methods to obtain regenerated cellulose

346 films and the different sources could affect the structure of the film, modifying its

347 properties (Minelli et al., 2010).

348 In the adsorption isotherm curve (Figure 1), 3 regions can be identified. In the

349 first region, aw = 0.17-0.20 the isotherm showed a convex shape. This initial stage

350 corresponds to the adsorption of water in the monolayer. This water is linked to the

351 binding sites (polar regions) on the surface of the film, due to the hydroxyl groups

352 present mainly in the PVOH and glycerol (P Cazon et al., 2018). In this region, the

353 moisture content reached the value of the Xm. In the following region, aw = 0.20-0.38, a

354 practically linear increase of moisture content is observed with the increase in water

355 activity. In this region the adsorption of water molecules takes place in multilayers; it is

356 water that is partially bound. In the last region, aw > 0.38, a significant increase in the

357 moisture content of the sample was observed with increasing aw. This steep increase

15
358 indicates the presence of free or condensed water filling the pores of the matrix

359 structure. In this region, swelling of the film occurs as the free volume increases due to

360 the presence of free water molecules inside the pores (Sothornvit & Krochta, 2005). The

361 moisture adsorption isotherm obtained was similar to that of other isotherms of edible

362 biopolymers with hydrophilic properties (Torres, Moreira, Chenlo, & Vázquez, 2012).

16
363

364 3.2. Water vapour permeability

365 The results of the water vapour permeability (WVP) can be useful for

366 understanding the possible mass transfer mechanisms, the interactions between water

367 molecules and polymers and the possible reorganization of the polymer matrix in the

368 biodegradable films (Bertuzzi, Castro Vidaurre, Armada, & Gottifredi, 2007). For this

369 reason, the water vapor permeability of the regenerated cellulose-glycerol-PVOH films

370 was measured at 30 °C in a wide range of aw, as shown in Figure 2. The results allowed

371 analyzing the behavior of the permeability properties of the developed films depending

372 on the environmental surrounding conditions. WVP is a constant of proportionality that

373 should be independent of the water vapor pressure gradient applied through the films.

374 However, in the case of hydrophilic materials, such as cellulose films, PVOH, starch,

375 chitosan, among others, their polar groups interact with water molecules of the

376 environmental moisture, resulting in important structural changes (Aguirre-Loredo et

377 al., 2016). These structural changes could modify the diffusion of water molecules

378 through the film matrix, producing variations of WVP. The measured WVP values of

379 the samples ranged from 5.15·10-10 to 5.44·10-9 g/m·s·Pa. Figure 2 shows that the WVP

380 increased as the RHeq increased, and consequently by increasing the moisture content of

381 the sample. Therefore, the permeability of the regenerated cellulose-glycerol-PVOH

382 films depended on the moisture content of the samples and on the environmental

383 moisture conditions.

384 The permeability values remained low until aw = 0.43. Similar behavior was

385 observed in regenerated cellulose films from several sources, keeping low permeability

386 values up to 60% RH (Bedane et al., 2015). In this region, the moisture adsorption on

387 the monolayer was completed, and the adsorption of the water molecules started taking

17
388 place on the multilayer (Figure 2). By increasing the RHeq of the samples, at aw higher

389 than 0.43, the WVP increased exponentially, enduring a significant increase as the RHeq

390 increased. At aw = 0.56 samples showed a WVP value of 1.65·10-9 g/m·s·Pa, reaching a

391 WVP value of 5.44·10-9 g/m·s·Pa at aw = 0.88. At these aw value the moisture content of

392 the samples was 7.90 and 18.91% on dry basis, respectively. In this region, the

393 multilayers began to saturate with water molecules slightly bound to the polymer

394 chains. As RHeq increased, free water could have filled the pores and voids in the

395 polymeric matrix, responsible for capillary condensation (Figure 2). The water adsorbed

396 on the multilayers of the polymer matrix had a plasticizing effect causing a swelling of

397 the polymer matrix and resulting in an increase of the free volume in the material

398 structure (Bertuzzi, Armada, & Gottifredi, 2007). The crystalline regions prevented

399 further swelling of the material (Aguirre-Loredo et al., 2016). The increase of the free

400 volume in the matrix resulted in a reorganization of the polymer network, losing the

401 packed structure and promoting the polymer chain mobility (Bedane et al., 2015). For

402 this reason, great mobility of the segments and high free volume resulted in a less

403 tortuous path, facilitating the diffusion of water vapour molecules through the

404 polymeric structure. Hence, the plasticizing effect of water increases the WVP values

405 (Sothornvit & Krochta, 2005), being more significant at high moisture content.

406 Similar behavior was observed in other type of polysaccharide films such as

407 micro fibrillated cellulose (Minelli et al., 2010), regenerated cellulose (Bedane et al.,

408 2015), cellophane (Del Nobile, Fava, & Piergiovanni, 2002) or chitosan (Aguirre-

409 Loredo et al., 2016).

410

18
411

412 3.3. Mechanical properties

413 The mechanical properties of the films previously conditioned in a wide range of

414 RH environments were measured. The adsorption isotherm, which relates the moisture

415 content with the RHeq, was used to evaluate the effect of moisture content on

416 mechanical properties. Results showed that the mechanical behavior of the films

417 depends strongly on the moisture content or on the RHeq of the surrounding

418 environment (p <0.05).

419 Figure 3 shows the results of the tensile properties of the films as a function of

420 the aw. Results showed values for TS ranged from 92.65 to 17.57 MPa, for %E ranged

421 from 5.88 to 19.85% and for YM ranged from 3639.09 to 227.89 MPa. The resistance to

422 rupture of the films decreased continuously with the increase of the aw (Figure 3a).

423 Although the decrease was continuous, 3 slopes were observed. In the first range (aw

424 0.11-0.32), TS values diminished slightly up to 76.31 MPa for films with a moisture

425 content of 5.61%.

426 In this region, the water molecules were adsorbed on the monolayer. The

427 moisture adsorbed on the monolayer did not have a significant effect on the resistance

428 to rupture of the samples. As aw increased up to the next region (0.43 < aw <0.56), there

429 was the first significant change in TS values. Thus, the initial significant modifications

430 took place when water molecules were absorbed at multilayer level. The water adsorbed

431 at this level began to manifest the initial plasticizing effects, forcing the reorganization

432 of the polymer chains (Sothornvit & Krochta, 2005). Along the second region, the TS

433 values remained slightly similar. In the last section, at high aw values, there was a

434 significant decrease of TS values. The TS values reached the lower value of 17.57 MPa,

435 decreasing TS more than 5 folds from the initial value, probably due to the capillary

19
436 condensation of water molecules in this region. The trend observed in the TS was

437 similar to that of YM, concurring the sections with the most significant modifications.

438 Until 0.32 aw, YM values diminished up to 2646.81 MPa, following by a slightly

439 decrease until 2311.14 MPa at 0.56 aw and observing a drastic decrease in the last

440 section, up to 227.8 MPa.

441 In the case of the elastic properties, %E values increased slightly by increasing

442 aw up to 0.43, from 5.88 to 8.13%, due the low plasticizing effect of water observed at

443 the monolayer level. However, the water adsorbed at multilayer level manifested a

444 significant plasticizing effect, reaching a %E value of 18.61%. The %E remained

445 slightly similar up to 0.75 aw. Nevertheless, at a maximum moisture content, the

446 elongation of the films was reduced to 10.76%. As observed in the resistance to rupture,

447 the condensed water considerably decreases the resistance to rupture of the films. At

448 this point, the samples were overplasticized, resulting in a decrease of the elongation

449 because of the weakness of film structure (Y. Zhang & Han, 2008).

450 The puncture test results showed BS values ranging from 525.47 to 3420.18 g

451 and elasticity values of DB ranged between 0.59 and 3.97 mm (Figure 4). The most

452 significant changes in puncture properties were observed in aw regions where the water

453 molecules either were adsorbed at multilayers or were free, filling the porous of the

454 film. The puncture results were in accordance with those observed in the tensile

455 properties. The BS and DB increased until aw = 0.75 reaching a value of 3420.18 g and

456 3.93 mm, respectively. In this case, the water molecules enhanced the resistance of the

457 films subjected to axial deformation, acting as a lubricant of the polymeric chains.

458 Water molecules eased the rearrangement of the polymer chains, reducing the

459 brittleness and enhancing the resistance to rupture. However, a weakening of the

460 structure was observed due the high moisture content at aw higher than 0.75, decreasing

20
461 the BS values but keeping constant the DB (3.97 mm). In this way, the films kept high

462 elasticity values although the resistance to rupture decreased.

463 Therefore, the data showed that water molecules manifested a plasticizing effect

464 on regenerated cellulose-glycerol-PVOH films, improving the mobility of the polymer

465 in the chains, decreasing TS and YM values as well as enhancing the elasticity

466 properties of the samples (E and DB). Previous works observed the plasticizing effect of

467 water molecules into the matrix of hydrophilic materials, decreasing the resistance to

468 rupture meanwhile increasing the elongation of films (Aguirre-Loredo et al., 2016).

469

470 3.4. Light barrier properties, color, transparency and opacity

471 The aw of the samples and the plasticizing effect of the water molecules affected

472 the optical properties of the composite blends. The transmittance properties depended

473 on the moisture content, which modified the UV-barrier properties, the color,

474 transparency and opacity of the films. Figure 5 shows the percentage of transmittance in

475 the UV-VIS region of each sample previously equilibrated at certain RHeq. The average

476 percentage of transmittance was calculated for each sample in the UVA, UVB and UVC

477 regions (Table 1) as well as the color, transparency and opacity values (Table 2).

478 As shown in Figure 5 and according to the data in Table 1, the variations of

479 transmittance values within each UV region were not drastically affected by the

480 moisture content. Transmittance values were in the 5.36-13.25, 4.25-10.02 and 2.09-

481 4.82 % range for the UVA, UVB and UVC regions, respectively. The values for each

482 water activity showed statistically significant differences. At low aw up to 0.575, the

483 transmittance properties of the samples increased slightly when the aw increased.

484 However, the trend changed at higher aw, decreasing to reach similar initial

485 values. Probably, the structural modifications due to the adsorption of water molecules

21
486 at the multilayer level resulted in a slight increase of the transmittance values (Bertuzzi,

487 Castro Vidaurre, et al., 2007), followed by a decrease of the transmittance at high aw

488 values, probably as a result of the condensed water into the voids of the films,

489 promoting an increase of the thickness and the optical properties of the water itself

490 (Litjens, Quickenden, & Freeman, 1999).

491 Regard the transparency and opacity, no significant variations were observed as

492 a function of the aw. Transparency values ranged between 11.30 and 18.67 meanwhile

493 opacity values ranged from 10.18 to 14.37. The color parameters L*, a* and b*, showed

494 similar tendency than the transmittance in the UV region (Table 2). Lightness, redness

495 and yellowish parameters increased up to 0.575 aw, followed by a decrease until

496 reaching similar values than the initial values. Values of a* parameters at aw of 0, 0.115

497 and 0.745 did not show statistically significant differences. Values of b* parameters at

498 aw of 0.745 and 0.905 did not show statistically significant differences. Results

499 indicated that the films developed have optima UV-barrier properties regardless of the

500 aw.

501

502 4. Conclusions

503 Results suggest that water molecules acted as a plasticizer on regenerated

504 cellulose-glycerol-PVOH films. The plasticizing effect resulted in a significant increase

505 in elongation properties analyzed in both tensile test and puncture tests. The change in

506 elongation was most noticeable at high aw values up to 0.75. Above 0.75 an

507 overplastification effect was observed, decreasing the elongation by weakening the

508 structure of the matrix. In the case of puncture resistance, the presence of water

509 molecules into the matrix facilitated the rearrangement of polymer chains, providing

510 greater resistance to rupture.

22
511 The rearrangement of the chain in the internal structure of the film was reflected

512 in an increasing of the permeability values at high moisture content. The excellent

513 barrier properties against UV radiation were not affected by the variations of the

514 moisture content.

515 The obtained results allow expanding the knowledge of the behavior of films

516 based on regenerated cellulose. This work is expected to be useful to predict the

517 suitability of regenerated cellulose-glycerol-PVOH composites depending on the

518 environmental conditions of the application.

519

520 Acknowledgements

521 A grant from CONACYT (Mexico) to author Patricia Cazón (#435948) is

522 gratefully acknowledged.

523

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729

31
730 Tables

Table 1. Values of the average percent transmittance UV-A (320−400 nm), UV-B

(280−320 nm) and UV-C (190-280 nm) light radiation and thickness of the films related

to the water activity of the samples. %T is value of average percent.

Water activity UV-A UV-B UV-C Thickness

%T %T %T (mm)

0.000 6.90±0.02b 5.45±0.01b 2.82±0.00b 0.082±0.007a

0.115 8.75±0.09d 6.58±0.10d 3.11±0.07c 0.075±0.017a

0.327 10.87±0.05e 8.38±0.01e 4.18±0.02e 0.081±0.009a

0.577 13.25±0.03f 10.02±0.02f 4.82±0.01f 0.071±0.008a

0.745 7.75±0.02c 6.28±0.02c 3.32±0.01d 0.077±0.006a

0.905 5.36±0.07a 4.24±0.05a 2.09±0.02a 0.075±0.010a

731 Different letters (a-f) in the same row indicate significant differences (P < 0.05).

732

32
 Table 2. Color, transparency and opacity properties of the samples related to its water

activity conditions. L*, a* and b* are the CIE parameters.

Water
L* a* b* Transparency Opacity
activity

37.52±0.06 0.68±0.01 4.04±0.02

b ab b
0.000 12.42 12.72

42.38±0.03 0.68±0.01 4.32±0.02

d ab c
0.115 14.98 12.29

0.327 47.06±0.01e 0.81±0.01

c
5.22±0.07
d
15.13 10.18

0.577 51.85±0.03f 0.90±0.01d 5.85±0.07e 18.67 10.33

0.745 39.18±0.05c 0.65±0.01a 3.92±0.01ab 13.70 13.02

0.905 33.31±0.02a 0.70±0.01b 3.87±0.01a 11.38 14.37

733 Different letters (a-f) in the same row indicate significant differences (P < 0.05).

734

33
735 Legend figures

736 Fig. 1. Water sorption isotherm of regenerated-glycerol-PVOH films analyzed at

737 30 °C. The solid line shows the fit obtained with the GAB model. X is the equilibrium

738 moisture content of the samples.

739 Fig. 2. Water vapour permeability of regenerated-glycerol-PVOH films analyzed

740 at 30 °C related to water activity (aw). The solid line shows the isotherm obtained with

741 the GAB model. X is the equilibrium moisture content of the samples.

742 Fig. 3. Mechanical properties of regenerated-glycerol-PVOH films analyzed at

743 30 °C as a function of water activity (aw). A) Tensile strength values at several aw. B)

744 Percentage of elongation values at several aw. C) Young’s modulus values at several aw.

745 Fig. 4. Puncture properties of regenerated-glycerol-PVOH films analyzed at 30

746 °C as a function of water activity (aw). A) Burst strength at several aw. B) Distance to

747 burst at several aw.

748 Fig. 5. UV-VIS spectra profile of regenerated-glycerol-PVOH films at 30 °C

749 evaluated at several water activity (aw).

750

34
751

752 Fig. 1

753

35
754

755 Fig. 2

756

36
757

758 Fig. 3

759

37
760

761 Fig. 4

762

38
763

764 Fig. 5

765

39
Highlights

• Properties of regenerated cellulose-glycerol-PVOH films depends on the

relative humidity.

• Water molecules showed a plasticizing effect modifying the mechanical

properties

• Water vapor permeability increased with the moisture content

• No significant variations with the moisture were observed in the UV-VIS

transmittance
 November 8nd, 2019

Manuel Vázquez, as the corresponding author of the manuscript Regenerated cellulose

films combined with glycerol and polyvinyl alcohol: Effect of moisture content on

the functional properties with author Patricia Cazón, Gonzalo Velazquez, Manuel

Vázquez, which we would like to be considered for publication in “Food Hydrocolloids”.

DECLARE

No potential conflict of interest is reported by the authors.

Sincerely,

Dr. Manuel Vázquez

Area of Food Science and Technology
University of Santiago de Compostela - Campus Lugo
Faculty of Veterinary Science
27002 Lugo
SPAIN
E-mail: manuel.vazquez@usc.es