Composite Films with UV-Barrier Properties Based on Bacterial

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Composite Films with UV-Barrier Properties Based on Bacterial Cellulose Combined with Chitosan and Polyvinyl Alcohol: Study of Puncture and Water Interaction Properties Patricia Cazón, Manuel Vazquez, and Gonzalo Velazquez Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00317 • Publication Date (Web): 29 Mar 2019 Downloaded from http://pubs.acs.org on March 30, 2019

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Composite Films with UV-Barrier Properties Based on Bacterial Cellulose Combined with Chitosan and Polyvinyl Alcohol: Study of Puncture and Water Interaction Properties

Patricia Cazón1,2, Manuel Vázquez2*, Gonzalo Velazquez1*

1Instituto

Politécnico Nacional. CICATA unidad Querétaro. Cerro Blanco No. 141. Colinas del Cimatario, Querétaro, 76090, México. 2Department

of Analytical Chemistry, Faculty of Veterinary, University of Santiago de Compostela, 27002-Lugo, Spain *Corresponding author: [email protected] (ORCID iD 00000002-0392-1724; [email protected]; phone +52 (442)-229-0804 Ext 81058.

ABSTRACT The present study describes the preparation and characterization of composite films from bacterial cellulose produced by Komagataeibacter xylinus combined with polyvinyl alcohol and chitosan. The unique bacterial cellulose structure provides an expanded surface area with high porosity, easing the combination with other soluble polymers by dipping. This blending method effectively reinforces the bacterial cellulose structure. Toughness, puncture strength, water solubility and swelling degree were measured to assess the effect of polyvinyl alcohol and chitosan on the analyzed 1 ACS Paragon Plus Environment

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properties. The morphology, optical and thermal properties were evaluated by scanning electron microscopy, UV-VIS spectral analysis, thermogravimetry and differential scanning calorimetry, respectively. Results showed that the films have good UV-barrier properties and higher thermal stability. Toughness values ranged from 0.26 to 7.18 MJ/m3, burst strength ranged from 58.88 to 3234.62 g and distance to burst ranged from 0.39 to 3.24 mm. Polyvinyl alcohol affected the water solubility and increased the swelling degree.

Keywords: Gluconoacetobacter xylinus, Acetobacter xylinus, toughness, burst strength, puncture strength, puncture force, deformation, water solubility, water retention DSC, SEM, UV protection.

INTRODUCTION Synthetic plastics have become increasingly dominant in the consumer marketplace since the mass production began in the 1940s. In 2012, the global plastic production reached 288 million metric tons, a 620% increase since 1975. The high growth is due to the suitable plastic properties for several applications, mainly, as packaging material which is nearly a third of the plastic production. Synthetic plastics have excellent mechanical properties suitable to keep the integrity of the packaging during handling, transport and storage. Besides, they own good transparency values, low cost and excellent oxygen/moisture barrier properties. Nevertheless, synthetic plastic has the drawback that it is not biodegradable. Thus, the complex waste management of the plastic packaging has led to serious current environmental problems 1,2. A recent study estimated that 275 million metric tons of plastic waste were generated in 192 coastal countries in 2010, with 4.8 to 12.7 million metric tons entering the ocean. The authors predicted that without an improved waste management system, the plastic waste available to pollute the ocean will be increased by an order of magnitude by 2025 1. In addition, there is a growing environmental concern about MICROPLASTICS. Degradation of the plastic waste in the environment causes the reduction of its size, turning it into small fragments of plastic. Due to their small size, marine invertebrates can ingest them throughout the foodchain. The introduction of toxins like microplastic to the base of the food chain possess a potential health risk to humans 1–3. To reduce the environmental problem derived from nonbiodegradable plastic, in recent years, research efforts has 2 ACS Paragon Plus Environment

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been focused in the developed, characterization and application of novel biopolymers from natural origin as an alternative to synthetic polymers intended for food packaging. The most relevant properties of these materials are their biodegradability and non-toxicity. These materials can be decomposed by the enzymatic action of bacteria, yeasts or fungi, preventing the accumulation in the environment 4. However, films based on biopolymers usually have some restrictions for certain applications, mainly due to the limited mechanical properties, high water vapour permeability or visual appearance compared to commercial synthetic polymers. Blending different biopolymers, plasticizers or natural extracts may improve or modify the properties of the biodegradable films expanding the potential applications and overcoming some limitations. Cellulose is the most abundant polymer on earth, making it an interesting raw material for development of new composites. It is the main structural component of plant cell wall, being wood the major resources for all cellulose products. Cellulose is also synthesized by tunicates and different microbes, such as algae, fungi and several aerobic non-pathogenic bacteria of the genera Agrobacterium, Sarcina, Rhizobium and Glucoacetobacter (formerly Acetobacter) 5. Komagataeibacter xylinus, initially named as Acetobacter xylinum and most widespread as Gluconoacetobacter xylinus 6,7 produces high amounts of cellulose, being one of the most commonly studied sources of bacterial cellulose (BC) 8,9. Unlike cellulose from vegetal sources, BC is produced in the pure form, devoid lignin and hemicelluloses. In addition, the BC has unique structural properties due to its nanoscale three-dimensional network structure, allowing to obtain porous films with high surface area. Accordingly, the crystallinity and mechanical strength of BC films are higher than those of vegetable cellulose 10–12. The most extensive studied applications for BC films are in the biomedical field. The suitability of BC has been evaluated as a biomaterial for artificial skin, artificial blood vessels, vascular grafts, scaffolds for tissue engineering, wound dressing, controlled drug delivery or dental implants 5,12. However, BC lacks certain properties, which limits its applications for food packaging. The preparation of BC composites incorporating other polymers or plasticizers, such as polyvinyl alcohol (PVOH) and chitosan, could improve the film properties and address these limitations. Besides, developed regenerated cellulose-based films manifested UV-light barrier properties 13. This UVbarrier properties was reinforced by the presence of chitosan 3 ACS Paragon Plus Environment


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in cellulose films. The development of films with UV-light barrier properties has a great interest and potential application in food to prevent or retard the oxidations of lipids, proteins or vitamins due the UV-light radiation. These oxidations generate undesirable flavors, color and odors and the loss of nutritional properties in food products that affect their quality and shelf-life 14. PVOH is a semicrystalline polymer, biodegradable with good optical and physical properties. Due to its properties, PVOH is extensively studied in combination with other biopolymers, such as gelatin 15, chitosan 16–18 and starch 19, among others. The addition of PVOH results in an improvement of the mechanical properties of the composites. PVOH is suitable for combining with cellulose to increase the physical, thermal and optical properties due to its high polar character, good mechanical properties and easy processability. Furthermore, the water solubility of PVOH allows the combination with BC by dipping bath. This is a viable strategy to reinforce the matrix while preserving the BC structure and improving the film properties 20,21. Chitosan is the second most abundant polysaccharide obtained from waste of the shell-fish industry. Due to its forming-film capacity, antimicrobial properties and availability, chitosan has received increased attention for commercial applications in the biomedical, food, and chemical industries 4,22,23. One of the most interesting properties of chitosan is its antimicrobial activity against a wide range of filamentous fungi, yeast, and bacteria, with potential applications in food packaging 24,25. Antimicrobial agents from natural sources are an alternative to chemicallysynthesized antimicrobial agents to increase shelf life and quality of foodstuff. Chitosan allows inhibiting the growth of microorganisms on the surface of the food in contact with the package or coating 26,27. Chitosan is soluble in slightly acidic solutions (pH below 6.5) 28. The compatibility between PVOH and chitosan has been reported 17,18,29. Hence, chitosan is suitable to combine with BC by dipping bath to develop BC films with antimicrobial properties. Freeze-dried BC–chitosan membranes has been studied Results showed that chitosan may penetrate into the BC pores and interact with the microfibrils, affecting the physico-chemical properties of the films. PVOH films reinforced with BC nanofibrils have also been reported 32,33. Results showed that the presence of BC changed the color and swelling degree of the composite films 33. The composite BCPVOH-chitosan film was analyzed as a drug-controlled releasing material for sodium salt of ibuprofen 34. However, the composite BC-chitosan-PVOH has not been characterized as biodegradable film yet aimed for applications in food 30,31.

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industry. In a previous study, the interaction of regenerated vegetable cellulose-PVA in presence of chitosan was analyzed. The author observed that the interaction between PVA-chitosan modified the properties of regenerated vegetable cellulose films with potential application as active food packaging 13,35. Considering the differences in the structure between BC and regenerated cellulose, BC could improve the interactions among the polymers, modifying the film properties of the composites, and extending the possible applications. The objective of this work was to develop biodegradable BC-based films with antimicrobial properties by adding chitosan and improve mechanical and visual appearance properties by incorporating PVOH to the formulation. A simple process to obtain the composites is presented to take advantage of the BC structure, avoiding complex regeneration and drying process of vegetal cellulose. Polynomial models were used to evaluate the effect of the share of PVOH and chitosan on the thickness, mechanical properties (toughness, burst strength, distance to burst), water solubility and swelling degree of the films. Besides, microstructure, optical and thermal properties of the BC-PVOH-chitosan films were evaluated.

EXPERIMENTAL SECTION Komagateibacter xylinus was obtained from the “Colección Española de Cultivos Tipo” (CECT). Extra pure anhydrous sodium bromide (99 %), sodium hydroxide (98 %) and D(+)glucose monohydrate (99% extra pure) were purchased from Acros organics (Geel, Belgium). Yeast extract was provided by Scharlau Microbiology (Barcelona, Spain). Full-hydrolyzed (>98%) polyvinyl alcohol with average molecular weight (Mw) of 30,000 g/mol and ester value of 12-25 were supplied by Merck (Billerica, MA, US). Chitosan (Mw 100000-300000) was purchased from Acros organics (Geel, Belgium). The chitosan solution at 1% was prepared using an aqueous solvent of 1% (v/v) of acetic acid at room temperature under agitation. The PVOH solution at 4% was prepared heating at 80 ºC under vigorous agitation for 2 h.



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Preparation of films. K. xylinus was cultured on a medium of 10% of glucose and 1% of yeast extract previously sterilized at 121ºC for 15 min. Inoculated Petri dishes with 75 ml of culture medium were statically incubated at 30 ºC for 8 days. After that time, the film suspended in the airliquid interface was removed and treated with 1% (w/v) of NaOH at 90 ºC for 1 h to inactivate bacterial cells. Then, BC films were washed with running distillate water until pH 7. Pure BC samples were dried at room temperature for 48 h. To obtain the composite films, the excessive water of wet BC films was removed by paper filter. The resulting films were immersed in a bath of the mixture chitosan-PVOH for 2 h. Finally, the BC-PVOH-chitosan films were dried in a Petri dish at room temperature for 2 days. The dried films were cut to specific sizes for each test. The samples were stored in desiccators with oversaturated salt solution of sodium bromide or silica gel for 5 days as required for each test. Scanning electron microscopy (SEM). Films were fixed on slides with adhesive carbon tape, metalized with Au, observed and photographed using a high-vacuum microscope (JEOL JSM6360LV, Jeol Ltd, Tokyo, Japan) at an accelerating voltage of 20 kV. Film thickness measurement. Films thickness (mm) was obtained using a Thickness Meter ET115S (Etari GmbH, Stuttgart, Germany). The measuring was carried out at 5 random locations on the surface and mean values were reported. The thickness of films was measured prior to other tests. Toughness and puncture properties. Toughness (T, MJ/m3) or tensile energy to break is the capability of a material to absorb energy during deformation up to fracture. A texturometer (TA-XTplus, Stable Micro System, UK) was used to measure the toughness following the standard method D-882 (ASTM). Ten samples of 15 × 100 mm were cut from each film formulation. The films were stored and equilibrated in desiccators with oversaturated salt solution of sodium bromide, ensuring 57% relative humidity at room temperature. The samples were stored for at least 5 days to ensure stable moisture content. The samples were fixed in the mechanical grips with initial separation of 40 mm. The test was carried out at a crosshead speed of 0.08 mm/s and the stress-strain during deformation was measured. T was calculated from the stress-strain plot, as the area under the stress-strain curve from 0% strain to the maximum strain at the breaking point. Puncture properties were analyzed by measuring the burst strength (BS) and distance to burst (DB), also denominated puncture force or puncture strength and puncture deformation, respectively. Four squared samples of each film 6 ACS Paragon Plus Environment

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(30 mm each side) were stored in desiccators with oversaturated salt solution of sodium bromide for 5 days. The samples were fixed on a film support rig (HDP/FSR) and submitted to perforation by a cylindrical probe (d = 3 mm), moving at 60 mm/min until rupture. Curves of force (g) vs deformation (mm) were recorded. BS was calculated as the maximum puncture force until the breaking point, and DB was reported as the maximum distance of deformation at the breaking point. Film solubility in water (%S). The water solubility of the films was determined by the gravimetric method. Film portions measuring 3×3 cm2 were cut and dried at 105 ˚C in a vacuum oven for 24 h and then weighed to the nearest 0.0001 g for the initial dry weight (W0). The dry samples were placed in glass beaker containing 100 mL of distilled water and shaken gently at 25˚C for 24 h. Then, the remaining undissolved film was dried at 105 ˚C for 24 h until constant weight to determine the final dry weight (Wf) 36,37. Tests for each type of film were carried out in triplicates. Solubility in water (%) was calculated by using the Equation 1: %𝑆(%) = (Eq. 1)

𝑊0 ― 𝑊𝑓 𝑊0

∙ 100

Swelling property (%W). The swelling or water retention was determined by the gravimetric method. Three samples of each film were cut into 3 x 3 cm2 and stored in desiccators with silica gel for 5 days. Dry samples were weighed (Wdry) to the nearest 0.0001 g using a precision balance. Then, each sample was immersed in an air-tight container with 100 ml of distilled water and stored at 25°C for 24 h allowing the samples reaching equilibrium. The excess surface water was removed from the swollen samples using filter paper. Wet samples were weighed (Wwet). The water retention (%W) of each sample was calculated using the Equation 2 17: %𝑊(%) = (Eq.2)

𝑊𝑤𝑒𝑡 ― 𝑊𝑑𝑟𝑦 𝑊𝑑𝑟𝑦

∙ 100



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Light barrier properties, transparency and opacity. The optical properties of the films were analyzed by measuring the transmittance of the samples between the wavelength from 200 to 800 nm using a spectrophotometer V-670 (Jasco Inc, Japan). The test was carried out in duplicate using transmittance mode at 2 nm intervals. The transparency of the films was calculated from the percent of transmittance at 600 nm using the Equation 3 38: 𝑇𝑟𝑎𝑛𝑠𝑝𝑎𝑟𝑒𝑛𝑐𝑦 = (Eq. 3)

(log % 𝑇600) 𝑥

Where %T600 is the percent transmittance at 600 nm and x is the film thickness (mm). The opacity of the films was calculated from de absorbance at 500 nm, following the Equation 4 39: 𝑂𝑝𝑎𝑐𝑖𝑡𝑦 = (Eq. 4)

𝐴𝑏𝑠500 𝑥

Where Abs500 is the absorbance at 500 nm and x is the film thickness (mm).

Thermal properties of films. The thermal stability of the developed films was analyzed by simultaneous thermogravimetry and differential scanning calorimetry using a TGA/DSC 1 (METTLER TOLEDO, Greifensee, Switzerland) equipment. The samples were placed in hermetic aluminum pans and the test was carried out at a heating rate of 10°C/min from 50 to 400°C, in atmosphere of N2 (50 mL/min). Statistical analysis. PVOH and chitosan concentration were considered as independent variables (denoted A and B, respectively) and the effect on the select dependent variables (thickness, T, BS, DB, %W and %S) were calculated. The set of experiments followed a complete factorial design. Cook's distance was used the detect outliers 40 and the BoxCox data transformation technique in order to reduce anomalies 41. The multifactor analysis of variance (ANOVA) was used. The statistical analysis was accomplished using Design Expert⁠® 10.0.6 software (Stat-Ease, Inc., Minneapolis, MN, US).

RESULTS AND DISCUSSION Table 1 shows the experimental design of BC-chitosanPVOH composite films. To avoid high viscosity baths, the


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maximum concentration of chitosan and PVOH was 1 and 4% (w/w), respectively.



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Table 1. Formulations assayed for the study of bacterial cellulose based films combined with chitosan and polyvinyl alcohol and experimental results for the thickness, mechanical and puncture properties, water retention and percentage of solubility of the films achieved. T is toughness, BS is burst strength, DB is distance to burst, %W is the percentage of water retention or swelling index and %S is the percentage of solubility. Chito san

PVOH

Thickn ess

% (w/w)

% (w/w)

mm

1

0

0

2

0

3

BS

DB

%W

%S

3

g

mm

%

%

2.08·1 0-2

0.26

58.88

0.39

364.7 8

9.37

2.0

5.07·1 0-2

1.16

543.0 2

0.97

224.5 2

46.9 1

0

4.0

6.62·1 0-2

2.48

1054. 04

1.37

164.4 9

41.9 6

4

0.5

0

2.36·1 0-2

1.12

489.9 8

1.31

157.7 9

4.53

5

0.5

2.0

3.98·1 0-2

1.29

1312. 33

1.60

345.9 9

32.0 8

6

0.5

4.0

5.04·1 0-2

5.94

2878. 19

2.75

368.3 5

36.8 2

7

1.0

0

2.99·1 0-2

1.92

1067. 35

1.71

96.16

8.14

8

1.0

2.0

5.17·1 0-2

3.41

2640. 13

2.36

478.7 3

29.7 1

9

1.0

4.0

5.71·1 0-2

7.18

3234. 62

3.24

397.0 9

33.1 3

Exp .

T MJ/m


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Scanning electron microscopy (SEM). The top, bottom and cross-section surfaces of the samples from the experiments 1, 5, 6, 8 and 9 were observed by scanning electron microscopy (SEM) (Figure 1). The selected experiments allowed analyzing the modifications on the microstructure of the bacterial cellulose due to the PVOH and chitosan addition. The microstructure of pure bacterial cellulose was continuous and porous (Figure 1a), with a laminated threedimensional structure, as showed in the cross-section (Figure 1a), as a result of the layer-by-layer process. The presence of chitosan and PVOH resulted in a smooth surface, decreasing the roughness and porosity (Figure 1b-e). This effect was more noticeable at higher PVOH concentration in presence of chitosan (Figure 1c and 1e). The bottom sides were smoother than the top because they were in contact with the Petri dish during the drying process. According to the cross-section micrographs, PVOH and chitosan increased noticeably the thickness and density of the film promoting a more compact structure. At high concentrations of chitosan and in presence of PVOH, the formation of an external chitosan-PVOH coating on the bottom and top sides was more evident (Figure 1d and 1e). This coating on the BC films was not that evident at low concentrations of chitosan. Increasing the chitosan content allowed improving the chitosan-PVOH interactions, resulting in a homogenous retention of the polymers on the BC surface. Previous studies reported that the chitosan may penetrate into the pores of BC, producing a denser and more compact matrix structure 30,31. Chitosan and PVOH also had a softening effect on the surface of the regenerated cellulose films 13,35.



Top

Bottom

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Cross-section

a)

b)

c)

d)

e)

Figure 1. Scanning electron microscopy of the top, bottom and cross section of the samples 1, 5, 6, 8 and 9 of the films. A) Pure bacterial cellulose. B) Bacterial cellulose, chitosan (0.5% w/w) and polyvinyl alcohol (2% w/w). C) Bacterial cellulose, chitosan (0.5% w/w) and polyvinyl alcohol (4% w/w). D) Bacterial cellulose, chitosan (1% w/w)


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and polyvinyl alcohol (2% w/w). E) Bacterial cellulose, chitosan (1% w/w) and polyvinyl alcohol (4% w/w).

The results obtained for dependent variables (thickness, T, BS, DB, %W and %S) are listed in Table 1. The effect of the PVOH and chitosan on the dependent variables were modelled using a second-order polynomial equation. Dependent variables were analyzed by ANOVA (Table 2).

Table 2. Analysis of variance (ANOVA) for each of the study dependent variables. Thickness

Source Model AChitosa n B-PVOH

Fvalue 15.03

pvalu e 0.00 46

3.10·1 0.95 0-3 74 30.06

0.00 15

Distance to burst

Source

Fvalue

pvalu e

Toughness Fvalue

pvalue

Burst strength

F-value

p-value

16.23

0.0038 82.25

< 0.0001

10.68

0.0171 79.1

0.0001

21.77

0.0034 85.4

< 0.0001

Swelling

Fvalue

pvalue

Solubility

F-value

p-value

57.93

0.00 01

10.46

0.023

53.77

0.0184

66.19

0.00 02

9.74

0.0355 26.14

0.0362

49.67

0.00 04

13.23

0.022

158.45

0.0063

AB

9.33

0.0925

A2

9.87

0.0881

B2

31.61

0.0302

Model AChitosa n B-PVOH



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Effect on the film thickness. The formulation of the films affected the thickness of the composite films. Thickness values ranged from 2.08·10-2 to 5.71·10-2 mm. Data fitted well to a linear model. The F-value of the model was 15.03 and the p < 0.05, being significant. According to the p-values of the model terms, only the PVOH concentration showed a significant effect on film thickness (p < 0.05). The chitosan concentration was not significant. Table 3 shows the fitting statistics values of r2, predicted r2, adjusted r2 and adequate precision for each variable. It is desirable that the difference between the predicted r2 and the adjusted r2 be less than 0.2, indicating that they are in reasonable agreement. The signal/noise ratio is measured by the adequate precision. A ratio higher than 4 is desirable. The r2 value was 0.83 and the predicted r2 was in reasonable agreement with the adjusted r2. The ratio of the adequate precision indicated an adequate signal. Table 3. Fit statistics of the model for each of the study dependent variable. Thickne ss

Toughnes s

Burst strength

Distance to burst

Swelling

Solubili ty

r2

0.83

0.84

0.97

0.95

0.89

0.99

Predicte d r2

0.62

0.65

0.91

0.89

0.41

0.85

Adjusted r2

0.78

0.79

0.95

0.93

0.80

0.97

Adequate precisio n

7.83

11.22

25.65

21.47

8.03

19.49

Equation 5 forecasts the values of thickness of the BC-PVOHchitosan films with the experimental chitosan and PVOH concentrations:

𝑇ℎ𝑖𝑐𝑘𝑛𝑒𝑠𝑠 (𝑚𝑚) = 2.66·10 ―2 + 3.37·10 ―4·𝐶ℎ𝑖𝑡𝑜𝑠𝑎𝑛 (%) + 8.29·10 ―3·𝑃𝑉𝑂𝐻(%) (Eq. 5)

According to the response surface (Figure 2), PVOH increased the thickness of the BC films as observed in the SEM images. According to the statistical analysis, the effect 14 ACS Paragon Plus Environment

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of the chitosan was not significant. However, it was observed by the SEM that higher chitosan content promoted the homogeneous retention of both polymers on both sides of the films. The homogeneous PVOH retention affected the thickness of the film.

Figure 2. Prediction of the model for the effect of the polyvinyl alcohol (PVOH) and chitosan on thickness.

Effect on mechanical and puncture properties of the films. The toughness values ranged from 0.26 to 7.18 MJ/m3. Data fitted well to a linear model. The F-value of the model was 16.23 and the p < 0.05, being significant. In this case both PVOH and chitosan showed significant model terms (p < 0.05). The r2 value was 0.84 and the predicted r2 was in reasonable agreement with the adjusted r2. The ratio of the adequate precision was higher than 4, indicating an adequate signal. Equation 6 predicts the values of toughness of the BC films with experimental chitosan and PVOH concentrations.

𝑇𝑜𝑢𝑔ℎ𝑛𝑒𝑠𝑠 (𝑀𝐽/𝑚3) = ―0.74 + 2.87·𝐶ℎ𝑖𝑡𝑜𝑠𝑎𝑛 (%) +1.03·𝑃𝑉𝑂𝐻(%) 6)

(Eq.

According to the response surface (Figure 3), chitosan and PVOH increased the toughness of the composite BC films. Pure BC showed a value of toughness of 0.26 MJ/m3. Pure 15 ACS Paragon Plus Environment


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regenerated cellulose T was 2.91 MJ/m3, exhibiting greater energy absorption capacity at the breaking point 13. The structural and thickness differences between BC films and regenerated cellulose films could be the main cause of the difference in T values. Pure regenerated cellulose films are threefold thicker than pure BC films. The thickness can be related to the amount of cellulose fibers that are interacting. Decreasing the thickness, the number of fibers that interact also decrease, modifying the mechanical performance of the films. Additionally, pure BC films showed an uncompact structure that could affect the resistance. Cellulose nanopaper prepared from softwood showed a decrease of toughness (denominated work to fracture) when the porosity of the films increased 42. However, the porous structure was an advantage to combine it with PVOH and chitosan by dipping in our study. The BC microstructure facilitated the interaction among the components and the penetration of the reinforcement material through the pores. Thereby, the addition of chitosan or PVOH increased the toughness values, being more significant the BC-PVOH effect or the presence of both polymers (BC-chitosan-PVOH). In literature, pure chitosan or PVOH films showed T values of 16.8 or 34.3 MJ/m3, respectively 16. In this study, the polymers with high T values reinforce the BC-based films. In addition, chitosan and PVOH are miscible probably promoted by strong intermolecular hydrogen bonds between both polymers. These interactions increased the Van der Waals forces into the BC matrix, resulting in increased T values. The reinforcement effect on mechanical properties due to the new formations of hydrogen bonds has been reported in kefiran-chitosan films 43, wheat starch-chitosan films 44 and PVOH-chitosan films 17,18. Despite, pure BC films had lower T values than pure regenerated cellulose films. Similar maximum T values were reached at PVOH 4% (w/w) and chitosan 1% (w/w) in BC film that those of regenerated cellulose films 13. In accordance with the obtained results, an improvement of the toughness was reported in BC hydrogels combined with polyacrylamide and gelatin by immersion 45.


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10

Toughness (MJ/m3)

8 6 4 2 0

4

3

2 PVOH (%)

1

0 0

1 0.8 0.6 0.4 0.2 Chitosan (%)

4000

Burst strength (g)

3000 2000 1000 0

4

3 2 PVOH (%)

1

0 0

1 0.8 0.6 0.4 Chitosan (%) 0.2

4 Distance to burst (mm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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3 2 1 0

4

3

2 PVOH (%)

1

0 0

1 0.8 0.6 0.4 0.2 Chitosan (%)

Figure 3. Prediction of the model for the effect of the polyvinyl alcohol (PVOH) and chitosan on toughness, burst strength and distance to burst.

Regard puncture properties, the characterization of these properties of the novel developed biopolymers has not been extensively studied. Puncture properties can provide important information about the resistance of the films in contact with the package corners or surfaces protuberance when it is used as food package.



Page 18 of 35

The burst strength ranged between 58.88 and 3234.62 g. Data fitted well to a linear model. The Box-Cox plot recommended transforming the data to square root of BS to get a better fit of the equation. The F-value of the model was 82.25 and p < 0.0001, being significant. All model terms were significant (p ≤ 0.0001). The r2 value was 0.97 and the predicted r2 was in reasonable agreement with the adjusted r2. The ratio of the adequate precision was higher than 4, indicating an adequate signal. Equation 7 estimates the response on the BS as a function of the concentration of chitosan and PVOH on the bacterial cellulose films.

𝐵𝑆 (𝑔) = 8.82 + 25.83·𝐶ℎ𝑖𝑡𝑜𝑠𝑎𝑛 (%) +6.71·𝑃𝑉𝑂𝐻(%) (Eq. 7)

Figure 3 shows a similar behavior of BS as compared to T. Chitosan and PVOH increased the BS values. The increase was more noticeable when the both polymers were added, probably due to the interaction BC-chitosan-PVOH into the film matrix. Pure chitosan films showed a BS value ranging from 1234 to 1892 g, as a function of the molecular weight 46. Pure PVOH film reported BS values ranging from 2703 to 3295 g 47. In the present study, the maximum value reached was 3234.62 g from a bath of 1% chitosan and 4% PVOH, indicating the good interaction BC-chitosan-PVOH. This effect was more noticeable in regenerated cellulose, reaching values of 6291 g, almost twice the puncture resistance 13. The values obtained were higher than those reported for other biopolymers, i.e., gelatin films (8.5 10.7 g) 48, kefiran-starch films (380 - 253 g) 49 or carboxymethyl cellulose–PVOH films (112.2 - 397.8 g) 50. The distance to burst (DB), ranging from 0.39 to 3.24 mm. Data fitted well to a linear model. The F-value of the model was 57.93 and p < 0.0001, being the model significant. All model terms were significant (p < 0.0001). The r2 value was 0.95 and the predicted r2 was in reasonable agreement with the adjusted r2. The ratio of the adequate precision indicated an adequate signal (greater than 4). Equation 8 forecasts the values of the DB as a function of the chitosan and PVOH concentrations.

𝐷𝐵 (𝑚𝑚) = 0.32 + 1.52·𝐶ℎ𝑖𝑡𝑜𝑠𝑎𝑛 (%) +0.33·𝑃𝑉𝑂𝐻(%) 8)

(Eq.


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The chitosan and PVOH had a similar effect (Figure 3) than that observed in the other mechanical properties. The DB values increased when the concentration of chitosan and PVOH increased, being more significant the increase in presence of the both polymers. Due to the good extensibility of pure PVOH and chitosan films, a plasticizing effect of the polymers was observed on BC films, increasing the deformation values 46,47,51. Regenerated cellulose-PVOHchitosan films showed a maximum puncture deformation of 2.52 mm 13, lower than that observed in the BC-chitosan-PVOH films. The structure of BC films promoted the interaction among the polymers, improving the plasticizing effect of PVOH and chitosan on BC films, allowing a higher deformation of the film. The plasticizing effect was also observed when PVOH was added into gelatin films 47 or chitosan-based films plasticized with glycerol 46.

Effect on the film solubility (%S). The percentage of the film solubility on water ranged from 9.37 to 46.91%. Data fitted well to a quadratic model. Trial 3 was ignored for ANOVA analysis because it was detected as an outlier in the Cook's distance test. The F-value of the model was 53.77 and p < 0.05, being significant. The chitosan, PVOH, and the quadratic effect of PVOH were significant model terms. The r2 value was 0.99 and the predicted r2 was in reasonable agreement with the adjusted r2. The ratio of the adequate precision indicated an adequate signal. Equation 9 forecasts the values of the film solubility as a function of the chitosan and PVOH concentrations.

%𝑆 (%) = 10.80 ―27.97·𝐶ℎ𝑖𝑡𝑜𝑠𝑎𝑛 (%) +23.0·𝑃𝑉𝑂𝐻(%) ―5.80·𝐶ℎ𝑖𝑡𝑜𝑠𝑎𝑛 (%) (Eq. 9) ·𝑃𝑉𝑂𝐻(%) +25.30·𝐶ℎ𝑖𝑡𝑜𝑠𝑎𝑛 (%)2 ―2.83·𝑃𝑉𝑂𝐻 (%)2

According to the response surface (Figure 4), the film solubility was mainly influenced by the PVOH content. Due to its hydrophilic and water-soluble nature, part of the PVOH was dissolved in water. A previous study reported an 65.5% of solubility for pure PVOH films 17. Pure BC films showed a low solubility value of 9%. Cellulose is insoluble in water 4. Thus, the value obtained for pure BC was probably due to the presence of soluble solids remained from the culture medium in the dry samples. BC-chitosan films did not show significant changes in the solubility values compared to pure BC samples. Chitosan was retained in the film because it is soluble at slightly acid pH. BC-chitosan-PVOH samples showed a slight decrease in the percentage of soluble matter 19 ACS Paragon Plus Environment

Biomacromolecules

compared to the BC-PVOH films. Probably, the cross-linking between the amine and hydroxyl resulted in a packed structure between PVOH and chitosan, limiting the interactions with water and reducing the PVOH solubility 17.

60 50

Solubility (%)

40 30 20 10 0

4 3 2 PVOH (%)

1 0 0

0.2

0.4

0.6

1

0.8

Chitosan (%)

800 600 400

Swelling (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 35

200 0

1 0.8 0.6 Chitosan (%) 0.4 0.2 0 4

3

2 PVOH (%)

1

0

Figure 4. Prediction of the model for the effect of the polyvinyl alcohol (PVOH) and chitosan on solubility and swelling of the films.


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Effect on the swelling property of the films (%W). The swelling or water retention values obtained ranged from 96.16 to 478.73 %. Data fitted well to a two-factor interaction mathematical model. Trial 9 was disregarded because it was identified as an outlier by the Cook's distance test. The Fvalue of the model was 10.46 and p < 0.05, being the model significant. All the terms in the model were significant (p < 0.05). The r2 value was 0.89. In this case, the predicted r² of 0.41 was slightly away to the adjusted r² of 0.80. However, the adequate precision obtained was greater than 4, indicating an adequate signal to noise ratio. Using the equation 9 is possible to estimate the values of the %W, as a function of chitosan and PVOH.

%𝑊 (%) = 335.28 ― 235.20·𝐶ℎ𝑖𝑡𝑜𝑠𝑎𝑛 (%) ―51.05·𝑃𝑉𝑂𝐻(%) +211.30·𝐶ℎ𝑖𝑡𝑜𝑠𝑎𝑛 (%)·𝑃𝑉𝑂𝐻(%) (Eq. 9)

The swelling behavior of films is affected by several factors including network density or hydration capacity of polymers 34. The swelling ratio of pure BC was about 5 times of its dry weight, yielding a capacity of water retention of 364.78%, higher than other hydrocolloid films 30. The porous and three-dimensional structure of BC allows a greater surface contact, easing the physical entrapment of water molecules inside the cellulose matrix. According to the response surface (Figure 4), the swelling behavior was influenced mainly by the chitosan. The swollen mass was drastically reduced from 364.78% (pure BC films) to 96.16% (BC-chitosan 1% film) when the chitosan concentration increased in the blend. As shown in the SEM images (Figure 1), the incorporation of chitosan resulted in a more compact structure and reduced the porosity. The packed BC-chitosan structure hindered the entrapment of water molecules inside the cellulose matrix, decreasing the water retention capacity. PVOH also decreased the water retention capacity, but at lower extent than chitosan. The pure BC swelling index was halved by combining with 4% PVOH. Although, PVOH contributed to the porosity reduction of the film as it is more hydrophilic than chitosan, due to its large number of hydroxyl groups 17. In contrast, the presence of both polymers, due to the chitosan-PVOH interactions, increased the water retention up to similar values than pure chitosan films. The intra- and intermolecular hydrogen bonds taking place between the PVOH and chitosan chains favored the swelling capacity. Results were in agreement with previous studies of BC–chitosan membranes for wound dressing applications 30 and chitosan-PVOH blends 16,17. 21 ACS Paragon Plus Environment


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Light barrier properties, transparency and opacity. One of the desired characteristics of packaging material is the protection of foods from the effects of light, especially the UV radiation 17. The light barrier properties of the films were analyzed in the region UV-A (315–400 nm), UV-B (280–315 nm) and UV-C (200–280 nm). The range of wavelength between 200 to 315 nm is the most susceptible region for lipid oxidation. Figure 5.a. shows the transmittances of the experiments 1, 5, 6, 8 and 9 in the range UV-A, UV-B and UV-C. The selected samples allowed analyzing the effect of the chitosan-PVOH interactions on the optical properties of pure BC. The average percentages of transmittance of the selected samples in each UV-light region are shown in table 4. Pure BC showed low transmittance values ranging from 0 to 2 % in the UV-C region, similar to pure regenerated cellulose 13. To increase the wavelength at the region UV-B and UV-C, increase the transmittance values of pure BC up at maximum value of 7.5% at 400 nm. Generally, the incorporation of PVOH and chitosan increased the transmittance values in these region as the wavelength increased. The maximum value raised in the UV-C region was 12.67% of transmittance for the experiment 5. However, when the chitosan concentration reached 1% (w/w), the transmittance was reduced to 4.80 %. Chitosan UV-barrier properties has been reported in previous studies 39,52 and being more noticeable at 1% concentration. Pure PVOH showed low UV barrier properties 17. These effect were also observed in the UV-B and UV-A region. The maximum transmittance value in the UV-B region was reached by the sample with higher PVOH and lower chitosan concentration, up 24%. Sample 8 with higher chitosan content and lower PVOH reached the minimum transmittance value, 9.2%. In the UV-C region, samples 6 and 8 showed again the maximum and minimum transmittance values, being 35.1% and 21.6%, respectively. For this reason, chitosan reinforced the UV radiation barriers of pure cellulose combined with PVOH.


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Figure 5. UV-VIS Spectra profile of selected films.

In addition to obtaining films with UV barrier properties, it is important that the developed polymers have a good visual appearance, associated with a high transparency and low opacity. It is possible to evaluate the visual appearance through the transmittance values in the UV-VIS region (280 – 800 nm), as shown in Table 4. PVOH significantly increased the transmittance of the samples, reaching the maximum values at the highest PVOH concentration. Experiment 9 and 6 showed 58.9 and 47.9 % of transmittance at 800 nm wavelength, respectively. At lower PVOH concentrations (2% w/w), the variation of chitosan content did not affect significantly the percentage of transmittance. The difference in transmittance between samples 5 and 8 was less than 1%. At higher PVOH and chitosan concentrations, the interaction between these polymers improved the optical 23 ACS Paragon Plus Environment


Page 24 of 35

properties in the region UV-VIS. Probably, chitosan facilitated the homogeneous retention of PVOH on both sides of the film and inside the matrix, modifying its optical properties values. Regenerated cellulose films with chitosan (1% w/w) and PVOH (4% w/w) showed a transmittance value of 38.31 % at 800 nm, lower than that of bacterial cellulosebased films 13. The unique structure of bacterial cellulose eased the cellulose-chitosan-PVOH interaction, resulting in an improvement in the optical properties in the UV-VIS region compared to vegetable cellulose.

Table 4. Optical properties of the selected samples. The average percentage of transmittance in the region UV-A (315– 400 nm), UV-B (280–315 nm) and UV-C (200–280 nm), transparency and opacity calculated of the selected samples. %T is percentage of transmittance, T600 is transmittance at 600 nm wavelength, %T600 is the percent transmittance at 600 nm, T500 is transmittance at 500 nm and Abs500 is absorbance at 500 nm. Exp.

% T

%T

%T

T60 0

%T60 0

T50 0

Abs50 Transpare Opacit 0 ncy y

UV-A

UV-B UV-C

6.05

3.30 1.00

0.1 0

9.72

0.0 9

1.06

47.58

50.85

5

22.9 4

15.9 6.35 6

0.3 2

31.6 9

0.2 9

0.53

37.69

13.32

6

29.5 4

17.7 5.72 0

0.4 3

43.0 3

0.4 0

0.40

32.42

7.98

8

15.3 9

7.04 2.43

0.2 9

29.4 4

0.2 6

0.58

28.44

11.19

9

29.6 0

14.4 4.28 1

0.5 2

51.6 8

0.4 7

0.33

30.01

5.81

1

Regarding transparency and opacity, Table 4 shows the obtained values for samples 1, 5, 6, 8 and 9. Transparency values ranged from 28.44 to 47.58 and opacity ranged from 5.81 to 50.85. Pure BC films showed higher transparency and lower opacity values than the other composites, but the transparency and opacity properties were strongly affected by the thickness. For this reason, although all the samples showed very high transparency values, it is important having low opacity values to obtain the best appearance properties. PVOH followed by chitosan, improved the appearance of the 24 ACS Paragon Plus Environment

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films by decreasing the opacity values in the visible region, as shown in Figure 6. The low transmittance values in the UV region due to the properties of the BC and chitosan, and the improved visual appearance due to the PVOH in the visible region, could make these composites an excellent option to apply as a food active packaging with light barrier properties suitable to prevent UV light-induced lipid oxidation.

a)

b)

c)

d)

Figure 6. Visual appearance of: a) Bacterial cellulose, chitosan (0.5% w/w) and polyvinyl alcohol (2% w/w); b) Bacterial cellulose, chitosan (0.5% w/w) and polyvinyl alcohol (4% w/w); c) Bacterial cellulose, chitosan (1% w/w) and polyvinyl alcohol (2% w/w); d) Bacterial cellulose, chitosan (1% w/w) and polyvinyl alcohol (4% w/w) films.



Page 26 of 35

Thermal properties of films (TGA-DSC). Thermogravimetry and differential scanning calorimetry were carried out simultaneously for each sample from the experiments 5, 6, 8 and 9, as shown in Figure 7. These measurements allowed assessing the effect of the PVOH-chitosan ratio on the thermal stability of the films. The thermograms showed a first endothermic peak in the 60 – 110 °C range, corresponding to the volatilization of water from the films samples 53. The volatilization of water assumed a weight loss between 4 - 5% of each sample, slightly higher than the weight loss of pure BC sample, due to the hydrophilic nature of PVOH and chitosan. The second endothermic peak at about 220 °C corresponds to the thermal degradation of PVOH 18. These peaks were larger and sharper when the PVOH concentration increased, and they were smoothed by increasing the chitosan content. At 220 ºC the decomposition of the samples ranged from 4 – 6%, being not significant. In addition, by increasing the concentration of PVOH, keeping the chitosan content constant, the proportion of degraded matter decreased slightly indicating that part of PVOH degraded at higher temperatures. Probably, the interaction between both polymers increased the thermal stability of the PVOH of the samples at 220 ºC. The onset temperature of thermal degradation of cellulose was 250 °C, in accordance with the data observed in previous works 9. The presence of chitosan and PVOH on BC films increased the thermal stability of the films. The third endothermic point was displaced up to 260 ºC, increasing the degradation temperature of the polymers. At 340 ºC, the weights loss was between 56.8 - 65.4 %, reaching lower values at higher PVOH and chitosan concentration, indicating that the interaction BC-PVOH-chitosan increased the thermal stability the sample. Regenerated cellulose-PVOH-chitosan also showed this increase in thermal stability due to the interaction among the polymers 13.


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Figure 7. Thermogravimetry and differential scanning calorimetry of selected films. a) Thermogram of the experiment sample 5, b) Thermogram of the experiment sample 6, c) Thermogram of the experiment sample 8 and d) Thermogram of the experiment sample 9.

CONCLUSIONS Combining BC with PVOH and chitosan allowed improving or modifying the mechanical and puncture properties, increasing 27 ACS Paragon Plus Environment


Page 28 of 35

the resistance and deformation at break of composite BC films. The porosity and three-dimensional structure of bacterial cellulose eased the penetration of chitosan and PVOH into the cellulose matrix, reinforcing the films. BCchitosan-PVOH manifested higher puncture properties than other polysaccharide films. The water solubility of the films increased when PVOH was added into the formulation. Pure BC films and BC-chitosan showed similar lower solubility values. Pure BC showed a capacity of water retention about 5 times of its dry weight, giving a swelling degree of 364.78%. Pure BC showed a water retention capacity much higher than other hydrocolloid films, with potential applications as food pads. Chitosan produced a significant decrease of the swelling degree. However, the presence of both reinforcement polymers (PVOH and chitosan), allowed reaching up similar degree values than pure BC. UV-VIS spectra showed the optimal optical barrier properties of BCbased films against UV-radiation. Adding PVOH improved the transparency and visual appearance of the films. TGA-DSC indicated the interaction among the polymers. The improved mechanical properties allowed developing cellulose-based films resistant to rupture with barrier properties to UVradiation and high degree swelling. The developed films could be useful in food industry as an alternative to synthetic film preventing the lipid oxidation in foods and as antimicrobial pads to inhibit the growth of microorganism.

ACKNOWLEDGEMENTS A grant from CONACYT (Mexico) to author Patricia Cazón (#435948) is gratefully acknowledged. The financial support for this project was provided by Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (ES) (Project # ED431B 2016/009). Authors would like to thank the use of RIAIDT-USC analytical facilities.

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Composite Films with UV-Barrier Properties Based on Bacterial

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