Effect of cellulose nanocrystals from sugarcane bagasse on whey protein isolate-based films

Food Research International 107 (2018) 528–535

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Effect of cellulose nanocrystals from sugarcane bagasse on whey protein isolate-based films

T

Sukyai P.a, Anongjanya P.b, Bunyahwuthakul N.b, Kongsin K.b, Harnkarnsujarit N.c, Sukatta U.b, ⁎ ⁎ Sothornvit R.d, , Chollakup R.b, a Biotechnology of Biopolymers and Bioactive Compounds Special Research Unit, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok 10900, Thailand b Kasetsart Agricultural and Agro-Industrial Product Improvement Institute, Kasetsart University, Chatuchak, Bangkok 10900, Thailand c Department of Packaging and Materials Technology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok 10900, Thailand d Department of Food Engineering, Faculty of Engineering at Kamphaengsaen, Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom 73140, Thailand

A R T I C L E I N F O

A B S T R A C T

Keywords: Cellulose nanocrystal Whey protein isolate Food packaging Sugarcane bagasse Film

Whey protein isolate (WPI) has been utilized as edible film or food packaging material. However, WPI films are hydrophilic due to highly polar amino acids which provide a moderate barrier to water vapor and low mechanical properties. To overcome these drawbacks, cellulose nanocrystals (CNCs) extracted from sugarcane bagasse were incorporated with whey protein. FTIR and TGA were used to confirm the changes in chemical structures and to observe the thermal properties, respectively. The CNCs had sizes of 200–300 nm and diameters of 20–40 nm using TEM and AFM technique, respectively. Different amounts of CNCs (0–8 wt% based on WPI) were added into whey protein solution and formed films. The lightness and transparency of the films tended to decrease with increasing WPI content. The water activity (aw) and water solubility of those films increased, whereas their water contact angle values decreased, implying that the film became more hydrophilic when the cellulose nanocrystal was added. The addition of CNCs increased the tensile strength and Young's modulus and reduced the water vapor permeability of WPI-based CNC films. However, the CNCs did not change the oxygen permeability of the film. Therefore, the obtained WPI films provided good mechanical performance and may be promising as an alternative product for film packaging.

1. Introduction

incorporation of cellulose nanocrystals (CNCs) in a biopolymer to improve these properties may be useful because CNCs have strong hydrogen bonding and a high surface area (Qazanfarzadeh & Kadivar, 2016). Thailand is the second largest sugar exporter in the world (Thailand: Sugar Annual | USDA Foreign Agricultural Service, 2017) and > 20 million tons of sugarcane bagasse (SCB) is created annually. Generally, SCB is used as boiler fuel to produce steam which in turn is used in the sugar production industry. Moreover, SCB is also utilized in ethanol and pulp production. SCB consists of approximately 40–50% cellulose (Hajiha & Sain, 2015). Therefore, SCB is an interesting source for CNC extraction. Acid hydrolysis is the most well-known process for CNCs extraction. It breaks and removes the disordered and amorphous regions of cellulosic fibers leaving well-defined crystals in the form of CNCs (Deepa et al., 2011; Habibi, Lucia, & Rojas, 2010). CNCs have been shown to be of benefit in food packaging by improving the mechanical and water barrier properties of alginate film (Sirviö, Kolehmainen, Liimatainen, Niinimäki, & Hormi, 2014).

Whey protein isolate (WPI) is a valuable by-product of the cheese production and it consists of protein content > 90% (w/w) (Mulvihill & Ennis, 2003). It has been studied for film formation and coating application. Several authors have investigated the properties of whey protein isolate films as transparency, flexibility, odorless, excellent barrier to oxygen transmission and providing moderate mechanical properties (Ramos et al., 2013; Sothornvit, Hong, An, & Rhim, 2010). Additionally, these films have demonstrated mechanical and barrier properties better than competitive protein-based (such as corn, zein, wheat, gluten and soy protein isolate) or polysaccharide-based (such as starch, cellulose, carrageenan and pectin) films (Ramos, Fernandes, Silva, Pintado, & Malcata, 2012). However, the limitation of whey protein isolate films to reach the expansive commercial applications was due to low mechanical and high water vapor permeability properties because of their hydrophilic nature (Khwaldia, Pérez, Banon, Desobry, & Hardy, 2004; Kim & Ustunol, 2001). Therefore, ⁎

Corresponding authors. E-mail addresses: [email protected] (R. Sothornvit), [email protected] (R. Chollakup).

https://doi.org/10.1016/j.foodres.2018.02.052 Received 18 September 2017; Received in revised form 29 December 2017; Accepted 20 February 2018 Available online 23 February 2018 0963-9969/ © 2018 Elsevier Ltd. All rights reserved.

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dimensional image of the CNCs. The solution of CNCs was diluted and dropped on the surface of a microscope slide. Then, it was allowed to dry at room temperature (RT) and analyzed. The dimension of the sample was measured using Gwyddion software.

Moreover, adding CNC and titanium dioxide nanoparticles increased the tensile strength, Young's modulus and water sensitivity of nanocomposite wheat gluten-based film (El-Wakil, Hassan, Abou-Zeid, & Dufresne, 2015). Qazanfarzadeh and Kadivar (2016) reported improved physical, mechanical and barrier properties of WPI film incorporated with oat husk nanocellulose. Therefore, to the best of our knowledge, this is the first report to extract CNCs from SCB for application in WPI films. Therefore, this study aimed to synthesize and characterize CNCs from SCB produced using acid hydrolysis and to investigate the effect of cellulose nanocrystals from sugar bagasse on the properties of nanocomposite whey protein film to determine its further utilization as a food packaging material.

2.4.3. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) The ATR-FTIR spectra of the CNCs were recorded using a spectrophotometer (Thermo Scientific Nicolet IR200, USA). The sample was heated at 50 °C until dried and cut or ground into small pieces. The samples were analyzed at wavenumbers in the range 400–4000 cm−1. 2.4.4. X-ray diffraction (XRD) The crystallinity of the sample after acid hydrolysis was obtained using an X-ray diffractometer (Bruker model D8 Advance, USA) with Cu Kα radiation. The condition was set at a speed of 5°/min and the sample was scanned at θ in the range 5–40°. The crystallinity index (Crl) calculation was determined using Eq. 1 (Segal, Creely, Martin, & Conrad, 1959). I002 is both crystalline and amorphous and Iam is an amorphous material.

2. Materials and methods 2.1. Materials The sugarcane bagasse (SCB) used in this study was obtained from Kaset Thai International Sugar Corporation Public Company Limited, Nakhonsawan, Thailand. Whey protein isolate (WPI) was purchased from Mighty International Co., Ltd., Thailand. Glycerol was provided from Sac Scienec-Eng Ltd., Part, Thailand. Sodium chlorite (NaClO2) was purchased from Ajax Finechem Pty., Ltd., New Zealand. Glacial acetic acid and sulfuric acid were bought from QRec Chemical Co., Ltd., New Zealand. Dialysis tubing with a 12–14 kDa molecular weight cutoff was used.

CrI (%) =

(I002− Iam ) x100 I002

(1)

2.4.5. Thermogravimetric analysis (TGA) The thermal properties of the fibre in each step were measured using a thermogravimetric analyzer (Mettler Toledo, TGA/SDTA 851e, Switzerland). Samples of 2–5 mg were weighed and heated from 25 to 600 °C at a rate of 10 °C/min under a nitrogen atmosphere.

2.2. Extraction of cellulose from SCB Cellulose extraction followed the methodology of Saelee, Yingkamhaeng, Nimchua, and Sukyai (2016). Sugarcane bagasse was treated with steam explosion at 195 °C for 15 min. Then, it was bleached around 7 times with 1.4% (w/v) sodium chlorite until the color of samples became white (Mandal & Chakrabarty, 2011). The untreated, steam-exploded and bleached SCB were chemically analyzed according to standard TAPPI methods (TAPPI, 2003) being alpha cellulose (T203 om-98), lignin (T222 om-98) and holocellulose (the combination of alpha-cellulose and hemicellulose) analysis using the acid chlorite method (Browning, 1967). The hemicellulose content was calculated using holocellulose minus the alpha cellulose content.

2.5. Preparation of cellulose nanocrystal combined with WPI for film application Whey protein isolate was prepared (5% by weight). The plasticizer used in this study was glycerol, which was 50% solid. The CNCs varied from 0 to 8% by wt. and then was added into the whey solution to form film. To control the solution under neutral conditions, 2.0 M of NaOH was used to adjust the pH to 7. The solution was properly mixed with an overhead stirrer (IKA RW 20 digital, Malaysia) in a water bath at 90 °C for 30 min and cooled down at RT for 15 min. The solution was degassed in a sonicator bath for 10 min and 30 g was poured into a Teflon mold (10 × 10 × 1.5 cm3), after which it was stored in a controlled incubator at 50 °C (Binder model FED115, Germany) for 15 h to form the film. The film was removed from the mold and controlled in the standard condition, (50% relative humidity, 25 °C and 48 h) before testing the film's characteristics. The WPI films at 0, 2, 5 and 8% CNC were named as WPI, WPI-2, WPI-5 and WPI-8, respectively.

2.3. Preparation of cellulose nanocrystals About 2 g of cellulose was dispersed in 40 mL of 60% (v/v) sulfuric acid (solid-liquid ratio 1:20) (Kumar, Negi, Choudhary, & Bhardwaj, 2014) at 45 °C for 75 min with an agitation speed of 700 rpm using a turbine. To stop the reaction, 400 mL of cold water was added. Acid removal for cellulose suspension was carried out using a centrifuge (TOMY MX-305, Japan) at 15,000 rpm and 4 °C for 15 min. Dialysis was applied against distilled water to remove acid residue until pH 7 was obtained. The sample was then dispersed in a sonicator bath (Elmasonic Model S100H, Germany) for 2 h at 30 °C and was kept at 4 °C for further use.

2.6. Characterization of whey protein film incorporated with cellulose nanocrystals 2.6.1. Water activity (aw) Film was analyzed for its water activity by keeping it in a desiccator that contained saturated magnesium nitrate (Mg(NO3)2) at 25 °C for at least 7 days. Then, 2 g of sample was measured using a water activity meter (Testo 650 Water Activity System, Testo Inc., USA).

2.4. Characterization of cellulose nanocrystals 2.4.1. Transmission electron microscopy (TEM) Transmission electron microscopy is an analytical method to show the general morphology of CNCs. The solution of CNCs was dropped into a carbon-coated copper grid, coated with 1.5% uranyl acetate for 5 min and analyzed using a transmission electron microscope (Hitachi model HT7700; Japan) with an electric potential for analysis of 80 kV.

2.6.2. Solubility First, the film was cut into rectangular (1 × 1 cm) pieces and dried in an oven at 70 °C for 24 h. The sample was placed into the tube filled with distilled water (10 mL) at RT for 24 h. Filter paper number 1 was prepared by drying at 150 °C for 1 h. and weighed after it had dried. Then, the film was poured onto the filter paper and dried at 105 °C for 3 h. Later, it was kept in a desiccator containing silica gel and weighed after drying. The % solubility was calculated using Eq. 2:

2.4.2. Atomic force microscopy (AFM) An atomic force microscope (Asylum Research model MFP-3D AFM (Bio), USA) was used to characterize the morphology and the 529

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%Solubility =

wt . of film before drying − wt . of film after drying x 100 wt . of film before drying

2.6.8. Mechanical properties The mechanical properties consisting of the tensile strength, Young's modulus and elongation were determined according to the ASTM D882 (2002b) method using a universal tensile testing machine (Shimadzu AGS5kN, Tokyo, Japan). The test conditions were set at 50 N for the load cell with a speed of 50 mm/min.

(2) 2.6.3. Color of film (L*, a*, b*) The color values (L*, a* and b*) of films were measured using a colorimeter (Lovibond AT100, USA) based on the CIELAB color system. The total color difference (ΔE*) compared with 0% CNC of WPI film was calculated using Eq. 3:

∆E ∗ =

(∆L∗2

+

∆a∗2

+

∆b∗2)1/2

2.7. Statistical analysis A completely randomized design was used to study the main factors (type and concentration of CNC). Three replications were used to determine each property. Data were subjected to one way analysis of variance (ANOVA) and Duncan's multiple range test was used to determine the significant difference between treatments at the 95% confidence interval. Analysis was performed using the SPSS 17.0 for Windows software (SPSS Inc., Chicago, IL, USA).

(3)

2.6.4. Transparency The transparency value of films was determined according to the ASTM D1746 (2015) method. The sample was cut into a rectangle (1.0 × 4.0 cm) and placed on the internal side of a spectrophotometer cell (Thermo Scientific GENESYS 10S UV–Vis spectrophotometer, USA). The transmittance (% T) of the film sample was measured at 600 nm and the transparency value was calculated using Eq. 4:

[log%T ] Transparency = t

3. Results and discussion 3.1. Characterization of cellulose nanocrystals Cellulose from sugarcane bagasse (untreated SCB) was extracted using steam explosion at 195 °C for 15 min and bleached with 1.4% w/v sodium chlorite. The chemical compositions in sugarcane bagasse (SCB) after each treatment are shown in Fig. 1. The treated SCB with the steam explosion and then the bleaching processes could eliminate hemicellulose and lignin leading to a high purity of cellulose. The result was in a similar range to a previous study which reported the cellulose extraction from sugarcane bagasse was 87.68 ± 0.95% (Saelee et al., 2016). After the acid hydrolysis process, CNCs were synthesized. The CNCs were short and needle-shaped (Fig. 2) after acid hydrolysis using TEM and AFM. The CNC size was in the range 200–300 nm and approximately 20–40 nm in diameter. The morphology of CNCs depends on the type of material and the preparation process. Generally, plant CNCs are around 100–250 nm in length and approximately 5–70 nm in diameter (Abdul Khalil et al., 2014). The crystallinity index (CrI) of the CNCs was measured using X-ray diffraction (XRD) and is presented as diffraction in Fig. 3. Generally, the CNC peak was at around 2θ = 16.5°, 22.5° and 33° assigned to planes (110), (200) and (004) respectively, representing the polymorphs of cellulose-I (Kumar, Negi, Bhardwaj, & Choudhary, 2013). The obtained CNCs had CrI = 68.28. This CrI value was similar to the value of 68.54% reported in previous work on CNCs from an SCB source (Lam, Chollakup, Smitthipong, Nimchua, & Sukyai, 2017a). The chemical structure of all treatments can be determined by functional groups using FTIR spectroscopy as shown in Fig. 4. A peak at 1735 cm−1 appeared in untreated SCB indicating the CeO stretching

(4)

where t is the film thickness (mm) and %T is the percentage of transmittance. 2.6.5. Water contact angle The films were measured for water contact angle using a contact angle goniometer (Dataphysics OCA20, Germany) equipped with a camera and using the image capture program of the SCA20 software. Samples were cut into rectangles (1 × 5 cm) before using a micro syringe to drop 0.5 μl of water droplets on the film samples. Contact angles were calculated by defining a circle around the drop and recording the tangent angle formed at the substrate surface. 2.6.6. Water vapor permeability (WVP) The WVP of the film was determined using a gravimetric modified cup method modified from the ASTM E-96 method (1995). The WVP was calculated using Eqs. 5 and 6:

WVTR = WVP =

(G / t ) A

(5)

WVTR × thickness (pA1 − pA2 )

(6) 2

where WVTR is the water vapor transmission rate (g/m .day), G/t is the ratio of loss of weight per time (g/day), A is the surface area of the sample (m2) (π × 0.0522/4 = 2.1237× 10−3 m2), WVP is the water vapor permeability (g.mm/m2.d.kPa) and pA1 and pA2 are the water vapor partial pressure inside and outside the cup, respectively (kPa). The film thickness (mm) was measured using a digital thickness gage (Mitutoyo, ID-C112XBS, Japan) and the values were averaged at different random locations on the film.

90

(Thickness x OTR) ∆P

Hemicellulose

% by weight

70

2.6.7. Oxygen permeability (OP) The OP was determined according to the ASTM D3985 (2002a) method, using an oxygen transmission rate analyzer (Systech Illinois 8500, Illinois, USA). The OP (cm3.mm/m2.d.kPa) was calculated by multiplying the thickness (mm) and oxygen transmission rate (OTR) and dividing by the pressure difference according to Eq. 7:

OP =

Lignin

80

α-cellulose

A

B

60

50

C a

40

30

a

20

b

b

10

c c

0 Untreated SCB

Steam-exploded SCB

Bleached SCB

Fig. 1. Chemical compositions of untreated sugarcane bagasse (SCB), steam-exploded SCB and bleached SCB. Error bars indicate standard deviation. Different letters (a, b, c) indicate results are significantly different at p < 0.05 using Duncan's multiple-range test.

(7)

where OP is the oxygen permeability (cm3.mm/m2.d.kPa), OTR is the oxygen transmission rate (cm3/m2.d) and ΔP is the pressure difference (kPa). 530

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Fig. 2. Morphology of cellulose nanocrystals (CNCs) from sugarcane bagasse using TEM at different magnifications: (A) 5,000× and (B) 10,000× and using AFM (C) 5 × 5 μm2 and (D) 1 × 1 μm2.

However, the spectra of CNCs and bleached CNCs were similar. Therefore, the acid hydrolysis did not affect the chemical structure of cellulose (Lam et al., 2017a). The thermal properties of SCB in each step were reported in the TGA graph as shown in Fig. 5. The onset of thermal decomposition (Ton) referred to the temperature of the beginning of degradation and the temperature of maximum decomposition (Tmax) indicating the maximum degradation temperature are shown in Table 1. Lignocellulose material was decomposed by heating in the range 200 to 400 °C due to the glycosidic bond composition resulting in water, carbon dioxide, alkanes and hydrocarbon derivatives. Since cellulose, hemicellulose and lignin have different chemical structures, they decomposed at different temperatures. Hemicellulose has an acetyl group which begins to decompose at 220–300 °C (Abraham et al., 2013; Manfredi, Rodríguez, Wladyka-Przybylak, & Vázquez, 2006; Shebani, van Reenen, & Meincken, 2008). Cellulose decomposes at 310–400 °C (Abraham et al., 2013; Deepa et al., 2011; Yang, Yan, Chen, Lee, & Zheng, 2007) and lignin decomposition has a wide temperature range beginning below 200 °C and persisting above 500 °C (Abraham et al., 2013) including phenyl group decomposition (Uma Maheswari, Obi Reddy, Muzenda, Guduri, & Varada Rajulu, 2012). The TGA graph (Fig. 5) indicated that the weight of all samples slightly decreased at 50–120 °C because of water evaporation and low molecular substances (Abraham et al., 2011). Untreated SCB started to degrade at around 260 °C (11.55% weight loss) because of hemicellulose decomposition and there was continuous degradation until 380 °C (73.20% weight loss). As seen in Table 1, Ton of steam-exploded SCB was higher than untreated SCB since explosion with steam

Intensity

22.5

16.5 33

2θ (degree) Fig. 3. X-ray diffraction of cellulose nanocrystals (CNCs) from sugarcane bagasse using acid hydrolysis.

vibration of acetyl and ester bonds in the lignin or hemicellulose but it was not observed in the steam-exploded SCB, bleached SCB and CNCs. This indicated that the hemicellulose and lignin had been eliminated. The band at 1249 cm−1 corresponded to the CeO out of plane stretching vibration of the aryl group in lignin and had a lower spectrum after the bleaching process using sodium chlorite (Kumar et al., 2014; Troedec et al., 2008). Furthermore, the peaks at 1604, 1515 and 1460 cm−1 represented aromatic ring vibration and deformation of CeH vibration in lignin (Alemdar & Sain, 2008; Kumar et al., 2014; Rosa et al., 2010; Sun, Xu, Sun, Fowler, & Baird, 2005) that appeared in untreated SCB but it was not detected in bleached SCB. This result ensured the success of lignin elimination in the bleaching process. 531

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1735 cm-1 1604 cm-1 1515 cm-1 1460 cm-1 1249 cm-1

P. Sukyai et al.

1.8 1.6

(D)

Absorbance

1.4 1.2 1.0

(C)

0.8

(B)

0.6

0.4 0.2

(A)

0.0 4000

3600

3200

2800

2400

2000

1600

1200

800

400

Wavenumber (cm-1) Fig. 4. FTIR spectrum: (A) untreated sugarcane bagasse (SCB) (B) steam-exploded SCB (C) bleached SCB and (D) cellulose nanocrystals (CNCs).

120

Residual Mass (%)

100

CNC (D) Bleached SCB (C)

80

Steam-exploded SCB (B) 60

Untreated SCB (A)

40

(D) (B)

20

(A) (C)

0 0

50 100 150 200 250 300 350 400 450 500 550 600 650

Temperature (°C) Fig. 5. TGA graph of (A) untreated sugarcane bagasse (SCB) (B) steam-exploded SCB (C) bleached SCB and (D) cellulose nanocrystals (CNCs).

so the bleached SCB had higher stability at high temperature (Shebani et al., 2008). However, the CNC degraded at a lower temperature starting at 220 °C (8.45% weight loss). This was due to its size being in the nanometer range which resulted in a greater surface area (Lam, Chollakup, Smitthipong, Nimchua, & Sukyai, 2017b; Quiévy et al., 2010; Zhao et al., 2013).

Table 1 Thermal properties of untreated sugarcane bagasse (SCB), steam-exploded SCB and bleached SCB. Sample

Ton (°C)

Weight loss (%)

Tmax (°C)

Weight loss (%)

Untreated SCB Steam-exploded SCB Bleached SCB CNC

260 320 310 220

11.55 19.89 20.90 8.45

380 390 370 260

73.20 73.36 79.08 20.20

3.2. Characterization of cellulose nanocrystal whey protein isolate based film

eliminated hemicellulose and some parts of the lignin. Bleached SCB began to degrade at 310 °C (20.90% weight loss) until 370 °C (79.08% weight loss). The bleaching process could get rid of other components,

From Table 2, it can be observed that adding 2–5% w/w CNC to whey protein isolate film could decrease the aw value (Table 2). This result was similar to the decrease in aw of 5% w/w CNC incorporated

Table 2 Thickness, water activity, water solubility and transparency of whey protein isolate (WPI) films at different cellulose nanocrystal (CNC) concentrations. Property

WPI film at different CNC content (%) 0

Water activity (aw) Water solubility (%) Water contact angle (°)

2 b

0.585 ± 0.004 43.82 ± 0.85b 70.71 ± 6.45a

5

0.567 ± 0.001 43.78 ± 1.08b 64.97 ± 3.26b

d

Different lowercase letters (a–d) in each row indicate significant difference (p < 0.05) using Duncan's multiple-range test. Values are mean of three replicates ± standard deviation.

532

8 c

0.577 ± 0.004 47.20 ± 1.81ab 60.45 ± 3.42b

0.604 ± 0.002a 51.10 ± 4.28a 56.83 ± 6.67c

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Table 3 Transparency and color of whey protein isolate (WPI) films at different cellulose nanocrystal (CNC) concentrations. WPI film at different CNC content (%)

Property

Transparency (AU nm) Color ⁎ L a⁎ b⁎ 1 ΔE⁎

0

2

5

8

14.32 ± 2.48a

13.50 ± 2.39ab

11.87 ± 0.89ab

11.62 ± 1.67b

89.67 ± 0.57a −0.78 ± 0.09ns 1.54 ± 0.23ns –

88.02 ± 0.11c −0.80 ± 0.06ns 1.65 ± 0.86ns 1.66

88.42 ± 0.84bc −0.77 ± 0.06ns 1.82 ± 0.42ns 1.29

88.85 ± 0.52b −0.73 ± 0.06ns 2.09 ± 0.32ns 1.00

Values are mean ± standard deviation. Different lowercase letters (a–c) in each row indicate significant difference (p < 0.05) using Duncan's multiple-range test. ns in the same row means indicates results are not significantly different at p < 0.05 using Duncan's multiple-range test. 1 Total color difference (ΔE*) was calculated from the color difference between 0% CNC film and other CNC films.

0.70

Absorbance

0.60

(E)

0.50 0.40

(D) 0.30

(C)

0.20

(B)

0.10

(A 0.00 4000

3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1) Fig. 6. FTIR spectrum of whey protein isolate (WPI) films with different cellulose nanocrystal (CNC) concentrations (% w/w of WPI): (A) CNC (B) 0 (C) 2 (D) 5 and (E) 8.

3.0

16

ns OP (x 10-12 m2/s.Pa)

WVP (x10-10 g/Pa.s.m)

14

a

12 b

10

b c

8 6 4

2.5

ns

2.0

ns

ns

1.5 1.0 0.5

2 0.0

0 0

2 5 Cellulose nanocrystals (%w/w of WPI)

0

8

2 5 Cellulose nanocrystals (%w/w of WPI)

8

Fig. 8. Oxygen permeability (OP) of whey protein isolate (WPI) films with different cellulose nanocrystal (CNC) concentrations. Error bars indicate standard deviation. ns indicates that results are not significantly different at p < 0.05 using Duncan's multiple-range test.

Fig. 7. Water vapor permeability (WVP) of whey protein isolate (WPI) films with different cellulose nanocrystal (CNC) concentrations. Error bars indicate standard deviation. Different letters (a, b, c) indicate results are significantly different at p < 0.05 using Duncan's multiple-range test.

water solubility (Table 2). This result was similar to the research of Reddy and Rhim (2014) who found that increasing the nanocellulose content (3–10%) in agar film increased the solubility of the film. Increasing the nanocellulose content could cause aggregation of nanocellulose particles and the higher amount of hydroxyl groups from the nanocellulose caused an increase in H-bonding to react with water. Therefore, the protein film tended to be more soluble in water.

into cassava starch based films (da Silva, Pereira, & Druzian, 2012). From our results, the addition of 2% w/w CNC could reduce the free water (aw) in the film. The interaction between water and the hydroxyl group of cellulose caused water in the gap to decrease. However, a higher CNC content in the film increased the aw value of the film due to the greater hydrophilicity of CNCs. Furthermore, adding more CNC content (> 5%) increased the film 533

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Table 4 Mechanical properties of whey protein isolate (WPI) films at different cellulose nanocrystal (CNC) concentrations. WPI film at different CNC content (%)

Property

Tensile strength (MPa) Elongation (%) Young's modulus (MPa)

0

2

5

8

2.30 ± 0.35c 46.07 ± 23.25a 57.56 ± 2.55c

3.41 ± 0.87b 20.82 ± 9.85b 104.89 ± 22.70b

3.49 ± 0.91b 26.54 ± 9.12ab 105.15 ± 9.14b

4.93 ± 0.49a 17.63 ± 3.93b 187.42 ± 11.28a

Stress (MPa)

Values are mean ± standard deviation. Different lowercase letters (a–c) in each column indicate significant difference (p < 0.05) using Duncan's multiple-range test.

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

due to CeO stretching vibration, CeN stretching vibration (Pereira, Souza, Cerqueira, Teixeira, & Vicente, 2010) and water molecules in the amorphous nanocellulose (Jayaramudu, Jeevan Prasad Reddy, Guduri, & Varada Rajulu, 2009). The WPI films with 2–8% w/w CNC had the same absorption peaks as the WPI film (Fig. 6). This might be due to the small amount of CNCs in whey protein film. Thus, the FTIR spectra of WPI film with 0–8% w/w CNC were not different. The higher CNC content significantly lowered the WVP value (p < 0.05) as shown in Fig. 7. The CNC particles might better disperse in the protein matrix due to the high aspect ratio and lower permeability of CNCs (Abdollahi et al., 2013; Huq et al., 2012; Pereda et al., 2011). Therefore, inclusion of CNCs in WPI film improved the WVP property of the WPI film due to the highly polar amino acids and the hydrophilic nature of WPI (Wakai & Almenar, 2015). The oxygen permeability (OP) property of films is presented in Fig. 8. Adding 2% CNC to WPI film tended to increase OP due to the CNCs being irregularly dispersed in the film because using a small amount of CNCs caused them to assemble themselves. Whereas increasing the CNC concentration to 5 and 8% resulted in decreased OP. Increasing the CNCs in the protein matrix caused increasing tortuosity that obstructed oxygen gas diffusion through the film (Abdollahi et al., 2013; Huq et al., 2012). The increased CNC content enhanced the film tensile strength and Young's modulus whereas the elongation value decreased (Table 4). Incorporation of 8% w/w CNC increased the tensile strength and Young's modulus approximately 2 and 3 times, respectively, compared with film without CNCs. There was a clear difference in the tensile strength as shown in Fig. 9. The tensile strength increased because of the greater H-bonds associated inter and intra with the CNC particles. Furthermore, the good dispersion of CNCs in the film matrix caused better connection among them, making the film stronger (Qazanfarzadeh & Kadivar, 2016). As expected, elongation decreased significantly (p < 0.05) because of the natural CNC strength and decreased flexibility of the protein matrix occurred due to the strong connection between the CNCs and protein (Bamdad, Goli, & Kadivar, 2006; Huq et al., 2012).

(D) (C) (B)

(A)

0

10

20

30

40

50

Strain (%) Fig. 9. Stress-strain curve of whey protein isolate (WPI) films with different cellulose nanocrystal (CNC) concentrations (% w/w of WPI): (A) 0 (B) 2 (C) 5 and (D) 8.

The increasing concentration of CNCs decreased the water contact angle value (Table 2). This result demonstrated that the film was more hydrophilic because of the OH– group in the CNC structure. The polarity around the structure could create H-bonding to react with water. Therefore, the higher CNC content resulted in greater hydrophilicity in the film. This result conformed to the research of Reddy and Rhim (2014) who reported agar film without CNCs had the highest contact angle and the increasing the CNC content in the film caused a decrease in the contact angle. Besides, Bahar et al. (2012) also found that the incorporation of cellulose nanowhiskers in polypropylene composite films resulted in the reduction of the contact angle in the film. Table 3 shows the transparency and the color of films. The transparency value of only WPI film was similar to that reported by other researches (Qazanfarzadeh & Kadivar, 2016; Sothornvit et al., 2010). It was found that adding more CNC content in WPI films tended to decrease the film transparency. The CNC particles dispersed in the protein film affected light scattering so that the light passing through the film decreased (Pereda, Amica, Rácz, & Marcovich, 2011). Another explanation could be the accumulation of cellulose particles obscured the light that shone through the structure of the protein film (Abdollahi, Alboofetileh, Rezaei, & Behrooz, 2013). The L* color of films was only significantly different in the WPI films with and without CNC (Table 3). The decrease in the L* value was due to the CNCs providing greater turbidity in the whey protein isolate film compared with 0% CNC film. However, the total color difference (ΔE*) of film at different CNC contents was not significantly different. The analysis of functional groups on the surface of whey protein isolate film with various CNCs by FTIR spectrophotometer is shown in Fig. 6. The FTIR spectra of the WPI film showed that the observed peak in the spectra at 3263 cm−1 was due to the OeH and NeH groups (Le Tien et al., 2000). However, the peak of the NeH group in the protein spectra was at 3254 cm−1 (Bandekar, 1992), with the difference due to the different components in the WPI film, especially the many hydroxyl groups of glycerol and the CNCs (Le Tien et al., 2000). The peak observed in the range 800 to 1150 cm−1 was glycerol occurring from CeC and CeO vibration. Five peaks were found at 850, 925, 995 1045 and 1117 cm−1 (Guerrero, Retegi, Gabilondo, & de la Caba, 2010). The presence of the peak between 1600 and 1700 cm−1 corresponded to the amide (I) vibration that related to the secondary structure of protein

4. Conclusion Sugarcane bagasse is one of the main forms of waste from the sugarcane juice extraction process in sugar production. Steam explosion and bleaching were applied to obtain high purity α-cellulose and this was used as a raw material in CNC production. The CNCs had good characteristics and high crystallinity. The addition of various amounts of CNCs into the WPI film could modify the film properties. The brightness and transparency of the film were slightly reduced. The increases in the aw value and water solubility as well as the decrease in the water contact angle indicated the increased hydrophilic nature of the film because the chemical structure of the CNCs included a hydroxyl group. The strength of film increased but the elongation decreased with the increasing CNC content. The barrier properties regarding water vapor and oxygen decreased with the increasing CNC content. Therefore, film with 8% CNC provided appropriate mechanical properties and barrier properties and can be utilized as a food packaging material. 534

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Acknowledgements

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