Accepted Manuscript

Use of seaweed and filamentous fungus derived polysaccharides in the development

of alginate-chitosan edible films containing fucoidan: study of moisture sorption,
polyphenol release and antioxidant properties

Mohamed Gomaa, Awatief F. Hifney, Mustafa A. Fawzy, Khayria M. Abdel-Gawad

PII: S0268-005X(18)30314-X
DOI: 10.1016/j.foodhyd.2018.03.056
Reference: FOOHYD 4367

To appear in: Food Hydrocolloids

Please cite this article as: Mohamed Gomaa, Awatief F. Hifney, Mustafa A. Fawzy, Khayria M. Abdel-
Gawad, Use of seaweed and filamentous fungus derived polysaccharides in the development of
alginate-chitosan edible films containing fucoidan: study of moisture sorption, polyphenol release and
antioxidant properties , Food Hydrocolloids (2018), doi: 10.1016/j.foodhyd.2018.03.056

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 ACCEPTED MANUSCRIPT

Sargassum latifolium

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Alginate & Fucoidan Chitosan

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Alg+Chit Alg+Chit+Ca Alg+Chit+Fuc Alg+Chit+Fuc+Ca
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Antioxidant properties
Kinetic modeling of moisture sorption and polyphenolic release
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1- Alginate, chitosan and fucoidan were effectively used for the
development of films.
2- Fucoidan blending decreased water solubility of the films.
3- The films exhibited good antioxidant properties.
4- The moisture sorption and migration of phenolic compounds were
fitted to Peleg's and Fick's laws.

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 ACCEPTED MANUSCRIPT
Use of seaweed and filamentous fungus derived polysaccharides in the 1

development of alginate-chitosan edible films containing fucoidan: study 2

of moisture sorption, polyphenol release and antioxidant properties 3

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Mohamed Gomaa*, Awatief F. Hifney, Mustafa A. Fawzy, Khayria M. 5

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Abdel-Gawad 6

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Botany and Microbiology Department, Faculty of Science, Assiut 8
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University, 71516 Assiut, Egypt 9
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* Corresponding author 10

M. Gomaa
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Botany and Microbiology Department, Faculty of Science, Assiut University, 12

71516 Assiut, Egypt 13

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e-mail: m_gomaa@aun.edu.eg 14
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Phone: 00201062104501 15

Fax: 002-088-2342708 16

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Use of seaweed and filamentous fungus derived polysaccharides in the 19

development of alginate-chitosan edible films containing fucoidan: study 20

of moisture sorption, polyphenol release and antioxidant properties 21

Abstract 22

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Alginate and fucoidan extracted from the brown macroalga Sargassum 23

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latifolium and chitosan derived from the filamentous fungus Aspergillus 24

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niger were used for the development of edible films with natural antioxidant 25

properties. The incorporation of fucoidan and/or Ca2+ into the alginate- 26

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chitosan films decreased water solubility, but increased film thickness, water 27
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vapor permeability and oxygen permeability. The developed films showed 28
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good barrier properties against ultraviolet light. The interactions between film 29

components were investigated using FTIR analysis which confirmed the

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presence of hydrogen bonded interaction. Kinetics of moisture sorption and 31

polyphenol release exhibited a good fit to Peleg's model. Film moisture 32

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content at equilibrium was increased by fucoidan blending. Additionally, the 33

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water vapor diffusion and polyphenol release were expressed in terms of 34

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effective diffusion coefficient based on simplified Fick's second law. The 35

developed films exhibited good antioxidant properties as measured by total 36

antioxidant assay, ferric reducing antioxidant power and hydroxyl radical 37

scavenging activity. Both film type and the type of the food simulant 38

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markedly affected the polyphenol release and the subsequent antioxidant 39

activity of the films. 40

Key words: Extraction, alginate chitosan films, Fick's second law, FTIR, 41

kinetics, Peleg's model. 42

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Chemical compounds studied in this article 43

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Sodium alginate (PubChem CID: 5102882); Fucoidan (PubChem CID: 44

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92023653); Chitosan (PubChem CID: 71853); Phloroglucinol (PubChem 45

CID: 359); Glycerol (PubChem CID: 753); Calcium chloride (PubChem 46

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CID: 5284359); Ethanol (PubChem CID: 702); Ascorbic acid (PubChem 47

CID: 54670067); Acetic acid (PubChem CID: 176). 48

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1. Introduction 49
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Nowadays there is a growing demand for biopolymer-based edible films and 50

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coatings since they are natural, renewable, nontoxic and biodegradable in 51

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comparison to synthetic ones. Therefore, the use of edible films may 52

contribute to the solution of environmental problems and play an important 53

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role in extending the shelf life of food products by preventing moisture loss, 54

oxidative rancidity and microbial spoilage (Benavides et al., 2012; Tavassoli- 55

Kafrani et al., 2016). Additionally, edible films with natural antioxidant 56

properties or films incorporated with antioxidants can supplement the 57

nutritional value of the food products (Talón et al., 2017). In these films, the 58

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incorporated compounds are released in a slow controlled process to maintain 59

an adequate concentration of these compounds for a certain period 60

(Jamshidian et al., 2012). Creating a successful antioxidant-active packaging 61

with the control release property has been the objective of many recent 62

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research studies. 63

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Brown seaweeds contain two main different types of polysaccharides in their 64

cell walls known as alginates and fucoidans. Alginate, the salt of alginic acid, 65

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are linear polysaccharides comprising (1‒4)-linked units of β-D-mannuronate 66

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(M) and α-L-guluronate (G) arranged at different proportions and different 67
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distributions in the chain as MM, MG and GG blocks (Fawzy et al., 2017). 68
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Alginate is a well known ionic polymer used mainly as stabilizing, 69

thickening, gelling and emulsifying agent in food industry and as a drug or 70

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enzyme carrier in pharmaceutical and medical industries (Tavassoli-Kafrani 71

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et al., 2016). 72
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Fucoidan is a fucose containing sulphated polysaccharide with wide 73

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promising biological applications, such as antioxidant, emulsifying, 74

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anticancer, anticoagulant, antimicrobial, anti-inflammatory and antidiabetic 75

activities (Hifney et al., 2016). However, it cannot form gels or films by 76

itself, but the mixture of fucoidan with other polymers may provide some 77

additional advantages (Venkatesan et al., 2014). 78

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On the other hand, chitosan, a deacetylated derivative of chitin, is composed 79

of β-(1-4)-linked N-acetyl-D-glucosamine and D-glucosamine units. It comes 80

from the exoskeleton of crustaceans or from the cell wall of fungi. However, 81

chitosan production from fungi grown under controlled conditions provides a 82

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promising potential for a consistent product, with high purity, well defined 83

physicochemical properties, and free of allergic shrimp protein (Bierhalz et

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84

al., 2016; Abdel-Gawad et al., 2017). Moreover, chitosan has a wide range of 85

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applications, most notably antioxidant, emulsifying, food supplement, 86

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antimicrobial, anticoagulant, and drug delivery systems (Pereda et al., 2012). 87
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Blending of alginate, fucoidan, and chitosan were reported as a promising 88

biocomposite for bone tissue regeneration (Venkatesan et al., 2014). Chitosan 89

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originated from mushroom were successfully mixed with alginate for the 90
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development of dense and porous membranes (Bierhalz et al., 2016). 91

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However, fucoidan blending with alginate and chitosan for the development 92
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of edible films was not reported yet at least regarding our knowledge. 93

Edible films developed from alginate suffer from strong hydrophilicity, 94

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which can be overcome by crosslinking with polyvalent cations such as Ca+2

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or polyamines such as chitosan (Rhim, 2004; Venkatesan et al., 2014). 96

Additionally, the development of edible films from commercial alginates did 97

not show good antioxidant properties, which may be enhanced by the 98

incorporation of exogenous antioxidant compounds such as natural plant 99

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extracts (Norajit et al., 2010). On the other hand, the use of crude alginate 100

extracts is suitable for the design of edible films with natural antioxidant 101

properties (Blanco-Pascual et al., 2014). Alginate and fucoidan are usually 102

conjugated with polyphenolic compounds during the extraction, which 103

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contribute to their bioactivities (Hifney et al., 2016). These phenols are 104

known as phlorotannins and are biosynthesized as secondary metabolites

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105

from the polymerization of phloroglucinol units. 106

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The present study aimed to develop edible films characterized by a natural 107

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antioxidant properties using crude alginate obtained from the brown 108
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macroalga Sargassum latifolium and chitosan from the filamentous fungus 109

Aspergillus niger, and to study the effect of fucoidan and/or Ca2+ additions on
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their physico-chemical and antioxidant properties. Additionally, the study 111

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aimed at evaluating the kinetics of moisture sorption and release of the co- 112
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extracted polyphenolic compounds from the films in solvents of different 113

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polarity. 114
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2. Materials and methods 115

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2.1. Seaweed material 116

The biomass of the brown seaweed Sargassum latifolium (Turner) C. Agardh 117

was collected during spring from the intertidal zone of Hurghada, Egypt (27° 118

12′ N, 33° 50′ E). 119

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2.2. Sequential extraction of alginate and fucoidan 120

The air-dried and milled algal biomass was subjected to two stage extraction 121

process. In the first stage, fucoidan was extracted from the macroalgal 122

biomass (1.5 % w/v) using citric acid solution (2 % w/v) (Hifney et al., 2016; 123

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Fawzy et al., 2017). After 2 h of extraction at 30 °C and 200 rpm, the 124

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liberated fucoidan was obtained by filtration followed by precipitation using 125

absolute ethanol (1:2 v/v), and left overnight at 4 °C. The precipitated crude 126

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fucoidan was recovered by centrifugation (6000 rpm, 15 min.). The residual 127

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biomass after fucoidan extraction was subjected to a second stage of 128
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extraction to isolate alginates. The alginic acids formed in the first acidic 129
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extraction process were converted into soluble sodium alginate by alkaline 130

treatment (1:40 g/mL) using 2 % Na2CO3 with shaking (200 rpm) for 3 h at 131
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40 °C. After filtration, alginate was precipitated using absolute ethanol (1:2 132
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v/v), and left overnight at 4 °C. The precipitated crude sodium alginate was 133
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recovered by centrifugation (6000 rpm, 15 min). The recovered fucoidan and 134

alginate were oven dried (60 °C, 24 h) and the yield was expressed as 135
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percentage in relation to the seaweed biomass. The fucose content of 136

fucoidan was determined by the cysteine-sulfuric acid method for methyl 137

pentoses using L-fucose as the standard, while its sulphate content was 138

measured by the gelatin-barium chloride method (Hifney et al., 2016). The 139

molecular weight of alginate was determined by an Ubbelohde capillary 140

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viscometer at 25 °C, and the mannuronic/guluronic acid (M/G) ratio was 141

determined by the partial acid hydrolysis method (Fawzy et al., 2017). 142

2.3. Fungal isolate and cultivation 143

Aspergillus niger Tiegh. was isolated by the surface sterilization method 144

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from the red alga Palisada perforata (Gomaa et al., 2015). The fungus was 145

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cultivated in potato dextrose broth medium in natural seawater and was 146

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incubated at 28 °C for 7 days with agitation (Abdel-Gawad et al., 2017). 147

2.4. Fungal chitosan extraction 148

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The extraction of chitosan from A. niger biomass followed the optimized 149

method reported by Abdel-Gawad et al. (2017). The milled biomass (1:40 150
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w/v) was deproteinized by NaOH treatment (1.0 M, 100 °C and 1 h), then 151
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centrifuged (6000 rpm, 15 min.) to separate the alkali-insoluble material 152

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(AIM). The AIM was washed with distilled water and recentrifuged until the 153
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pH was neutral. Chitosan was extracted from the AIM using 10% v/v acetic 154

acid (1:40 w/v) at room temperature for 6 h on a rotary shaker (200 rpm), 155
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then the acid insoluble residue was discarded by vacuum filtration. The crude 156
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chitosan was precipitated from the filtrate by raising the pH to 9.0 with 4 M 157

NaOH solution and collected by centrifugation. The fungal chitosan 158

molecular weight was determined by an Ubbelohde capillary viscometer at 159

25 °C, while its degree of deacetylation was determined by UV 160

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spectrophotometry using dual standards as described previously (Abdel- 161

Gawad et al., 2017). 162

2.3. Film preparation 163

All the films were developed using oven dried polysaccharides (60 °C, 24 h). 164

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Aqueous solutions of sodium alginate were prepared in distilled water at 25 165

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°C and 200 rpm to make a homogenous solution. Secondly, fungal chitosan 166

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was dissolved in 1% acetic acid solution and was carefully mixed with the 167

alginate solution (1:2 v/v) and homogenized at 500 rpm for 1 h at room 168

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temperature. The concentration of alginate and chitosan in the film forming 169
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solutions was kept at 2% and 1% w/v, respectively. For the preparation of 170
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alginate-chitosan-fucoidan films, 100 mg fucoidan was homogenized with 171

the alginate solution prior to the addition of chitosan. The mixture was
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homogenized at 500 rpm for 1-2 h at room temperature. Films cross-linked 173

with calcium ions were developed by dropwise addition of CaCl2 solution (10 174
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mL/90 mL polymer solution) to give a final concentration of 0.01 g CaCl2/g 175

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alginate. The mixture was homogenized at 25 °C and 500 rpm for 1 h, then 176
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glycerol was added as a plasticizer (0.3 g/g alginate), and the film forming 177

solutions were further homogenized for 1 h at the same conditions. 178

Four different films were developed from alginate and chitosan: alginate- 179

chitosan film (Alg+Chit), alginate-chitosan film crosslinked by CaCl2 180

(Alg+Chit+Ca), alginate-chitosan films blended with fucoidan 181

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(Alg+Chit+Fuc), and alginate-chitosan films blended with fucoidan and 182

crosslinked by CaCl2 (Alg+Chit+Fuc+Ca). Film forming solution (25 mL) 183

was cast into polyethylene Petri dish and dried at 30 °C. Films were detached 184

and conditioned in a desiccator at 25 °C and 52 % relative humidity (RH) at 185

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least one week prior to analysis. 186

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2.4. Film analysis 187

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2.4.1. Film thickness 188

The film thickness was measured with a 0–25 mm manual micrometer with 189

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an accuracy of ±0.01 mm in 15 random locations for each film. 190

2.4.2. Film color, light transmittance and opacity 191

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Color measurement of films was performed as described by Razavi et al. 192

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(2015). The light transmittance of the films was recorded at 200, 280, 400, 193
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600 nm against air as a blank in a UV‒vis spectrophotometer. The opacity of 194

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the film (UA/mm) was calculated by dividing the absorbance value at 600 nm 195

by the film thickness (mm). 196

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2.4.3. Water solubility 197

The solubility of the films in water was measured from the immersion of the 198

films in distilled water at 25 °C for 24 h without agitation. The insoluble film 199

fraction was collected by centrifugation (6000 rpm, 10 min) and oven dried 200

(105 °C, 24 h). The water solubility (WS %) was calculated using the 201

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expression [(W0 ‒ Wf)/W0] × 100, where W0 and Wf were the initial and the 202

final dry weight of the film sample. 203

2.4.4. Water vapor permeability (WVP) 204

The water vapor permeability (WVP) of the films was determined 205

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gravimetrically using the method described by Zhang et al. (2016). The film 206

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samples were equilibrated in a desiccator containing NaCl saturated solution 207

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(75 % RH). The film samples were fixed with elastics at the top of a 208

weighing bottle containing 5.0 g of anhydrous CaCl2. Subsequently, all the 209

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bottles covered with the films were placed into a desiccator containing NaCl 210
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saturated solution (75 % RH) at 25 °C. The weight change of the bottles was 211
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determined at 1 h intervals and over a 7 h period. The water vapor 212

transmission rate per unit area (WVTR, g/s m2), and WVP (g/m s Pa) were
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calculated using the following equations: 214

WVTR = K/A (1) 215

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WVP = WVTR × D / ∆P (2) 216

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Where K (g/s) is the slope obtained by linear regression analysis of the plot 217

between weight of the moisture gain (m) and time (t), A is the area of the 218

exposed film surface (m), D is the average film thickness (m), and ∆P is the 219

water vapor pressure difference between the two sides of the film. 220

2.4.5. Oxygen permeability (OP) 221

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Adsorption method of deoxidizing agent proposed by Zhang et al. (2016) was 222

used to determine the oxygen permeability of the films. The deoxidizing 223

reagent is composed of reduced iron powder, NaCl, and activated carbon in 224

the proportion of 0.5: 1.5: 1.0 g. Activated carbon can adsorb and transfer 225

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oxygen into iron; whereas NaCl can promote the reaction of oxygen and iron 226

through transferring electron. The reduced iron can be converted into

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227

Fe(OH)2 by reacting with water vapor at 90% RH under the promotion of 228

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NaCl and activated carbon. 3.0 g of the deoxidizing agent was put in a 229

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weighing bottle, and subsequently the bottles were covered by the film 230
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samples (pre-equilibrated at 90% RH in a desiccator with saturated BaCl2 in 231

its bottom), and fixed with elastics. The bottles were maintained at 90% RH 232
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for 48 h at 25 °C. The OP (Q) was calculated using Eq. (3), and was 233
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converted into a normalized constant film thickness, d, of 100 µm (Q100) 234

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using Eq. (4) (Jost et al., 2014). 235

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Q = (mf ‒ mi)/ (t × A) (3) 236

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Q100 = Q × (d/100) (4) 237

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Where mf is the final weight of the weighing bottle after 48 h; mi is the initial 238

weight of the weighing bottle; t is time (s), and A is the area of the exposed 239

film surface (m2). 240

2.4.6 Fourier transform infrared spectroscopy (FT-IR) 241

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FT-IR spectra of the films was recorded using Nicolet IS 10 FT-IR 242

spectrophotometer in the 4000–400 cm‒1 region by accumulation of 32 scans 243

at 4 cm‒1 resolution. 244

2.4.7. Moisture sorption 245

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The films were dried to a constant weight at 90 °C and placed in a desiccator 246

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maintained at 75 % RH and 25 °C. The changes in the weight of the films 247

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were determined at regular time intervals and the moisture content (Mt) of the 248

films as a function of time was determined using equation (5): 249

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Mt (%) = [(Wt ‒ W0) / W0] × 100 (5) 250

Where W0 is the initial dry weight of the film sample, and Wt is the weight of 251
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the film sample at fixed time (t). 252

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2.4.8. Determination of total phenolic contents of the films 253

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Film samples (100 mg dry weight) were dissolved in 3% acetic acid solution 254
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at 50 °C for 2 h. The total phenolic contents of the dissolved film were 255
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determined by Folin-Ciocalteau reagent as described by Hifney et al. (2017) 256

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with slight modification. Dissolved film sample (100 µL) was mixed with the 257

Folin-Ciocalteau reagent (100 µL) and 200 µL of Na2CO3 (20 %). The tubes 258

were incubated in the dark at room temperature for 1 h, then the total volume 259

was completed to 1 mL with distilled water, and the absorbance was 260

measured at 760 nm using a spectrophotometer. The total phenolic content 261

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was expressed as mg phloroglucinol equivalent/ g film sample (mg PGEq/g 262

film). 263

2.5. Release of polyphenolic compounds from the films 264

Three different solvents were used to study the release of polyphenolic 265

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compounds from the films: water, ethanol (10 % v/v), and acetic acid (3 % 266

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v/v), which act as a simulant for aqueous, alcoholic and acidic food products, 267

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respectively (Jamshidian et al., 2012). The release experiment was performed 268

at 25 °C for 24 h (Talón et al., 2017). Aliquots of 100 µL of samples were 269

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taken at different film-solvent contact times and the total phenolic content 270
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was determined as described in Section 2.4.8. 271
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2.6. Mathematical modeling of moisture sorption and polyphenols release 272

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Two data analysis models: Peleg's equation and Fick's diffusion model were 273
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used to analyze the moisture sorption and polyphenol release experimental 274
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data. 275

The first mathematical design was based on Peleg's equation (Peleg, 1988): 276
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Mt = M0 + [t / (K1+K2t)] (6) 277

For moisture sorption data, Mt represents the moisture content after time t, 278

and M0 is the initial moisture (M0 = 0). For polyphenols release data, Mt 279

represents phenolic content released at time t, and M0 is the initial phenolic 280

content in the solvent (M0 = 0). The constants K1 and K2 are the Peleg's rate 281

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constant and Peleg's capacity constant, respectively. The Peleg's rate 282

constant, K1, relates to the initial mass change (R0) of any component: 283

R0 = 1 / K1 (7) 284

while the Peleg's capacity constant, K2, relates to the contents at equilibrium 285

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(M∞): the maximum attainable moisture content or maximum phenolic 286

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contents. 287

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M∞ = M0 + (1 / K2) (8) 288

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The linear relationship of Eq. (6) is: 289
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t / (Mt ‒ M0) = K1+K2t (9) 290
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The linear regression analysis of the relationship between t / (Mt ‒ M0) vs. t 291

offers a simple way to calculate K1 and K2

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The second model is based on the Fick's second law of diffusion reported by 293

Crank (1979) and simplified for long-term diffusion (Mt / M∞ ˃ 0.6) using 294
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equation (10) (Cran et al., 2010): 295

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Mt / M∞ = 1 ‒ (8/π2) exp (‒π2 Dt/l2) (10) 296

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where D is the diffusion coefficient and l is the film thickness. 297

The plot of ln (1 ‒ Mt / M∞) versus time (t) should yield a straight line; the 298

diffusion coefficient (D) was calculated from the slope and film thickness (l) 299

using equation (11): 300

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D = Slope (l2 / π2) (11) 301

2.7. Antioxidant activity of the films 302

Aliquots of the film samples from the release experiment (Section 2.5.) were 303

used to determine the antioxidant activity of the films in different assays. 304

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2.7.1. Total antioxidant capacity (TAC) 305

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TAC was determined by the phosphomolybdenum assay reported by Hifney 306

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et al. (2016). Total antioxidant activity was expressed as the number of grams 307

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equivalent to ascorbic acid (g AAEq/g film) from the standard curve. 308
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2.7.2. Ferric reducing antioxidant power (FRAP) 309
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The FRAP of the films was determined by the ability to reduce Fe (III)- 310

ferricyanide complex to its Fe (II) form and measuring the formation of Perl's
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Prussian blue at 700 nm as described by Fawzy et al. (2017). The FRAP 312

values were determined against an ascorbic acid calibration curve and 313
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expressed as mg equivalents of ascorbic acid per gram of film. 314

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2.7.3. Hydroxyl radical scavenging activity (HRSA) 315

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Hydroxyl radicals were generated from FeSO4 and H2O2 and detected by their 316

ability to hydroxylate salicylate according to Hifney et al. (2016). HRSA was 317

expressed in percentage in relation to a control (without sample). 318

2.8. Statistical analysis 319

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Microsoft Windows Excel 2016 and SPSS software (IBM SPSS Statistics for 320

Windows, Version 25; IBM Corp., Armonk, NY, USA) were used to analyze 321

the resulting data. The analysis of variance (ANOVA) was performed and 322

Duncan's multiple range test was used to evaluate significant differences (p < 323

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0.05) among data of the film samples. 324

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3. Results and discussion 325

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The macroalgal biomass of Sargassum latifolium was used as a natural, safe 326

and renewable source for alginate and fucoidan, while the biomass of the 327

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filamentous fungus Aspergillus niger was used as a source for chitosan. 328
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Under the extraction conditions, fucoidan yield was 10.1 ± 0.2 %, containing 329
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49.08 ± 2.24 mg fucose/g fucoidan, and 13.33 ± 0.3 % sulphate. The yield of 330

the extracted alginate was 44.26 ± 1.5 %, which had a molecular weight of
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1.9 ± 0.2 × 105 Da, and M/G ratio of 0.57 ± 0.1. On the other hand, chitosan
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yield was 7.00 ± 0.2 %, with a molecular weight of 2.70 ± 0.3 × 104 Da and 333
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its degree of deacetylation was 83.64 ± 2.0 %. 334

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3.1. Film thickness 335

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All the developed films were easily manageable, flexible and non-sticky in 336

nature and without bubbles (Fig. 1). The thickness of the films ranged 337

between 0.107‒0.143 mm (Table 1). The addition of Ca2+ and/or fucoidan 338

significantly increased the film thickness in comparison to Alg+Chit film. 339

This effect could be attributed to the binding of Ca2+ ions to the alginate in 340

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the film matrix as well as the total solid matter of the film forming solution 341

(Benavides et al., 2012). 342

3.2. Optical properties 343

The values of the color parameters, L* (lightness/darkness), a* 344

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(redness/greenness) and b* (yellowness/blueness), are listed in Table 1. All 345

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the films had high L* and b*, while a* values were low. The incorporation of 346

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Ca2+ and/or fucoidan to the alginate-chitosan films significantly reduced L* 347

and b* values, but increased a* values (Table 1, p < 0.05). The change in the 348

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surface color of the films is an indicator for the development of crosslinking 349
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in the film molecules (Rhim, 2004). The obtained results indicated that 350
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Alg+Chit+Fuc film had the lowest lightness value, which was increased by 351

Ca2+-crosslinking in Alg+Chit+Fuc+Ca film.

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The light transmission of the films was measured in the UV range (200 and 353

280 nm) and visible region (400 and 600 nm). The values of light 354
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transmission at 600 nm were used to calculate the film opacity (Table 1). All 355
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the films exhibited no light transmission in the UV region (200 and 280 nm), 356
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providing an effective UV barrier. In the visible region, the light transmission 357

at 400 nm was zero, and reached 21.45 % and 17.20 % at 600 nm for 358

Alg+Chit and Alg+Chit+Ca films. The incorporation of fucoidan into the 359

films significantly reduced the light transmission at 600 nm to 12.05 and 12.5 360

% for Alg+Chit+Fuc and Alg+Chit+Fuc+Ca films, respectively (Table 1). 361

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This effect can be attributed to the interaction of fucoidan with the alginate 362

and chitosan in the film matrix as well as higher thickness. On the other hand, 363

the addition of Ca2+ and/or fucoidan to the alginate chitosan films exhibited 364

no significant impact on the film opacity (p ˃ 0.05, Table 1). 365

PT
The color variation and opacity of the edible films are of major importance 366

RI
since they directly influence the visualization and acceptance of food 367

products. For instance, opaque films could not be used for food products that 368

SC
should be easily visible through the package (such as minimally processed 369

U
foods) (Salgado et al., 2010). However, these intense colored films could find 370
AN
applications where the visibility of the product is not important or when the 371
M

color provide an additional usefulness, as in the case of plastics used in 372

agriculture or in light sensitive products (such as dairy products, baby foods, 373
D

soy-based sauces, and nutritional or medicinal products) (Cian et al., 2015; 374
TE

Salgado et al., 2010). The developed films in the present study could 375
EP

potentially retard lipid oxidation of food (induced by UV light) due to their 376

excellent UV light absorbance properties. 377

C
AC

3.3. Film's water susceptibility 378

Water sensitivity of polysaccharide-based films is one of the major problems 379

which has limited their application. Water sensitivity of the developed films 380

was evaluated using water solubility, water vapor permeability and moisture 381

sorption tests. The water solubility (WS) of Alg+Chit film was 89.53 %, 382

19
 ACCEPTED MANUSCRIPT
which exhibited no significant changes by Ca+2-crosslinking (Alg+Chit+Ca 383

film, Table 2). In contrast the incorporation of fucoidan significantly reduced 384

the WS of the blend films to 76.15 and 68.65 % for Alg+Chit+Fuc and 385

Alg+Chit+Fuc+Ca films (Table 2). Comparatively, the WS values were 386

PT
lower than that of alginate-fucoidan films reported by Gomaa et al. (2018). 387

Generally, films with high solubility is usually useful for the development of

RI
388

food coatings and food encapsulation, where the food and the film are 389

SC
consumed together. While, films with low solubility are usually used for food 390

U
packaging (Campos et al., 2011). 391
AN
Additionally, the water vapor permeability (WVP) of the films is a crucial 392
M

property for many practical applications of the edible films. The WVP values 393

of the developed films are listed in Table 2. The incorporation of Ca2+ and/or 394
D

fucoidan significantly increased the WVP values. The high WVP values for 395
TE

films containing fucoidan (Alg+Chit+Fuc and Alg+Chit+Fuc+Ca) should be 396

EP

attributed to the strong hydrophilic nature of alginate and fucoidan due to 397

their OH and COOH groups. According to Norajit et al. (2010) the water 398
C
AC

vapor transmission generally occurs through the hydrophilic portion of the 399

film and depends on the hydrophilic–hydrophobic ratio of the film 400

constituents. Additionally, the WVP of the films is a consequence of WS and 401

diffusion rate of water in the film matrix which, in turn, is dependent on the 402

partial pressure of water vapor (Gomaa et al., 2018). 403

20
 ACCEPTED MANUSCRIPT
3.4. Oxygen permeability 404

Oxygen permeability measurements were performed at 90 % relative 405

humidity. The general trend of the WVP values was observed for the OP 406

values. The increase of OP by Ca2+ and/or fucoidan incorporation could be 407

PT
related to the general increase of the WVP of the films. Indeed, at high water 408

RI
activity, the water sorption capacity strongly increased in the films leading to 409

the formation of water clusters. Masclaux et al. (2010) reported that the 410

SC
formation of water clusters may lead to a drastic decrease of the cohesive 411

U
energy density of the polymer matrix, which increased oxygen permeability. 412
AN
Additionally, at high RH, hydrophilic films tend to lose their gas barrier 413
M

properties due to the increase of polymer chain mobility (Forssell et al., 414

2002). 415
D
TE

3.5. FTIR analysis 416

A polyelectrolyte mixture is formed in the aqueous solutions of alginate and 417

EP

chitosan in which the negatively charged carboxyl groups of alginate possess 418
C

a strong electrostatic interaction with the positively charged amine groups of 419
AC

chitosan. The addition of fucoidan and/or CaCl2 to the alginate-chitosan 420

mixture make more complex interactions possible. The FTIR spectra were 421

used to determine the specific absorption bands of the blend films (Fig. 2). 422

The FTIR spectra of the crude alginate, fucoidan, and chitosan extracts were 423

21
 ACCEPTED MANUSCRIPT
reported previously (Hifney et al., 2016; Fawzy et al., 2017; Abdel-Gawad et 424

al., 2017). 425

The broad band at 3418.71‒3431.86 cm‒1 is attributed to the overlapping of 426

O‒H or N‒H stretching vibrations. In Ca-crosslinked films, this band was 427

PT
shifted towards a lower wavenumber, indicating the formation of more 428

RI
intermolecular hydrogen bonds (Venkatesan et al., 2014). The small band 429

between 1634-1640 cm‒1 is ascribed to asymmetric stretching vibrations of 430

SC
carboxylate groups (COO‒) of alginate with a contribution of N‒H bending 431

U
band (amide I band) of chitosan (Fawzy et al., 2017). This band was shifted 432
AN
towards a lower wavenumber in Ca-crosslinked films in comparison to 433

uncross-linked films. Ca+2 addition into the films induced alginate gelation. A
M

434

significant peak is also observed at 1562.17-1567.75 cm-1, which indicates 435

D

the amide II absorption band of chitosan (Abdel-Gawad et al., 2017). 436

TE

Additionally, the symmetric stretching vibrations of carboxylate groups 437

EP

(COO‒) of alginate appeared between 1413.48-1416.52 cm-1. The 438

corresponding characteristic frequency of fucoidan (i.e. the sulphate groups) 439

C
AC

is not clearly detectable in the films containing fucoidan. This may be due to 440

the low concentration of fucoidan (100 mg) in the films. It is worth to 441

mention here that during the film preparation, the increase of fucoidan 442

concentration (>100 mg) induced a strong gelation, which prevented the 443

formation of suitable, uniform, easily manageable films. 444

22
 ACCEPTED MANUSCRIPT
3.6. Moisture sorption 445

The results of the moisture sorption behavior of the developed films until 446

reaching equilibrium moisture content are depicted in Fig. 3a. The moisture 447

sorption was more rapid at the initial stages and declined with increasing time 448

PT
until the moisture equilibrium was attained. The data obtained from the 449

RI
sorption kinetic curve were fitted to Peleg's equation (Eq. 10), and the 450

sorption curve equations and their constants (k1 and k2) were calculated as 451

SC
shown in Table 3. The k1 values is related to the initial moisture adsorption 452

U
rate (R0) of the developed films. The higher k1 value indicates the lower 453
AN
adsorption rate. The k2 values is related to the moisture content of the film 454
M

that was absorbed when the film reached equilibrium (M∞). The higher k2 455

value indicates less absorbed moisture. The coefficients of determination in 456

D

all cases were very high (R2 > 0.99), indicating excellent fitting of the 457
TE

equations to the experimental data (Fig. 3b). A significant variation in the 458
EP

k1 values was found between Alg+Chit+Ca and Alg+Chit+Fuc films, with the 459

former having low initial moisture adsorption rate. The decrease in R0 is very 460
C
AC

important, considering that the films could be applied for packaging of 461

perishable foods with a short shelf-life. On the other hand, fucoidan 462

containing films (Alg+Chit+Fuc and Alg+Chit+Fuc+Ca films) exhibited 463

lower k2 values (Table 3), but Alg+Chit+Fuc+Ca film exhibited the highest 464

moisture content at equilibrium (M∞). Generally, the M∞ values are increased 465

23
 ACCEPTED MANUSCRIPT
by fucoidan blending, which could be attributed to the hydrophilic nature of 466

fucoidan molecules. 467

The effective diffusion coefficients (D) of water vapor in the films were 468

calculated based on the Fick's second law of diffusion simplified for the long- 469

PT
time diffusion (Mt/M∞ ˃ 0.6) and the results are listed in Table 3. The highest 470

RI
effective diffusion coefficient (D) of the water vapor was recorded in 471

Alg+Chit+Fuc film (20.16 × 10‒6 mm2/min) and significantly (p < 0.05) 472

SC
decreased by Ca2+ crosslinking (Alg+Chit+Fuc+Ca film) or in films without 473

U
fucoidan blending (Alg+Chit and Alg+Chit+Ca films). It was reported that 474
AN
the water sorption in films containing hydrophilic polysaccharides is a 475
M

complex process due to the presence of specific interactions between the 476

hydrophilic sites of the polymer and the water molecules which delays water 477
D

diffusion through the films (Pereda et al., 2012). The higher moisture content 478
TE

at equilibrium (M∞) in Alg+Chit+Fuc+Ca makes water molecules less mobile 479

EP

and decreased D values in comparison to Alg+Chit+Fuc film. 480

C

3.7. Release of polyphenols and antioxidant activity of the films 481

AC

Crude polysaccharide extracts from brown algae are usually co-extracted 482

with a small proportion of polyphenolic compounds, which contribute to their 483

antioxidant properties (Hifney et al., 2016). The total phenolic content in 484

Alg+Chit and Alg+Chit+Ca films was 5.81±0.43 mg PGEq/g film, which 485

was increased to 6.18±0.31 mg PGEq/g film by the incorporation of 486

24
 ACCEPTED MANUSCRIPT
fucoidan. The release of polyphenolic compounds from the films was 487

evaluated in three different food simulants (water, 10 % ethanol, and 3 % 488

acetic acid) (Fig. 4). Several processes could influence the release of active 489

compounds from a polymeric matrix including: (1) related polymer factors 490

PT
such as polymeric swelling, which allows the water diffusion, relaxation of 491

polymer network, molecular weight distribution, density, and orientation,

RI
492

which eventually determine sizes and shapes of the microcavities and their 493

SC
distribution, (2) related polyphenols factors such as their molecular size, 494

U
shape, density, polarity, and solubility, and (3) factors related to the 495
AN
interaction of polymeric matrix with the polyphenols, such a plasticizing or 496

antiplasticizing effect (Buonocore et al., 2003; Jamshidian et al., 2012; Talón 497
M

et al., 2017). In the current study, the release of polyphenols is dependent on 498
D

different factors such as liquid migration to the film matrix and the polymer 499
TE

solubility and diffusion of the active compound through film matrix to the 500
EP

food simulating liquid. This last phenomenon is not only due to mass transfer 501

but it is the result of different factors such as the specific interactions 502
C

between the polyphenolic compounds and the polysaccharides. The 503

AC

incorporation of fucoidan decreased the percentage of polyphenols release 504

from the films into all the solvents (Fig. 4). In addition, acetic acid solution 505

showed a maximum release of polyphenolic compounds, due to the high 506

solubility of chitosan in acidic media leading to dissociation of the hydrogen 507

bonding involving the amino groups, and consequent facilitation of the 508

25
 ACCEPTED MANUSCRIPT
entrance of solvent into the film matrix, increasing the release rate of 509

polyphenols. Talón et al. (2017) reported that the release of polyphenols from 510

starch-chitosan films was influenced by solvent polarity and pH with a 511

maximum release in acetic acid solution. 512

PT
Peleg’s kinetic model was used to describe the release of polyphenolic 513

RI
compounds over processing time. Table 4 shows the values of the Peleg’s 514

constants (K1 and K2) and the corresponding values of initial rate of phenolic 515

SC
mass transfer (R0 = 1/ K1) and equilibrium phenolic content (M∞ = 1/ K2). The 516

U
release of polyphenols from the developed films in the three different 517
AN
solvents exhibited a good fit to Peleg's equation with high R2 values (R2 ˃ 518
M

0.99, Fig. 5), indicating a good reliability of the model. The R0 values were in 519

the following order: acetic acid (3 %) > ethanol (10 %) > water for all the 520
D

films, except for Alg+Chit+Fuc+Ca film which showed higher R0 values in 521
TE

ethanol (10 %). Different film formulations exhibited a significant influence 522
EP

on the initial rate of polyphenols release with higher R0 values for Alg+Chit 523

and Alg+Chit+Ca films in solvents of higher polarity (water and acetic acid, 524
C
AC

3 %). Additionally, the maximum concentration of polyphenols (M∞) was 525

delivered in acidic media, due to the higher solubility of chitosan at low pH. 526

The M∞ values calculated using Peleg's model coincide with the experimental 527

data in Fig. 4. The ability of Peleg's model to predict the moisture value at 528

equilibrium when time tends to infinity is important since it can estimate long 529

26
 ACCEPTED MANUSCRIPT
range of values from data obtained from relatively short duration (Peleg, 530

1988). 531

The effective diffusion coefficients (D) of the polyphenols in the films were 532

estimated from the release kinetics by using Eq. (10) and are given in Table 533

PT
4. The D values were in the order of 10‒6 mm2/min and influenced by both 534

RI
film type and solvent. All the films exhibited similar diffusion properties in 535

aqueous solution since no significant variations in the D values were 536

SC
observed (Table 4). Polyphenolics released from Alg+Chit and Alg+Chit+Ca 537

U
films in both ethanol (10 %) and acetic acid (3%) showed similar trend, with 538
AN
the higher D values recorded for Alg+Chit+Ca film. However, D values for 539
M

the films containing fucoidan (Alg+Chit+Fuc and Alg+Chit+Fuc+Ca) 540

followed the following order: ethanol (10 %) > water > acetic acid (3 %). 541
D
TE

Table 5 shows the antioxidant activity of the released active compounds from 542

the films after 24 h of contact with different food simulants (water, ethanol 543
EP

10% and acetic acid 3%). The antioxidant potential of the films was 544
C

evaluated using three different assays: total antioxidant capacity (TAC), 545
AC

ferric reducing antioxidant power (FRAP) and hydroxyl radical scavenging 546

activity (HRSA). 547

The results of the TAC assay suggested that the films had strong TAC in 3% 548

acetic acid solution, which is attributed to the higher solubility and the 549

antioxidant properties of chitosan in acidic solution, and subsequent higher 550

27
 ACCEPTED MANUSCRIPT
release of phenolic compounds. No significant variation (p > 0.05) in the 551

TAC was found between aqueous and ethanolic solvents, except for 552

Alg+Chit+Fuc+Ca film which exhibited a significant variation (p < 0.05) 553

between the studied solvents (Table 5). Alg+Chit and Alg+Chit+Ca films 554

PT
showed similar TAC for each of the three studied solvents. On the other 555

hand, the incorporation of fucoidan, in Alg+Chit+Fuc and Alg+Chit+Fuc+Ca

RI
556

films, significantly decreased the TAC, with the later having the lowest 557

SC
values (Table 5). This behavior is related to the strong interaction of the film- 558

U
forming polymers, which was interrupted in acetic acid and resulted in the 559
AN
enhancement of the TAC. 560
M

In solvents of high polarity (water and acetic acid 3%), the developed films 561

exhibited a higher FRAP, except for Alg+Chit+Fuc+Ca film which showed 562
D

higher FRAP in acetic acid solution only. The higher reducing ability is 563
TE

associated with the presence of a higher content of reductones, which could 564
EP

react with free radicals to terminate radical chain reactions (Hifney et al., 565

2016). Differences in the solubility of the films in different solvents, as well 566
C
AC

as the total amount of the polyphenols released, can explain the different 567

FRAP observed in each case. Fawzy et al. (2017) reported a decrease of 568

FRAP of alginate at acidic conditions, but the higher activity of chitosan at 569

low pH may be responsible for the enhanced FRAP in acetic acid solution. 570

28
 ACCEPTED MANUSCRIPT
Additionally, the incorporation of fucoidan into the films (Alg+Chit+Fuc and 571

Alg+Chit+Fuc+Ca films) significantly decreased the HRSA (p < 0.05, Table 572

5) in aqueous solution, which is concomitant with solubility decrease. The 573

HRSA of the developed films was significantly reduced in 3 % acetic acid 574

PT
solution. This behavior is not consistent to the TAC and FRAP results, which 575

is related to the different antioxidant mechanisms, since TAC and FRAP

RI
576

measure the reducing activity, while HRSA measure the radical scavenging 577

SC
antioxidant properties. 578

U
4. Conclusion 579
AN
Naturally activated edible films with promising antioxidant properties were 580
M

developed based on alginate, chitosan and fucoidan blending. The results 581

showed that fungal derived chitosan is suitable for the development of

D

582
TE

alginate-chitosan films, which may limit the use of animal derived chitosan in 583

the future since fungal chitosan is free of allergenic shrimp protein. 584
EP

Additionally, Ca+2 and or fucoidan incorporation into alginate-chitosan films 585

C

decreased water solubility but increased film thickness, WVP and OP. 586
AC

Kinetics of moisture sorption and polyphenol release were effectively fitted 587

to Peleg’s mathematical model, which allowed the calculation of equilibrium 588

values when time tend to infinity. Alg+Chit+Ca exhibited the lowest initial 589

moisture sorption rate, while Alg+Chit+Fuc+Ca showed the highest 590

equilibrium moisture content. The maximum polyphenol delivered was 591

29
 ACCEPTED MANUSCRIPT
found in acetic acid solution, due to higher solubility of chitosan. Generally, 592

films exhibited enhanced TAC and FRAP in acidic media, but the HRSA was 593

decreased. Additionally, the effective diffusion of water vapor and 594

polyphenolic compounds were calculated based on simplified Fick’s law. The 595

PT
current study highlighted the use of seaweed and fungal derived 596

polysaccharides in a cost-effective and ecofriendly development of edible

RI
597

films. 598

SC
References 599

U
Abdel-Gawad, K. M., Hifney, A. F., Fawzy, M. A., & Gomaa, M. (2017). 600
AN
Technology optimization of chitosan production from Aspergillus niger 601
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biomass and its functional activities. Food Hydrocolloids, 63, 593-601. 602
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Barrie, J. A., & Platt, B. (1963). The diffusion and clustering of water vapour 603
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in polymers. Polymer, 4, 303-313. 604

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Benavides, S., Villalobos-Carvajal, R., & Reyes, J. E. (2012). Physical, 605

mechanical and antibacterial properties of alginate film: Effect of the 606

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crosslinking degree and oregano essential oil concentration. Journal of Food 607
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Engineering, 110(2), 232-239. 608

Bierhalz, A. C., Westin, C. B., & Moraes, Â. M. (2016). Comparison of the 609

properties of membranes produced with alginate and chitosan from 610

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mushroom and from shrimp. International journal of biological 611

macromolecules, 91, 496-504. 612

Blanco-Pascual, N., Montero, M. P., & Gómez-Guillén, M. C. (2014). 613

Antioxidant film development from unrefined extracts of brown seaweeds 614

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Laminaria digitata and Ascophyllum nodosum. Food Hydrocolloids, 37, 100- 615

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Buonocore, G. G., Del Nobile, M. A., Panizza, A., Corbo, M. R., & Nicolais, 617

L. (2003). A general approach to describe the antimicrobial agent release 618

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Campos, C. A., Gerschenson, L. N., & Flores, S. K. (2011). Development of 621

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Technology, 4(6), 849-875. 623

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Cian, R. E., Salgado, P. R., Drago, S. R., & Mauri, A. N. (2015). Effect of 624

glycerol and Ca+2 addition on physicochemical properties of edible 625

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carrageenan/porphyran-based films obtained from the red alga, Pyropia 626

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columbina. Journal of applied phycology, 27(4), 1699-1708. 627

Cran, M. J., Rupika, L. A. S., Sonneveld, K., Miltz, J., & Bigger, S. W. 628

(2010). Release of naturally derived antimicrobial agents from LDPE 629

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Crank, J. (1979). The mathematics of diffusion. Oxford, UK: Oxford 631

university press. 632

Fawzy, M. A., Gomaa, M., Hifney, A. F., & Abdel-Gawad, K. M. (2017). 633

Optimization of alginate alkaline extraction technology from Sargassum 634

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latifolium and its potential antioxidant and emulsifying 635

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properties. Carbohydrate polymers, 157, 1903-1912. 636

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Forssell, P., Lahtinen, R., Lahelin, M., & Myllärinen, P. (2002). Oxygen 637

permeability of amylose and amylopectin films. Carbohydrate 638

Polymers, 47(2), 125-129.

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Gomaa, M., Hifney, A. F., Fawzy, M. A., Issa, A. A., & Abdel-Gawad, K. M. 640
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(2015). Biodegradation of Palisada perforata (Rhodophyceae) and Sargassum 641

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sp. (Phaeophyceae) biomass by crude enzyme preparations from algicolous 642

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fungi. Journal of applied phycology, 27(6), 2395-2404. 643

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Gomaa, M., Fawzy, M. A., Hifney, A. F., & Abdel-Gawad, K. M. (2018). 644

Use of the brown seaweed Sargassum latifolium in the design of alginate- 645
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fucoidan based films with natural antioxidant properties and kinetic modeling 646
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of moisture sorption and polyphenolic release. Food Hydrocolloids, DOI: 647

10.1016/j.foodhyd.2018.03.053. 648

Hifney, A. F., Fawzy, M. A., Abdel-Gawad, K. M., & Gomaa, M. (2016). 649

Industrial optimization of fucoidan extraction from Sargassum sp. and its 650

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potential antioxidant and emulsifying activities. Food Hydrocolloids, 54, 77- 651

88. 652

Hifney, A. F., Fawzy, M. A., Abdel-Gawad, K. M., Issa, A. A., & Gomaa, M. 653

(2017). In vitro comparative evaluation of antioxidant activity of 654

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hydrophobic and hydrophilic extracts from algicolous fungi. Journal of 655

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Aquatic Food Product Technology, 26(1), 124-131. 656

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Jamshidian, M., Tehrany, E. A., & Desobry, S. (2012). Release of synthetic 657

phenolic antioxidants from extruded poly lactic acid (PLA) film. Food 658

Control, 28(2), 445-455.

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Jost, V., Kobsik, K., Schmid, M., & Noller, K. (2014). Influence of 660
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plasticiser on the barrier, mechanical and grease resistance properties of 661

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alginate cast films. Carbohydrate polymers, 110, 309-319. 662

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Masclaux, C., Gouanve, F., & Espuche, E. (2010). Experimental and 663
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modelling studies of transport in starch nanocomposite films as affected by 664

relative humidity. Journal of Membrane Science, 363(1-2), 221-231. 665

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Norajit, K., Kim, K. M., & Ryu, G. H. (2010). Comparative studies on the 666

characterization and antioxidant properties of biodegradable alginate films 667

containing ginseng extract. Journal of Food Engineering, 98(3), 377-384. 668

Peleg, M. (1988). An empirical model for the description of moisture 669

sorption curves. Journal of Food science, 53(4), 1216-1217. 670

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Pereda, M., Amica, G., & Marcovich, N. E. (2012). Development and 671

characterization of edible chitosan/olive oil emulsion films. Carbohydrate 672

Polymers, 87(2), 1318-1325. 673

Razavi, S. M. A., Amini, A. M., & Zahedi, Y. (2015). Characterisation of a 674

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new biodegradable edible film based on sage seed gum: Influence of 675

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plasticiser type and concentration. Food Hydrocolloids, 43, 290–298. 676

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Rhim, J. W. (2004). Physical and mechanical properties of water resistant 677

sodium alginate films. LWT-Food Science and Technology, 37(3), 323-330. 678

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Salgado, P. R., Ortiz, S. E. M., Petruccelli, S., & Mauri, A. N. (2010). 679

Biodegradable sunflower protein films naturally activated with antioxidant 680

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compounds. Food Hydrocolloids, 24(5), 525-533. 681

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Talón, E., Trifkovic, K. T., Vargas, M., Chiralt, A., & González-Martínez, C. 682
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(2017). Release of polyphenols from starch-chitosan based films containing 683

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thyme extract. Carbohydrate Polymers, 175, 122-130. 684

Tavassoli-Kafrani, E., Shekarchizadeh, H., & Masoudpour-Behabadi, M. 685

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(2016). Development of edible films and coatings from alginates and 686

carrageenans. Carbohydrate polymers, 137, 360-374. 687

Venkatesan, J., Bhatnagar, I., & Kim, S. K. (2014). Chitosan-alginate 688

biocomposite containing fucoidan for bone tissue engineering. Marine 689

drugs, 12(1), 300-316. 690

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Zhang, P., Zhao, Y., & Shi, Q. (2016). Characterization of a novel edible film 691

based on gum ghatti: Effect of plasticizer type and 692

concentration. Carbohydrate polymers, 153, 345-355. 693

694

PT
Figure captions 695

RI
Fig. 1: Optical images of the developed films. (a) Alginate+Chitosan film, (b) 696

SC
Alginate+Chitosan+Ca2+ film, (c) Alginate+Chitosan+Fucoidan film, (d) 697

Alginate+Chitosan+Fucoidan+Ca2+ film. 698

U
AN
Fig. 2: FTIR spectra of the films. (a) Alginate+Chitosan film, (b) 699

Alginate+Chitosan+Ca2+ film, (c) Alginate+Chitosan+Fucoidan film, (d) 700

M

Alginate+Chitosan+Fucoidan+Ca2+ film. 701

D

Fig. 3: (a) Moisture sorption of the developed films at 25 °C and 75 % RH as 702

TE

a function of time, and (b) Application of Peleg's model on moisture sorption 703
EP

data of all the films. 704

C

Fig. 4: Percentage of total phenolic compounds released from the films at 25 705
AC

°C in the three studied solvents. 706

Fig. 5: Application of Peleg's model on the release of phenolic compounds 707

from the films in different food simulants. 708

709

35
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Table 1
Thickness, color parameters, light transmission (T600), and opacity of the developed films from unrefined polysaccharides from Sargassum
latifolium and chitosan from Aspergillus niger

PT
Thickness Color parameters Opacity
Film Type T600 (%)
(mm) L* a* b* (UA/mm)

RI
c
Alg+Chit 0.107±0.007 57.47±3.57a 10.72±2.06c 56.81±5.01a 21.45±2.05a 6.28±0.39a
Alg+Chit+Ca 0.134±0.007b 50.00±4.41c 15.16±3.04ab 51.97±2.91bc 17.20±0.42b 5.71±0.08a

SC
Alg+Chit+Fuc 0.143±0.010a 46.33±4.02d 16.91±2.67a 50.31±2.79c 12.05±2.33c 6.44±0.59a
Alg+Chit+Fuc+Ca 0.136±0.009ab 54.78±3.70b 13.94±2.59b 53.96±3.96b 12.50±1.70c 6.65±0.43a

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Table 2
Water solubility (WS %), water vapor permeability (WVP (×10‒10 g H2O/m s Pa)),
and oxygen permeability (OP (×10‒3 g/100µm m2 s)) of the developed films from
unrefined polysaccharides from Sargassum latifolium and chitosan from Aspergillus
niger

WVP (×10‒10 g OP (×10‒3

Film Type WS (%)
H2O/m s Pa) g/100µm m2 s)

PT
Alg+Chit 89.53±3.76a 6.10±0.36b 5.53±0.14b
Alg+Chit+Ca 87.81±1.99a 8.45±0.01a 8.09±0.09a
Alg+Chit+Fuc 76.15±5.41b 9.22±0.72a 8.56±0.44a
68.65±4.72b 8.92±0.46a 8.18±0.51a

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Alg+Chit+Fuc+Ca

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Table 3
Parameters of Peleg's model at 25° C for moisture sorption, (K1 (min %‒1) is Peleg's
rate constant and related to the initial sorption rate (R0), K2 (%‒1) is Peleg's capacity
constant and related to the amount of water adsorbed at equilibrium (M∞)), and the
corresponding diffusion coefficient (D × 10‒6 mm2/min)

Peleg's model D (× 10‒6

Film Type
K1 K2 R0 M∞ mm2/min)

PT
ab
Alg+Chit 1.02±0.15 0.0245±0.0002a 0.98±0.12ab 40.82±0.33a 5.06±0.63b
a
Alg+Chit+Ca 1.19±0.16 0.0244±0.0002a 0.84±0.17a 40.98±0.34a 4.14±0.47b
Alg+Chit+Fuc 0.80±0.24b 0.0225±0.0003b 1.26±0.22b 44.44±0.59b 20.16±2.0a

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Alg+Chit+Fuc+Ca 0.93±0.17ab 0.0206±0.0002c 1.08±0.07ab 48.54±0.47c 4.99±0.65b

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All values are mean ± standard deviation
a,b,c
Different letters in the same column indicate significant difference among
different films (p < 0.05).

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Table 4
Parameters of Peleg's model at 25° C for polyphenolic release from the films in
different food stimulants, (K1 (min/ mg PGEq/g film) is Peleg's rate constant and
related to the initial release rate (R0), K2 (g film /mg PGEq) is Peleg's capacity
constant and related to the amount released at equilibrium (M∞)), and the

Peleg's model D (× 10‒6

Film Type
mm2/min)

PT
K1 K2 R0 M∞
Solvent: water
Alg+Chit 8.04±0.42c,1 0.226±0.008c,2 0.12±0.006c,1 4.42±0.15c,2 4.53±0.07a,1
d,1
Alg+Chit+Ca 5.80±0.21 0.225±0.004c,2 0.17±0.006d,1 4.44±0.08c,2 4.92±0.05a,1

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Alg+Chit+Fuc 13.59±0.45b,1 0.428±0.008b,2 0.07±0.002b,1 2.34±0.05b,2 5.67±0.04a,2
Alg+Chit+Fuc+Ca 24.07±1.12a,1 0.747±0.021a,1 0.04±0.002a,1 1.34±0.04a,1 4.32±0.02a,2
Solvent: ethanol (10%)

SC
Alg+Chit 4.01±0.09a,2 0.481±0.002c,1 0.25±0.06a,2 2.08±0.01c,1 3.21±0.05b,2
Alg+Chit+Ca 4.36±0.25a,2 0.481±0.005c,1 0.23±0.01a,2 2.08±0.02c,1 25.4±1.74c,2
a,2
Alg+Chit+Fuc 3.60±0.28 0.584±0.005b,1 0.28±0.02a,2 1.71±0.02b,1 13.9±1.09d,3

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Alg+Chit+Fuc+Ca 0.91±0.38b,3 0.760±0.007a,1 1.10±0.56b,3 1.32±0.01a,1 5.64±0.04a,3
Solvent: acetic acid (3%)
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Alg+Chit 2.74±0.81a,3 0.166±0.002c,3 0.37±0.12d,3 6.03±0.06c,3 3.39±0.04b,2
b,3
Alg+Chit+Ca 1.08±0.13 0.172±0.002b,3 0.93±0.11c,3 5.83±0.08b,3 26.9±3.96a,2
Alg+Chit+Fuc 3.05±0.81a,2 0.160±0.002d,3 0.33±0.01b,2 6.24±0.06d,3 3.32±0.05b,1
a,2
0.184±0.001a,2 0.29±0.06a,2 5.45±0.04a,2 2.49±0.03b,1
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Alg+Chit+Fuc+Ca 3.41±0.67
corresponding diffusion coefficient (D × 10‒6 mm2/min)
D
TE

All values are mean ± standard deviation

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a,b,c
Different letters in the same column indicate significant difference among
different films (p < 0.05).
1,2,3
Different numbers in the same column indicate significant difference among
C

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Table 5
Total antioxidant capacity (TAC, g AAEq/g film), ferric reducing antioxidant power (FRAP, mg AAEq/g film), and hydroxyl radical scavenging

PT
RI
TAC FRAP HRSA
Film Type
Water Ethanol Acetic acid Water Ethanol Acetic acid Water Ethanol Acetic acid
a,2 a,2
0.51±0.010a,1 1.69±0.10a,1 1.01±0.01a,2 1.57±0.28a,1 99.51±1.0a,1 93.79±1.5a,2 90.36±2.0a,2

SC
Alg+Chit 0.27±0.019 0.23±0.026
Alg+Chit+Ca 0.34±0.046a,2 0.28±0.016a,2 0.48±0.001a,1 1.85±0.18a,1 1.12±0.02a,2 1.50±0.05a,1 99.61±2.0a,1 95.99±0.4a,1 84.76±5.6ab,2
Alg+Chit+Fuc 0.14±0.014b,2 0.16±0.006b,2 0.46±0.038a,1 0.97±0.15b,1 0.63±0.03b,2 0.84±0.12b,1 96.41±2.2b,1 94.76±1.8a,1 87.64±0.4ab,2

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Alg+Chit+Fuc+Ca 0.02±0.001c,3 0.09±0.011c,2 0.35±0.020b,1 0.31±0.02c,2 0.39±0.05c,2 0.66±0.18c,1 82.85±0.7c,2 88.90±1.2b,1 83.01±0.6b,2
activity (HRSA, %) of the developed polysaccharide films

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Different letters within the same column indicate significant difference among different film formulations (p < 0.05).
123
Different numbers in the same row indicate significant difference among different solvents (p < 0.05).

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