Development of Water-Triggered Chitosan Film Containing

Feb 15, 2017 - 4−6 to prepare a chitosan film containing β-cyclodextrin, resveratrol−β-cyclodextrin inclusion (RCI), was developed, using glucam...
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Development of Water-triggered Chitosan Film Containing Glucamylase for Sustained Release of Resveratrol Dongliang Zhang, Yanfei Cao, Chengye Ma, Shanfeng Chen, and Hongjun Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05380 • Publication Date (Web): 15 Feb 2017 Downloaded from http://pubs.acs.org on February 23, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Development of Water-triggered Chitosan Film Containing Glucamylase for Sustained Release

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of Resveratrol

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Authors:

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Dongliang Zhang, Yanfei Cao, Chengye Ma, Shanfeng Chen, Hongjun Li*

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Address:

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School of Agricultural Engineering and Food Science, Shandong University of Technology, No.12

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Zhangzhou Road, Zhangdian District, Zibo, Shandong Province, Chian.

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Corresponding author:

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Hongjun Li

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Affiliation: School of Agricultural Engineering and Food Science, Shandong University of

13

Technology

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Contact details:

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Address: School of Agricultural Engineering and Food Science, Shandong University of

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Technology, No.12 Zhangzhou Road, Zhangdian District, Zibo, Shandong Province, Chian.

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Phone number: +86-5332786382-88

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E-mail address: [email protected]

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Abstract

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There was a paradox to incorporate enzyme into edible chitosan film that chitosan was

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dissolved in acid solution and enzyme activity was maintained in mild condition. Method for

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maintaining the pH of the chitosan solution at 4–6 to prepare chitosan film containing

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β-cyclodextrin, resveratrol–β-cyclodextrin inclusion (RCI) was developed, glucamylase and acetic

25

acid. A considerable amount of resveratrol was released by the glucamylase-incorporated film

26

within 15 days, and the maximum amount released was 46% of the total resveratrol content. The

27

highest resveratrol release ratio (released resveratrol/total resveratrol) was obtained in the film

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with 6 mL of RCI. Scratches and spores were generated on the surface of the glucamylase-added

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film immersed in water (GAFW) for 7 days because of β-cyclodextrin hydrolysis during film drying

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and water immersion. RCI and β-cyclodextrin were extruded from the film surface and formed

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teardrops, which were erased by water on the GAFW surface but appeared on the

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glucamylase-added film without water immersion (GAF). The bubbles generated by the reaction

33

of acetic acid and residual sodium bicarbonate were observed in both glucamylase-free films

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immersed in water (GFFW) for 7 days and without water immersion (GFF). The FT-IR spectra

35

illustrated that covalent bond was not generated during water immersion and β-cyclodextrin

36

hydrolysis. The crystal structure of chitosan was destroyed by water immersion and

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β-cyclodextrin hydrolysis, resulting in the lowest chitosan crystallization peak at 22°. The

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increasing of water holding capacity determined by EDX presented the following order: GAF,

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GFFW, GFF and GAFW.

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Key words: Chitosan film; Glucamylase; Resveratrol–β-cyclodextrin inclusion; Sustained release;

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Resveratrol

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Running title

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Sustained-release of resveratrol by glucamylase-incorporated chitosan film

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Introduction

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Bioactive edible films, containing natural or synthetic active compounds, are alternative

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preservatives used to inhibit microbial growth and extend the shelf life of food.1, 2 Microbial

48

growth follows four typical development phases, namely, lag phase, logarithmic phase, stationary

49

phase and decline phase. The first three steps commonly occur during food storage.3, 4 Scholars

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have developed a sustained-release film to delay or restrict the first three stages of antimicrobial

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growth; the film also functions as an antioxidant during release of bioactive compounds.

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Numerous film-forming materials containing bioactive compounds have been investigated.5, 6

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Chitosan, a non-toxic, biocompatible and biodegradable material derived from chitin, comprises a

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cationic linear polysaccharide of randomly distributed β-(1-4)-linked D-glucosamine and

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N-acetyl-D-glucosamine.7 Chitosan is one of the most abundant compounds that can be obtained

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from natural renewable sources, such as crustaceans, insects and fungi.8 Chitosan has received

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considerable research interest because of its antimicrobial activity. Chitosan inhibits both

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gram-negative

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dysenteriae, Vibrio spp. and Salmonella typhimurium) and gram-positive bacteria (Listeria

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monocytogenes, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus, Lactobacillus

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plantarum, Lactobacillus brevis and Lactobacillus bulgaricus).9-11 Furthermore, chitosan film

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exhibits satisfactory mechanical properties, good appearance (adequate gloss and transparency)

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and adequate water and gas barrier properties.12 Chitosan film has been extensively studied to

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improve its functional properties; chitosan can be combined with other bioactive compounds

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because of its highly hydrophilic behaviour and antimicrobial and excellent film-forming

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properties.13, 14

bacteria

(Escherichia

coli, Pseudomonas

aeruginosa, Shigella

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Resveratrol, a phenolic antioxidant found in many sources, including grapes, wine, peanuts,

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mulberries and soy, exhibits stronger antioxidant activity and inhibits microbial growth when

69

compared with the food benchmark.15-18 Resveratrol is a candidate compound for protecting the

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vascular walls from oxidation, inflammation, platelet aggregation and thrombus formation by

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regulating cellular signalling, enzymatic pathways, apoptosis and gene expression.19 Despite the

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potential health benefits of resveratrol, its use as a functional ingredient in the food industry is

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limited because of its poor water solubility, low bioavailability and chemical instability.20

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Resveratrol is highly soluble in ethanol, moderately soluble in triacylglycerol oils and insoluble in

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water. As such, incorporation of high levels of resveratrol into aqueous-based food products is

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difficult.21 Thus, application of resveratrol in food remains limited; the amount of active

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compounds released to the product is crucial and should be controlled to avoid spoilage and

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undesirable collateral problems.22 In this regard, scholars have developed sustained-release

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systems for many applications, especially for packaging.23 These systems mainly contain a matrix,

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entrapping materials, guest compounds and ingredients for improving physical properties.24, 25

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Cyclodextrins are cyclic oligosaccharides that consist of 6 (α-cyclodextrin), 7 (β-cyclodextrin), 8

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(γ-cyclodextrin) or more glucopyranose units attached by α-(1,4) glucosidic bonds.26

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Beta-cyclodextrin, one of the most common entrapping materials used in the food industry, is an

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enzymatically modified starch molecule and is structured similarly to a hollow truncated cone

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forming a complex with various guest molecules; β-cyclodextrin can improve the bioavailability of

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water-insoluble compounds by increasing their solubility.15, 17, 26 Encapsulation of β-cyclodextrin

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requires the application of various kinds of driving forces, but the release process is difficult to

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control.27, 28

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In this study, a chitosan film was developed to release resveratrol from β-cyclodextrin through

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hydrolysis of the β-cyclodextrin loop; the film comprised resveratrol-β-cyclodextrin inclusion

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complex (RCI), pH-adjusting ingredients, agents for improving physical properties and

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glucamylase. Chitosan was dissolved in several acidic aqueous solutions or dimethylsulfoxide

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solution, which contained inedible ingredients or had very low pH to maintain the activation of

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compounds.29 The utilization of chitosan was restricted by its solubility in few common solvents.

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Though many documents showed that some formulas were investigated to obtain mild solutions,

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these solutions require complicated protocols for solution preparation and many elements and

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inedible compounds in solutions, corrosive solvents and environmentally unfriendly ingredients.30

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Therefore, a solution of acetic acid and sodium bicarbonate with high pH was developed to

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provide a mild environment for activation of enzyme and release of resveratrol. The solution

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allows suspension of enzymatic hydrolysis of β-cyclodextrin in the dry film and triggering enzyme

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activity in water.

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Materials and methods

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Materials

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Medium molecular weight chitosan (CAS, 9012-76-4, deacetylation degree >90%, Bide

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biotechnology Ltd., Shanghai China), resveratrol (CAS, 501-36-0, Baishun Biotechnology Ltd.,

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Shanghai, China), β-cyclodextrin (CAS, 68168-23-0, Zhongtai Food Ltd., Henan, China),

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glucamylase (CAS, 9032-08-0, 105 U/g, Aobokangxing Biotechnology Ltd., Beijing, China) were

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used to prepare a film-forming dispersion at the optimum temperature of 65 °C and optimum pH

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of 4.0–6.0, ethanol (CAS, 64-17-5, >99.5%,

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64-19-7,

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5949-29-1, >99.5%, Aladdin Ltd., Shanghai, China), sodium bicarbonate (CAS, 144-55-8, >99.8%,

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Aladdin Ltd., Shanghai, China) and water (distilled water).

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Film preparation

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pH determination of pre-preparation solution

>99.5%,

Baishun

Biotechnology

Aladdin

Ltd.,

Ltd., Shanghai, China), acetic acid (CAS, Shanghai,

China),

citric

acid

(CAS,

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Pre-preparation solution was used to dissolve the main components (chitosan, glucamylase,

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resveratrol-β-cyclodextrin complex and agents for improving physical property) of the film. Five

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pre-prepared solutions were maintained at 40 °C, and pH was measured at residual volumes of

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100, 80, 60, 40, 20, 10 and 5 mL after moisture evaporation. Table 1 shows the compositions of

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the five pre-preparation solutions.

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Preparation of RCI

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An inclusion complex was prepared through a simple procedure with 1:2 molar ratio of

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β-cyclodextrin and resveratrol in the final solution. Resveratrol was dissolved in ethanol (35

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mg/mL). Briefly, 10 mL of the dissolved solution was obtained and dispersed into 90 mL of

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β-cyclodextrin (1% wt) aqueous solution (5 mL/min flow rate) under stirring with an

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electromagnetic stirrer for 15 min. The solution was subjected to ultrasound treatment for 15

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min and stirred for another 15 min with the electromagnetic stirrer. The insoluble sediment

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(resveratrol) was removed by filtration.

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Standard curves of resveratrol and glucose

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Absorbance of resveratrol solutions (0, 40, 80, 100, 200, 400, 800, 1000, 2000, 5000, 8000,

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10000, 15000 and 20000 μg/mL dissolved in 50% ethanol) was determined by

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spectrophotometer at 306 nm to establish a standard curve.

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Reducing sugar content was measured by 3,5-dinitrosalicylic acid assay.31 The absorbance of

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aqueous glucose solutions (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg/mL) were

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determined to establish a standard curve.

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Evaluation of the hydrolysis degree of β-cyclodextrin

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Certain amount of β-cyclodextrin was dissolved in 0.05 mol/L acetic acid–sodium acetate

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buffer (pH = 4.5) to obtain 1.6, 2.5, 4, 6, 8 and 10 mg/mL solutions (procedure was depicted in

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2.2.5). Glucamylase (1g) was added in 100 mL of water, stirred using an electromagnetic stirrer

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for 10 min and then filtered. β-Cyclodextrin solution (5 mL for each solution) was mixed with

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glucamylase solution (1 mL) and incubated at 5 °C and 25 °C. After 3 days (72 hours), 1 mL each of

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the above-mentioned reaction solutions were mixed with 0.5 mL of 3,5-dinitrosalicylic acid

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reagent and incubated at 100 °C for 5 min. Absorbance of appropriately diluted reaction mixture

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was determined by spectrophotometer at 540 nm. The content of reducing residues in the

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hydrolysate was determined using the amount of glucose. An index (ratio of β-cyclodextrin and

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reducing residue) was used to evaluate the degree of β-cyclodextrin hydrolysis.

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Film formation

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The film-forming dispersion was prepared by the following procedures. Water (30 g) was mixed

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with chitosan (1 g) with an electromagnetic stirrer until the homogeneous dispersion of chitosan

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was obtained. The chitosan dispersion was added with 10 mL of acetic acid (1 mol/L), 10 mL of

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water, 5 mL of sodium bicarbonate (0.5 mol/L) and 1 mL of glycerol and homogenised with 5 min

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stirring interval. The homogenised solution was added with water to obtain a total volume of 85

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mL. A certain volume of RCI solution and 1% glucamylase (prepared as described in 2.2.4) were

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blended with the homogenised solution in sequence. Water was added to obtain a final volume

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of 100 mL.

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The modified film was obtained by casting the film-forming dispersion onto a

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polytetrafluorethylene (PTFE) plate (15 cm diameter). The dispersion was dried in a sealed

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container with saturated aqueous solution of magnesium nitrate at 40 °C and 50% relative

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humidity for 36 h in the dark to protect against light.

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Film characterization

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Film thickness

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A handheld micrometre (Mitutoyo, Japan) was used to measure the film thickness in five

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different points of each film.

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Mechanical properties

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Food texture analyser (TMS-2000, USA) was used to assess the mechanical properties of the

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film. The films were cut into rectangular strips (1 cm width and 2 cm length), conditioned at 25oC

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and 50% humidity for 48 h before testing, and promptly mounted and stretched at a rate of 50

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mm/min until breaking. Tensile strength (TS) and percentage of elongation (E%) at the breaking

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point were determined from stress–strain curves, which were obtained from force–deformation

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data. Shear force was tested by the crosscut of double layers of film strips. The results of these

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three physical indicators were divided by average film thickness to reduce the deviation induced

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by inconsistent thickness. The experiments were performed in five replicates for each film.

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Optical properties

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Colour coordinate Colorimeter (CM-3600A, Konica Minolta, Japan) and ultraviolet-visible

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spectrometer (UV-2102PCS, Unico Instrument Ltd. China) were employed to determine the

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optical properties of the film. Total colour difference (ΔE) was calculated as follows:32  = ∗ +  ∗ +  ∗

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Absorbance and transmittance value After immersion in water for 1 hour, the film was cut

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to fit the cuvette and attached on the side near the light source (blanking with empty cuvette).

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Absorbance and transmittance were recorded at every 24 nm wavelength from 250 nm to 994

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nm. Final values were calculated by dividing the film thickness.

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Determination of sustained-release resveratrol and reducing sugar

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Films containing different volumes (2, 4, 6, 8 and 10 mL) of RCI solution were prepared.

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Glucamylase solution was replaced by water in control groups (glucamylase-free films). The films

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were submerged separately in 50 mL of water for 15 days, and lixivium was replaced by 50 mL of

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distilled water every 2 days. The contents of the reducing sugar (glucose content) and resveratrol

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in the lixivium were determined by the method described in the part of standard curves of

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resveratrol and glucose.

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Scanning electron microscopy (SEM)

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The prepared RCI solution (6 mL, following the part of preparation of RCI) was added to

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fabricate a film. Glucamylase was inactivated by 5 min of boiling and was used in control groups

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(glucamylase-free films). Dry films were stored directly in a sealed package for 7 days or

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immersed in water for 5 days and dried for 2 days. The films were analysed by SEM, X-ray

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diffraction, Fourier transform infrared spectroscopy (FT-IR), energy-dispersive X-ray spectrometer

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(EDX) and X-ray spectrometer analyses to investigate differences between films containing

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non-hydrolysed and hydrolysed β-cyclodextrin. The morphology of the film was observed using

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SEM with an FEI Sirion 200 microscope (PHILIPS Ltd., Netherlands).

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X-ray diffraction

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X-ray scattering measurements of the samples were performed with an X-ray diffractometer

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(D8-ADVANCE, Bruker AXS, Germany) equipped with a copper tube operated at 35 kV and 30 mA

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with Cu radiation of 0.154 nm wavelength. Diffractograms were obtained by scanning from 3° to

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50° at a rate of 0.5°/min, with a step size of 0.02.33

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Film stability determination by FT-IR spectra

202

The FT-IR spectra of the samples were recorded using a Nicolet 5700 spectrophotometer

203

(Thermo Nicolet 5700, USA). The powdered samples were mixed separately with an analytical

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grade KBr and then pressed into discs. The spectra of the samples were recorded in the region of

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4000–400 cm-1, with a total of 32 scans. The baseline was adjusted against a KBr background.34

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Energy-dispersive X-ray spectrometer (EDX) analysis

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Energy-dispersive X-ray spectrometer (Oxford INCA Energy, UK) with 128 eV energy resolution

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was used to determine the proportion of elements (C and O). The electron beam was focused on

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the location where the elemental composition should be determined. X-ray signals were

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collected by EDX because of the interaction between the primary electron and the sample.

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Results and discussion

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pH determination of pre-preparation solution

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Glucamylase exhibited hydrolysis activity in dispersions with appropriate pH, and chitosan was

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insoluble in solution with high pH. Furthermore, the evaporation of the film-forming dispersion

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during drying induced pH change, which was important to the enzyme activity in the final film.

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Thus, the pH change of the pre-preparation solution was measured (Figure 1).

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The same increasing pH trends accompanying the volume shrinkage were observed in five

218

pre-preparation solutions. The pH of the solution containing acetic acid, citric acid (1 mL) and

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sodium bicarbonate (7 mL) initiated at 4.03 possessed the lowest position in the entire

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evaporation process (HAC+LCA+HSOB in Figure 1), which was unsuitable to maintain the enzyme

221

activity. The terminal pH values of HAC+HSOB and HAC+SA solutions even reached around 5.6

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which was equal to the pH of carbon dioxide saturated solution. Considering the rise of pH and

223

the evaporation of acetic acid in film formation, solutions with relative high pH were abandoned

224

(HAC+HSOB and HAC+SA in Figure 1).. Finally, solutions of HAC+LSOB and HAC+SA+HCA were

225

suitable for the preliminary pre-preparation solutions, however, films made by 10 mL citric acid

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and 2 mL citric acid (HAC+SA+HCA) contained much more inorganic salt which resulted that film

227

was broken in drying process. A solution comprising acetic acid and 5 mL of sodium bicarbonate

228

(0.5 mol/L) was selected in this trial for maintaining the enzyme activity (HAC+LSOB).

229

Evaluation of β-cyclodextrin hydrolysis degree

230

The reducing groups engaged in estimating the hydrolysis degree of starch were investigated.35

231

The ratio of the reducing residue and β-cyclodextrin (mol/mol) was used to investigate the

232

optimum additive proportion of β-cyclodextrin and glucamylase. A low hydrolysis ratio (reducing

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residue and β-cyclodextrin ratio) induced by a low substrate–enzyme ratio was observed when

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the substrate–enzyme ratio was less than two (Figure 2) denoting an optimum substrate–enzyme

235

ratio of 2:1. Moreover, the amount of reducing residue elevated subsequently with an increase in

236

substrate–enzyme ratio exceeding or equating to two. Thus, there was no significant difference

237

between the hydrolysis degrees at different points. Considering an elongation of

238

sustained-release time of the film, the substrate-enzyme ratio of the film was less than 2:1, with

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sequential addition of 4, 6, 8 and 10 mL of RCI solution.

240

Mechanical properties

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The physical properties of a film, including anti-shearing and deformation resistance, were not

242

the critical parameters for the sustained-release film, but they referred to the operability in

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manufacture and storage. Shear force and elongation percentage were divided by film thickness

244

and the relative values were obtained, which sharply decreased at 4 mL of RCI addition. Tensile

245

strength decreased with a higher slope from 0 mL to 6 mL of RCI solution addition than with the

246

solution from 6 mL to 10 mL (Figure 3).

247

Both β-cyclodextrin and oligosaccharide (produced by hydrolysis of β-cyclodextrin) were

248

physical-improvement ingredients in film-forming.36,

37

249

hydrogen bonds between NH3+ of the chitosan backbone and OH− of the β-cyclodextrin and

250

oligosaccharide led to an improved physical properties of the film at a certain RCI addition

251

amount.38 However, opposite result was observed in the chitosan film where the glucamylase

252

hydrolysis (a dynamic process throughout the film-forming process) destroyed the cross-linked

253

β-cyclodextrin-chitosan, resulting in reduced physical properties of the film (lines in Figure 2).

254

Optical properties

The formation of inter-molecular

255

Absorbance value The absorption peaks of five films appearing from 260 nm to 380 nm were

256

elevated following the increase in RCI addition, which agreed with the absorption peak of

257

resveratrol (Figure 4A).39 Other absorption peaks at 250 nm that existed in all solutions including

258

the RCI-free solution were induced by chitosan.40 The combination of resveratrol, β-cyclodextrin

259

and chitosan did not change the absorption property of each ingredient. The interlinkage among

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the chitosan groups was supposed to reduce the absorbance at 250 nm, because the absorbance

261

values were decreased accompanying the decrease in the RCI addition which is a

262

structure-destroyed ingredient. In determining the film absorbance in wavelength from 250 nm

263

to 994 nm, there was no significant difference between the absorbance values of the six samples

264

in the wavelength from 418 nm to 994 nm and a visible difference was observed from 260 nm to

265

380 nm.

266

Colour coordinate Values of ΔE approximately overlapped with lightness (L*) suggesting that

267

the major difference in optical property was lightness. The colour of the packaging is an

268

important factor in terms of general appearance and consumer acceptance. The results of the

269

measurements performed on colour were expressed in accordance with the CIELAB system, the

270

rectangular coordinates (L*, a* and b*), and the total colour difference (ΔE).41 The values of ΔE

271

approximately overlapped with lightness (L*) indicating that the major difference in optical

272

property was lightness. The main difference was that films with higher content of RCI had lighter

273

colour as indicated by the L* value and similar changes were observed in a*, b* and ΔE (Figure

274

4B). The yellowness (b∗) possessed a high increase in colour compared with the variation of b*

275

(redness) demonstrating that the film acquired yellow colour with an increase in RCI.

276

Nevertheless, RCI, a white colour ingredient, was not regarded as yellowness-contributor. The

277

colour change was possibly attributed to glucamylase which is an ingredient with a yellow colour.

278

RCI breaking of the chitosan-chitosan interlinkage and packaging the glucamylase was supposed

279

to elevate the yellowness of chitosan and glucamylase. The film incorporated with more

280

β-cyclodextrin demonstrated an increase in the whiteness induced by the light reflecting the

281

property of β-cyclodextrin.

282

Sustained-release of resveratrol and reducing sugar

283

The amount of resveratrol release, monitored within 15 days, was the critical factor in the

284

sustained-release film, and glucose content was also determined to confirm the degree of

285

β-cyclodextrin hydrolysis which opened the inclusion of β-cyclodextrin and released resveratrol.

286

The resveratrol release amount of glucamylase-added and glucamylase-free films decreased

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following the immersion time (Figure 5A and B). Glucose release amount of glucamylase-added

288

also possessed the decline trend as resveratrol (Figure 5C), and almost no glucose was detected

289

in glucamylase-free films.

290

Induced by the hydrolysis of glucamylase, glucamylase-added films released nearly four times

291

more resveratrol than control groups with the same volume of RCI solution. A vast descent of

292

resveratrol release amount was detected among the films with different volumes of RCI solution

293

(Figure 5A), however no descent of glucose release amount was observed in glucamylase-added

294

films. This contradiction suggested that the immobilization of enzyme and substrate effected the

295

enzyme hydrolysis in films. Polysaccharides (heptose, hexose, pentose, etc.), produced by

296

hydrolysis in films with higher content of substrate (6, 8 and 10 mL of RCI solutions) was

297

immobilised in the film, leading to decreased amount of reducing groups (Figure 5C). The

298

decrease in glucose amount and the constant resveratrol release in film with 2 mL of RCI solution

299

demonstrated that the RCI number close to the enzyme decided the amount of released

300

resveratrol by opening the surrounding of β-cyclodextrin. A stable glucose release including

301

traces was detected after 9 days in glucamylase-added films, but the resveratrol amount still

302

decreased and was higher than glucamylase-free films with the same additive volume of RCI

303

solution. Similar resveratrol release trend in the glucamylase-free films suggested that the

304

weakened inclusion function of β-cyclodextrin induced by water evaporation, acetic acid

305

volatilization and interlinkage with other groups forced β-cyclodextrin to release the resveratrol

306

(Figure 5B). An acidic environment contributing to the hydrolysis of β-cyclodextrin also played a

307

role in sustained-release of the resveratrol both in glucamylase-free and glucamylase-added

308

films.42 It was difficult for acid environment to induce the generation of small molecular weight of

309

reducing sugar that could escape from the interlinkage with other macromolecular( such as the

310

chitosan), which resulted that almost no reducing sugar was released by glucamylase-free films.

311

Glucamylase-added films with 2, 4, 6, 8 and 10 mL of RCI solution released 0.288, 0.844, 2.04,

312

2.29 and 3.05 mg of resveratrol, respectively, within 15 days, corresponding to 18.46%, 27.06%,

313

43.59%, 36.63% and 39.11% of the total resveratrol. Meanwhile, 15.95, 21.38, 21.82, 22.82 and

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26.58 mg of glucose were released consecutively from those groups, indicating that 0.0886,

315

0.1187, 0.1212, 0.1267 and 0.1476 mmol reducing residual were detected. The total amount of

316

β-cyclodextrin added in the film was 0.0845 mmol. These data revealed that low hydrolysis

317

degree of β-cyclodextrin still had the ability of holding resveratrol together with the constraint

318

force of chitosan. The film containing 6 mL RCI solution needs further experiments due to the

319

resveratrol release ratio of the film and the trace amount of resveratrol for maintaining

320

antibacterial and antioxidant functions.43

321

Morphologies of films

322

GAFW revealed a coarse surface with scratches and pores (arrow heads in Figure 6A, B and C)

323

whereas GFFW displayed a slippery and uneven surface with bubbles inside (arrow heads in

324

Figure 6D, E and F).

325

Carbon dioxide, dissolved in film-forming dispersion and produced by the chemical action of

326

acetic acid and residual sodium bicarbonate in the film-forming process, was entrapped by the

327

interlinkage of chitosan, β-cyclodextrin and other ingredients, which generated the bubbly

328

surface of glucamylase-free film. The buoyancy of bubbles acting on the flexible film surface at

329

the beginning of film solidification was supposed to be the cause of uneven surfaces of

330

glucamylase-free films (arrow heads in Figure 6 D, E, F, J, K and L). Meanwhile, the film’s surface

331

was more quickly converted to solid than inside, and the carbon dioxide bubbles moving up the

332

film-forming dispersion were blocked by the solid surface of the film causing the bubbles to move

333

closer to the surface of GFFW (arrow heads in Figure 6 D, E and F). Escape of entrapped bubbles

334

through the breakage formed by the hydrolysis of β-cyclodextrin in film solidification and

335

immersion process generated the scratches and pores on the surface of GAFW (Figure 6 A, B and

336

C).

337

GAF obtained a coarse surface with teardrop-shaped objects (Figure 6G, H and I), however, GFF

338

showed a slippery surface (Figure 6J, K and L). Escape of carbon dioxide bubbles in film-forming

339

process, accompanying with the hydrolysis of β-cyclodextrin, opened the film surface with pores

340

and allowed the unconsolidated dispersion to escape by the extrusion pressure of film

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solidification, this process induced the formation of teardrop-shaped objects (Figure 6G, H and I).

342

The bubbles hardly escape across the solid surface of the film without the hydrolysis of

343

glucamylase leading to the absence of the teardrop-shaped objects on the surface of GFF (Figure

344

6J, K and L).

345

X-ray diffraction

346

The X-ray diffraction analysis was also performed to determine the film stability and the results

347

are illustrated in Figure 7. Water decreased the crystallization peak area and height and broke the

348

crystal structure through the dissolution of the β-cyclodextrin and hydration of chitosan;

349

furthermore, β-cyclodextrin hydrolysis induced by glucamylase aggravated the crystal structure in

350

GAFW which had the lowest peak in four samples (Figure 7A). Two scattered peaks was found in

351

GFFW (circle and square of Figure 7B) and GAF (circle and square of Figure 7C). Water had strong

352

ability in alleviating crystal structure showing that the crystallization peaks of films without water

353

immersion had higher peaks at 22° than films with water immersion.

354

Three

crystal

structures

including

resveratrol

crystal,

β-cyclodextrin

crystal

and

355

resveratrol-β-cyclodextrin crystal were predictably generated by the simple chemical reactions in

356

film preparing, water immersion and drying. The hydrolysed β-cyclodextrin could not form a

357

crystal structure because of the high hydrolysis degree of β-cyclodextrin, proved by the release of

358

0.1212 mmol reducing residual from 0.0845 mmol β-cyclodextrin. Resveratrol-β-cyclodextrin

359

displays a pattern in the 5°–30° area where the peaks assigned to resveratrol almost

360

disappeared;44 the chitosan peaks at 12° was enlarged and disappeared with the addition of

361

β-cyclodextrin.45-47 These results coincided with the X-ray diffraction pattern of GFF that

362

contained resveratrol-β-cyclodextrin and β-cyclodextrin (Figure 7D). This result also specified that

363

peaks of GFFW and GFF between 10° and 20° was generated by resveratrol monomers which had

364

crystal

365

resveratrol-β-cyclodextrin inclusion to create new crystallization peaks in 10°–20° was impossible

366

because no peak was observed in GFF (rectangle in Figure 7D).

367

peaks

at

6°,

16°,

19°

and

22°,48

Furthermore,

the

β-cyclodextrin

and

The peaks of resveratrol-β-cyclodextrin inclusion crystal and β-cyclodextrin crystal in GAF could

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not generate under the hydrolysis of glucamylase in drying process and without the mobility in

369

water immersion. Nevertheless, resveratrol was released by hydrolysis of glucamylase and

370

restricted by the formation of film solid surface generated in drying process. The pores on the

371

film surface of GAF caused by the β-cyclodextrin hydrolysis allowed the outflow of mobilisable

372

resveratrol and β-cyclodextrin hydrolysis resulting in the peak of GAF between 10° and 20° to be

373

lower than the peak of GFFW at the same position. The extrusion in GAF process observed by

374

SEM in 3.4 allowed the formation of tighter structure than other films, which demonstrated that

375

β-cyclodextrin and resveratrol-β-cyclodextrin inclusion had opposite functions on the crystal

376

formation of chitosan film (Figure 7C). Resveratrol, released from β-cyclodextrin in 7 days water

377

immersion, was restricted in film for no resveratrol releasing spores on the film surface, therefore

378

resveratrol crystallization peak in GFFW was higher than GAF (circles in Figure 7B and C). Linkages

379

of β-cyclodextrin–chitosan and chitosan–chitosan could not recover and obtain the same regular

380

crystal structure as before in the second drying process (after 7 days immersion). This

381

unrecoverable structure of chitosan linkages led to a high chitosan film peak at 22° in GFF and a

382

low one in GFFW. No peak was found in GFF between 10° and 20° for the homogeneous scatter

383

and immobilization in film (rectangle in Figure 7D), conversely, resveratrol release, β-cyclodextrin

384

hydrolysis and dissolution of β-cyclodextrin hydrolysate destroyed the structure of GAFW and

385

resulted the low peak at 22° and no peak in 10°–20° (Figure 7A).

386

Film stability determination by FT-IR

387

Chemical reactions including the resveratrol β-cyclodextrin inclusion, hydrogen bond

388

generation between β-cyclodextrin and chitosan, occurred between sodium bicarbonate and

389

acetic acid, and hydration. No chemical reactions of covalent bond generation or break among

390

the main ingredients of films including chitosan, resveratrol, β-cyclodextrin and glucamylase were

391

observed during film preparation, except the hydrolysis of α-1, 4-glucosidic bond which induced

392

hydroxyl generation. In particular, peaks of hydroxyl produced from β-cyclodextrin hydrolysis and

393

hydrogen bond between β-cyclodextrin and chitosan were located at the same position with

394

hydroxyl and hydrogen bond of chitosan aqueous solution, which were investigated by many

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researchers and generation of chemical bonds was easily deduced by FT-IR spectra.49, 50 Thus,

396

films including glucamylase-added and glucamylase-free films were prepared and tested to

397

determine the film stability.

398

The stability of GAFW, compared with GFF and GAF, was determined by FT-IR spectra. No peaks

399

disappeared after resveratrol release and β-cyclodextrin hydrolysis in GAFW that contained same

400

peaks as other films with no disappeared and generated peaks (Figure 8), which suggested that

401

no covalent bond generated or broke and the hydrolysis of α-1, 4-glucosidic bond and the

402

hydroxyl generation were the dominating reactions. The peaks at the same position had the same

403

width illustrated that water immersion and reactions in water immersion did not enhance or

404

weaken the bonding.

405

The peaks of films at 3357.513 cm−1 (-OH stretching), 2921.673 and 2871.532 cm−1 (-CH

406

stretching), 1423.231 cm−1 (-COO- stretching of acetic acid), 1386.589 cm−1 (-OH bending),

407

1058.754 and 1025.961 cm−1 (skeletal vibration) were identified, and -NH2 bending at 1641.151

408

and 1529.939 cm−1 supposed to be caused by the generation of hydrogen bond between -NH2

409

and -OH (β-cyclodextrin and chitosan).49,

410

gradually stronger in sequence, which represented the bond density. Resveratrol-β-cyclodextrin

411

inclusion was linked with main skeleton of chitosan by the hydrogen bond. Almost no hydration

412

and hydrolysis happened after drying in GFF (Figure 8GFF), whereas β-cyclodextrin dissolution

413

and hydration brought down the bond dentistry of GFFW (Figure 8GFFW). Hydration and

414

hydrolysis of β-cyclodextrin destroyed the crystal structure in GAF, especially when the

415

hydrolysate was held in films (Figure 8GAF). The film structure was rearranged and formed in

416

water by releasing the hydrolysate and resveratrol, which obtained higher bond density in the

417

drying process after water immersion (Figure 8GAFW).

418

EDX analysis

51

The peaks in GAF, GAFW, GFFW and GFF were

419

Film water-holding capacity determined by C/O ratio was measured by EDX after drying at

420

40 °C for 36 hours (Table 2 and Figure 9). The linkage of β-cyclodextrin-chitosan, formed in

421

film-drying process, was broken by hydrolysis of β-cyclodextrin resulting the loose film structure

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which could not afford enough hydrogen bonds to hold water, which was the reason that atomic

423

percent of C in GAFW was higher than other films (Table 2). More water combined with chitosan

424

and β-cyclodextrin in water immersion and decreased the C/O ratio of GFFW. Hydrolysed

425

β-cyclodextrin, combining more water than β-cyclodextrin and resveratrol-β-cyclodextrin

426

inclusion in GAF, also displayed a lower C/O ratio than GAFW and GFF (Table 2). Without any

427

interference of water and enzyme, the film obtained a compact structure by connecting

428

β-cyclodextrin and chitosan and rarely allowed the hydration. Water-holding capacity was

429

decreased in the order, GAF, GFFW, GFF and GAFW.

430

Conclusions

431

Chitosan film containing glucamylase-induced resveratrol release was developed and detected.

432

The pH of a solution, a crucial factor for maintaining glucamylase activity and dissolving chitosan,

433

was determined following the evaporation of pre-preparation solution and the results showed

434

that 10 mL acetic acid (1 mol/L) and 5 mL sodium bicarbonate (0.5 m/L) could obtain proper pH

435

and decrease the effect of high inorganic salt content on the film’s physical property.

436

Substrate–enzyme ratio, used to determine the addition amount of β-cyclodextrin and

437

glucamylase, was determined by hydrolysis degree of β-cyclodextrin, and substrate-enzyme ratio

438

of the film should be less than 2:1 (2, 4, 6, 8 and 10 mL). The physical properties were reduced

439

following the increase of RCI, conversely, absorbance value at 250 nm to 400 nm and colour

440

coordinate increased.52 The difference in the four film morphologies was mainly induced by

441

water immersion and/or glucamylase by destroying film surface and hydrolysing β-cyclodextrin

442

which also had an effect on the film’s crystal stability and water-holding capacity.

443

Glucamylase improved the release amount of resveratrol and glucose within 15 days

444

experimental period. GAFW with 2, 4, 6, 8 and 10 mL of RCI solution released 18.46%, 27.06%,

445

43.59%, 36.63% and 39.11% of total resveratrol, respectively. Meanwhile, 15.95, 21.38, 21.82,

446

22.82 and 26.58 mg of glucose were released from those groups. The film with 10 mL RCI

447

released less than 1/3 resveratrol at the 15th day than the 1st day. Though the release amount of

448

resveratrol was detectable, β-cyclodextrin did not maintain its activity for 15 days. Incomplete

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hydrolysis of β-cyclodextrin still had the inclusion function and released resveratrol at the last

450

days of the experiment period. Improvement of glucamylase activity was critical for

451

enzyme-added film and two possible methods were designed for the further research,

452

activity-maintaining ions (Ca2+, Mg2+), usage of thermostability enzyme and sustained-release

453

enzyme by the inclusion.

454 455

Acknowledgement

456

This work was supported by

457

1. Development of Science and Technology Support Plan of Shandong (2013GSF12108).

458

2. National Natural Science Foundation of China, Project supported by the National Natural

459

Science Foundation of China, 31471676.

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food-grade industrial waste in the obtaining active biodegradable films for packaging. Industrial Crops and Products 2016, 87, 218-228. 2.

Silva, Â.; Duarte, A.; Sousa, S.; Ramos, A.; Domingues, F. C., Characterization and antimicrobial

activity of cellulose derivatives films incorporated with a resveratrol inclusion complex. LWT - Food Science and Technology 2016, 73, 481-489. 3.

Thatoi, H.; Dash, P. K.; Mohapatra, S.; Swain, M. R., Bioethanol production from tuber crops

using fermentation technology: a review. International Journal of Sustainable Energy 2016, 35. 4.

Khan, M. A.; Abbasi, B. H.; Shah, N. A.; Yücesan, B.; Ali, H., Analysis of metabolic variations

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Kakaei, S.; Shahbazi, Y., Effect of chitosan-gelatin film incorporated with ethanolic red grape

seed extract and Ziziphora clinopodioides essential oil on survival of Listeria monocytogenes and chemical, microbial and sensory properties of minced trout fillet. LWT - Food Science and Technology 2016, 72, 432-438. 9.

Helander, I. M.; Nurmiaho-Lassila, E. L.; Ahvenainen, R.; Rhoades, J.; Roller, S., Chitosan disrupts

the barrier properties of the outer membrane of Gram-negative bacteria. International Journal of Food Microbiology 2001, 71, 235-244. 10. Liu, H.; Du, Y.; Wang, X.; Sun, L., Chitosan kills bacteria through cell membrane damage. International Journal of Food Microbiology 2004, 95, 147-155. 11. Hong, K. N.; Na, Y. P.; Lee, S. H.; Meyers, S. P., Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. International Journal of Food Microbiology 2002, 74, 65-72. 12. Sánchez-González, L.; Chiralt, A.; González-Martínez, C.; Cháfer, M., Effect of essential oils on properties of film forming emulsions and films based on hydroxypropylmethylcellulose and chitosan. Journal of Food Engineering 2011, 105, 246-253. 13. Liu, F.; Avena-Bustillos, R. J.; Chiou, B. S.; Li, Y.; Ma, Y.; Williams, T. G.; Wood, D. F.; Mchugh, T. H.; Zhong, F., Controlled-release of tea polyphenol from gelatin films incorporated with different ratios of free/nanoencapsulated tea polyphenols into fatty food simulants. Food Hydrocolloids 2016, 62, 212-221. 14. Wang, X.; Lou, T.; Zhao, W.; Song, G., Preparation of pure chitosan film using ternary solvents and its super absorbency. Carbohydrate Polymers 2016, 153, 253-257. 15. Busolo, M. A.; Lagaron, J. M., Antioxidant polyethylene films based on a resveratrol containing Clay of Interest in Food Packaging Applications. Food Packaging and Shelf Life 2015, 6, 30-41. 16. Agnes M. Rimando, †; Muriel Cuendet; Cris[an Desmarchelier; Rajendra G. Mehta; John M. Pezzuto, a.; Duke†, S. O., Cancer Chemopreven[ve and Antioxidant Activities of Pterostilbene, a

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physicochemical, morphological, thermal and crystalline properties of starches separated from different Dioscorea opposita cultivars. Food Chemistry 2006, 99, 38-44. 34. Zarski, A.; Ptak, S.; Siemion, P.; Kapusniak, J., Esterification of potato starch by a biocatalysed reaction in an ionic liquid. Carbohydrate Polymers 2016, 137, 657-663. 35. F.R.I.C., I. A. P. M. S. P. D., Joint action of α-and β-amylases. II. influence of β: α ratio and of temperature on reducing group production from starch. Journal of the Institute of Brewing 1949, 55, 298–306. 36. Rabbani, M. M.; Yang, S. B.; Park, S. J.; Oh, W.; Yeum, J. H., Characterization of Pullulan/Chitosan Oligosaccharide/Montmorillonite Nanofibers Prepared by Electrospinning Technique. Journal of nanoscience and nanotechnology 2016, 16, 6486-93. 37. Sun, X.; Sui, S.; Ference, C.; Zhang, Y.; Sun, S.; Zhou, N.; Zhu, W.; Zhou, K., Antimicrobial and Mechanical Properties of β-Cyclodextrin Inclusion with Essential Oils in Chitosan Films. Journal of Agricultural and Food Chemistry 2014, 62, 8914-8918. 38. Xu, Y. X.; Kim, K. M.; Hanna, M. A.; Nag, D., Chitosan-starch composite film: preparation and characterization. Industrial Crops & Products 2005, 21, 185-192. 39. Zhang, J.; Mi, Q.; Shen, M., Resveratrol binding to collagen and its biological implication. Food Chemistry 2012, 131, 879-884. 40. Adam, T.; Hashim, U.; Dhahi, T. S., Silicon Nanowire Surface Preparation Using Chitosan. Advanced Materials Research 2015, 1109, 350-354. 41. Bourtoom, T.; Chinnan, M. S., Preparation and properties of rice starch–chitosan blend biodegradable film. Food Science & Technology 2008, 41, 1633-1641. 42. Hassan, M. A.; Suleiman, M. S.; Najib, N. M., Improvement of the in vitro dissolution characteristics of famotidine by inclusion in β-cyclodextrin. International Journal of Pharmaceutics 1990, 58, 19-24. 43. Duarte, A.; Martinho, A.; Luís, Â.; Figueiras, A.; Oleastro, M.; Domingues, F. C.; Silva, F., Resveratrol encapsulation with methyl-β-cyclodextrin for antibacterial and antioxidant delivery applications. LWT - Food Science and Technology 2015, 63, 1254-1260. 44. Lu, Z.; Chen, R.; Fu, R.; Xiong, J.; Hu, Y., Cytotoxicity and inhibition of lipid peroxidation activity of resveratrol/cyclodextrin inclusion complexes. Journal of Inclusion Phenomena and Macrocyclic Chemistry 2012, 73, 313-320. 45. Dan, Y. U.; Ling-Ling, W. U.; Yang, J.; Wang, J. F., Preparation of β-CD/CS membrane and its application to wastewater treatment. Dyeing & Finishing 2013. 46. Luo, Y.; Pan, X.; Ling, Y.; Wang, X.; Sun, R., Facile fabrication of chitosan active film with xylan via direct immersion. Cellulose 2014, 21, 1873-1883. 47. Tripathi, S.; Mehrotra, G. K.; Dutta, P. K., Preparation and physicochemical evaluation of chitosan/poly(vinyl alcohol)/pectin ternary film for food-packaging applications. Carbohydrate Polymers 2010, 79, 711-716. 48. Zhang, Y.; Song, H.; Shang, Z.; Chen, A.; Huang, D.; Zhao, H.; Du, H., Amino acid-PEGylated resveratrol and its influence on solubility and the controlled release behavior. Biological & Pharmaceutical Bulletin 2014, 37, 785-93. 49. Das, S.; Subuddhi, U., Cyclodextrin Mediated Controlled Release of Naproxen from pH-Sensitive Chitosan/Poly(Vinyl Alcohol) Hydrogels for Colon Targeted Delivery. Industrial & Engineering Chemistry Research 2013, 52, 14192-14200. 50. Ji, J.; Hao, S.; Liu, W.; Zhang, J.; Wu, D.; Xu, Y., Preparation and evaluation of O -carboxymethyl

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chitosan/cyclodextrin nanoparticles as hydrophobic drug delivery carriers. Polymer Bulletin 2011, 67, 1201-1213. 51. Anirudhan, T. S.; Divya, P. L.; Nima, J., Synthesis and characterization of novel drug delivery system using modified chitosan based hydrogel grafted with cyclodextrin. Chemical Engineering Journal 2016, 284, 1259-1269. 52. Sun, X.; Sui, S.; Ference, C.; Zhang, Y.; Sun, S.; Zhou, N.; Zhu, W.; Zhou, K., Antimicrobial and Mechanical Properties of β-Cyclodextrin Inclusion with Essential Oils in Chitosan Films. Journal of Agricultural & Food Chemistry 2014, 62, 8914-8.

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Legends of figures

602

Figures:

603

Figure 1. Determination of the change of pre-preparation solution pH following volume decrease.

604

Figure 2. Hydrolysis degrees of β-cyclodextrin measured at 25 oC and 5 oC. (Same letters mean

605

statistical difference in different proportion of β-cyclodextrin and glucamylase, p < 0.01)

606

Figure 3. Mechanical properties of film. A, relation between RCI volume and shear force. B, film

607

tensile strength at different RCI addition. C, film elongation at different RCI addition. (Letters

608

mean statistical difference in different addition volumes of RCI, p < 0.05)

609

Figure 4. Optical properties of six films. A, absorbance value from 250 nm to 994 nm. B, Colour

610

coordinate.

611

Figure 5. Release amount of resveratrol and glucose traced within 15 days (the volumes of 2, 4, 8,

612

and 10 mL were the addition volumes of RCI in film preparation). A, release amount of resveratrol

613

in glucamylase-added films. B, release amount of resveratrol in glucamylase-free films. C, release

614

amount of glucose in glucamylase-added films.

615

Figure 6. Morphologies of films observed using SEM. Pictures of first line (A, B, and C) were the

616

morphologies of GAFW. Pictures of second line (D, E, and F) were the morphologies GFFW.

617

Pictures of third line (G, H, and I) were the morphologies of GAF. Pictures of third line (J, K, and L)

618

were the morphologies of GFF. The first, second, and third row were pictures with 1000, 2000,

619

and 4000 times magnification.

620

Figure 7. Film stability determination by X-ray diffraction. A was the FT-IR spectra curve of GAFW.

621

B was the FT-IR spectra curve of GFFW. C was the FT-IR spectra curve of GAF. D was the FT-IR

622

spectra curve of GFFS.

623

Figure 8. Film stability determination by FT-IR spectra.

624

Abstract Graphic

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Figure 1

626

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Figure 2

628

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Figure 3

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Figure 4

632

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Figure 5

634

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Figure 6

636

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Figure 7

638

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Figure 8

640

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Tables:

642

Table 1. Formulas of pre-preparation solutions (Acetic acid concentration was 1 mol/L , sodium acetate

643

concentration was 1 mol/L, citric acid concentration was 1 mol/L and sodium bicarbonate concentration was 0.5

644

mol/L).

Water

Acetic acid

Sodium acetate

citric acid

Sodium bicarbonate

(mL)

(mL)

(mL)

(mL)

(mL)

HAC+HSOB

83

10

0

0

7

HAC+LSOB

85

10

0

0

5

HAC+LCA+HSOB

82

10

0

1

7

HAC+SA

80

10

10

0

0

HAC+SA+HCA

78

10

10

2

0

Solutions

645

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

646

Page 34 of 35

Table 2. Energy dispersive spectrometer of films GAFW Element Atomic percent

GFFW

GAF

GFF

C

O

C

O

C

O

C

O

63.53

36.47

59.79

40.21

59.39

40.61

61.12

38.48

647

ACS Paragon Plus Environment

Page 35 of 35

Journal of Agricultural and Food Chemistry

648

Abstract Graphic

649

ACS Paragon Plus Environment