Research Article Received: 13 October 2013
Revised: 24 February 2014
Accepted article published: 10 March 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6644
Effect of Maillard reaction products on the physical and antimicrobial properties of edible films based on 𝝐-polylysine and chitosan Yingying Wang, Fuguo Liu, Chunxuan Liang, Fang Yuan and Yanxiang Gao* Abstract BACKGROUND: Edible films based on Maillard reaction products (MRPs) of 𝝐-polylysine and chitosan, without the use of any plasticiser, were prepared by solution casting. The effect of Maillard reaction parameters (reaction time and the ratio of polylysine/chitosan) of 𝝐-polylysine and chitosan on the structure, moisture content, water solubility, total colour difference and mechanical properties of edible films formed by MRPs were systematically evaluated. RESULTS: Scanning electron microscopy confirmed that edible films prepared by the MRPs of 𝝐-polylysine and chitosan through the Maillard reaction exhibited a more compact and dense structure than those from the mixture of biopolymers without the presence of MRPs. The tensile strength and % elongation values of films from the mixture were decreased significantly with the rise of 𝝐-polylysine (P < 0.05). The moisture content of the films was not significantly affected by Maillard reaction, whereas water solubility was decreased and total colour difference was increased significantly (P < 0.05) with the extension of Maillard reaction time. In addition, antimicrobial activity of chitosan films against E. coli and S. aureus. could be achieved by incorporating 𝝐-polylysine into chitosan. CONCLUSION: These films can ensure food quality and safety, especially for coating highly perishable foods, such as meat products. © 2014 Society of Chemical Industry Keywords: 𝜖-polylysine; chitosan; Maillard reaction; structure and functional properties; edible film
INTRODUCTION Chitosan, a linear cationic polysaccharide, has been proved to be non-toxic, biodegradable, biofunctional, biocompatible1 – 3 and has also been well known for its good film-forming and antimicrobial properties.3 – 6 One of the reasons for the inhibitory microbial activity of chitosan was that the polycationic amino groups interacted with negatively charged microbial cell menbranes and changed the membrane permeability, leading to the leakage of intracellular constituents of the microorganisms.7,8 The other reason was the formation of a film over the surface of the cell membrane, hindering nutrients from passing into the cell.9 However, only chitosan film could never display any antimicrobial effect against Escherichia coli and Staphlococcus aureus, and even the contact area under film discs on an agar surface was not clear.10,11 The development of improving functional property of edible films is an active research field. Antimicrobials could be possibly enhanced by chitosan-based films and released in a controlled manner.12 Antimicrobial agents, such as potassium sorbate, nisin, garlic oil, thyme essential oil and so on, have been added to chitosan films.10,13 The incorporation of those additives other than cross-linking agents generally lowered tensile strength,14 increased water vapour transmission15 and even changed the appearance of films. Umemura and Kawai,16,17 who investigated the Maillard reaction between chitosan and cellulose or hemicellulose model compounds, found that better tensile properties of J Sci Food Agric (2014)
chitosan films were obtained when the compounds were added at 20 wt%. Su et al.18 studied the structure and properties of carboxymethyl cellulose–soy protein isolate blend edible films cross-linked by Maillard reactions. These authors found that the crystallinity of the soy protein isolate was greatly reduced by Maillard reactions, the water sensitivity of the films was decreased and the mechanical properties were improved with increasing carboxymethyl cellulose content. 𝜖-Polylysine, which is a homo-poly-amino acid characterised by the peptide bond between carboxyl and 𝜖-amino groups of L-lysine, is water soluble, biodegradable, edible and non-toxic and shows a wide spectrum of antimicrobial activity.19 Chitosan films lacking polylysine were ineffective against E. coli, but the incorporation of polylysine into chitosan films could give fully formed zones against E. coli.20 However, much less understanding has been obtained in terms of the effect of Maillard reaction between 𝜖-polylysine and chitosan on the functional property of edible films, such as antimicrobial property, physical characteristics and mechanical properties. To the best of our knowledge, no
∗
Correspondence to: Yanxiang Gao, P. O. Box 112, No.17 Qinghua East Road, Haidian District, Beijing 100083, China. E-mail: [email protected] Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China
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www.soci.org information is available concerning the influence of the Maillard reaction between 𝜖-polylysine and chitosan onthe antimicrobial property of edible films. Therefore, the objective of this study was to enhance the antimicrobial property of edible films based on chitosan by incorporating 𝜖-polylysine. Physical characteristics and mechanical properties of the edible films were evaluated. Furthermore, film microstructures were characterised by scanning electron microscopy and the effect of Maillard reaction products on film structure is also discussed.
EXPERIMENTAL Materials 𝜖-Polylysine (purity of 97.0%) was purchased from Silver Elephant Bio-engineering Co., Ltd (Zhejiang, China). Chitosan (YK111018213) with a deacetylation degree of 91.3%, moisture and ash contents of 70.7 g kg−1 and 8.4 g kg−1 , respectively, was purchased from Golden-Shell Biochemical Co. Ltd, (Zhejiang, China). All other chemicals used were of analytical grade, unless otherwise stated.
Bacterial strains and maintenance The bacterial strains used in this study were Escherichia coli CICC 10302 and Staphylococcus aureus CICC 10786. These bacteria were obtained from the China Center of Industrial Culture Collection (CICC, Beijing, China) and kept at 4 ∘ C on nutrient agar slants. Overnight cultures of bacterial strains were grown and agitated at 140–150 rpm in an incubator shaker for 24 h in brain–heart infusion (Merck Co., Darmstadt, Germany) at 37 ∘ C.
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Moisture content. Moisture content was determined by drying small film-strips in an oven at 105 ∘ C until constant weight (dry sample weight). Moisture content was calculated by dividing weight loss by the original weight. Samples were measured at least in triplicate.24 Film solubility in water. Film solubility (S) was measured using the modified method proposed by Gontard et al.25 Films were cut into pieces (3 × 4 cm) and dried at 105 ∘ C until constant weight (W 0 ). Each film sample was immersed in 100 mL of distilled water for 24 h at 25 ± 2 ∘ C. After that, the remaining pieces of the film were dried at 105 ∘ C until constant weight (W 1 ). Triplicate measurements were performed for each type of the film and the average was taken as the result. The solubility (%) in water was calculated as follows: S=
W0 − W 1 × 100 W0
(1)
Surface colour Film colour was determined by using a colorimeter (DC-P3; Beijing Xingguang Color Measurement Instruments Co., Beijing, China) with the method by Hosseini et al.15 Measurements were taken as an average of at least eight points of each sample by placing the film sample over the standard white plate. Total colour difference (ΔE) was calculated as follows: √ (2) ΔE = (L∗ − L)2 + (a∗ − a)2 + (b∗ − b)2
where L*, a* and b* are values of the white plate, and L, a and b are values of the samples measured. Methods Film preparation A reported method21,22 with minor modifications was applied to prepare film samples. Some mixtures of 𝜖-polylysine and chitosan in the weight ratios of 1:5, 1:10 and 1:15 were dissolved in distilled de-ionised water under moderate stirring. The solution was first frozen hard at −18 ∘ C, and then lyophilised at a shelf temperature of −25 ∘ C, and a pressure of 0.2 mBar for 35 h in a freeze-dryer (Alpha 1–2 LD Plus Martin Christ Company, Osterode am Harz, Germany). The frozen dried mixture of 𝜖-polylysine and chitosan was incubated at 70 ∘ C under 79% relative humidity in a desiccator containing saturated KBr solution23 at the bottom for given times (0, 5 and 10 h). After the incubation, the reaction product was dissolved in 1% (v/v) acetic acid at a concentration of 10 g kg−1 , followed by vacuum filtration to eliminate insolubles and bubbles. After that, 20 g of each film-forming solution was poured on Plexiglas plates (8.0 × 8.0 cm). After being dried at room temperature for 24 h, all films were stored at 25 ∘ C and 60% relative humidity in a constant temperature and humidity chamber (Yi-heng Scientific Apparatus Co., Shanghai, China). These films were equilibrated for at least 48 h before being subsequently removed from the casting surface for further evaluation.
Evaluation of film characteristics Thickness. Film thickness was determined using a digital micrometer (Chengdu Chengliang Co., Chengdu, China). For each film, the values presented were an average of at least 15 random measurements.
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Mechanical properties Tensile tests were performed using a texture analyser (TMS-Pro; Food Technology Corporation, Sterling, VA, USA) according to ASTM26 with a head speed of 1 mm s−1 . At least five samples (6 × 40 mm) of each film were tested. Tensile strength (in MPa) was calculated by dividing maximum force by film cross-section (thickness × width). The % of elongation at break was calculated as (length extended/original length) × 100. Puncture tests were conducted with a cylindrical probe (2 mm in diameter) at speed of 1 mm s−1 . The puncture strength (in N mm−1 ) was calculated as puncture force/thickness of the film. Puncture deformation (in mm) was the fall distance of the probe when the film was punctured. Scanning electron microscopy The cross-sections of the films were examined with scanning electron microscopy (JSM-6360; JEOL Techniques, Tokyo, Japan). The specimens were mounted on bronze stubs using double-sided tape and then coated with a layer of gold. All samples were observed at an acceleration voltage of 10 kV. Antimicrobial assay Antimicrobial activity tests of edible films were performed using the agar diffusion method.27 The edible films were cut into 21-mm diameter discs and placed on Mueller–Hinton agar (Merck) plates and the plates were then incubated at 37 ∘ C for 24 h. Agar plates had been previously seeded with 0.1 mL of inoculum
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J Sci Food Agric (2014)
The structure and properties of edible films formed by the conjugate of 𝜖-polylysine and chitosan containing the indicator microorganisms in the range of 105 to 106 CFU mL−1 . The diameter of inhibitory zones surrounding film discs was then measured with a sliding caliper in triplicate. The antibacterial zone was the area of inhibition surrounding film discs, which was calculated as (whole zone area − film disc area). Statistical analysis The data were analysed using SPSS 16.0. Duncan’s multiple range test (P < 0.05) was used to detect the differences among the mean values of the film properties.
RESULTS AND DISCUSSION Physical properties of films The thickness, moisture content, solubility in water (S) and total colour difference (ΔE) of individual and composite films are shown in Table 1. Film thickness depended on the nature and composition of films.28 Compared with the pure chitosan film, the thickness of composite films was significantly (P < 0.05) increased with an 𝜖-polylysine–chitosan ratio of 1:5. This result was in agreement with those reported by Abugoch et al.,28 Di Pierro et al.29 and Sebti et al.30 Moisture content of composite films was similar to that of pure chitosan film (Table 1). However, the thickness and moisture content of 𝜖-polylysine–chitosan (1/10) samples were decreased with the extension of Maillard reaction time. This could be explained by the formation of covalent bonds by 𝜖-polylysine and chitosan, leading to a compactness of the film network. Chitosan film had a low solubility in water (S) at room temperature after 24 h of dipping in water. However, the incorporation of 𝜖-polylysine into chitosan film formulations increased its water solubility significantly (P < 0.05), from 26.1 ± 0.6% to 74.1 ± 3.0%. This could be explained by considering that chitosan exhibited a hydrophobic nature for the presence of N-acetyl groups on the chitosan backbone,31 and when hydrophilic 𝜖-polylysine was presented, the film network disintegrated, resulting in faster diffusion of water into films. The solubility of chitosan in acidified polyol was substantially high. Polyols such as polyethylene glycol and glycerol-2-phosphate have been reported to aid the formation
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of water-soluble chitosan at neutral pH.32 The solubility was also related to film-forming polymer content and nature (Table 1). The solubility was decreased with the extension of reaction time except for the 𝜖-polylysine and chitosan ratio of 1:15. After 10 h of Maillard reaction, the solubilities of the MRPs with 𝜖-polylysine and chitosan ratios of 1:5 and 1:10 were reduced to 65.2 ± 1.1% and 36.0 ± 1.6% from 74.1 ± 3.0% and 52.8 ± 1.7%, respectively. The more compact and dense structures (Fig. 1D) of composite films were unfavourable for the diffusion of water into films and resulted in a decrease in the solubility.15 Generally, the formation of MRPs is accompanied by a colour change during the Maillard reaction. The total colour difference (ΔE) of films is shown in Table 1. Pure chitosan film was more transparent and had a slightly yellow appearance, and its colour difference was 5.2 ± 0.5. Visually, the transparency of composite films was reduced continuously with the progress of Maillard reaction. From Table 1, it could be found that the ΔE value at the reaction time of 10 h was significantly (P < 0.05) higher than that at 0 h. Therefore, the solubility and total colour difference of composite films depended on the extent of the Maillard reaction. Mechanical properties of the films The effect of incorporating 𝜖-polylysine and the MRPs on the mechanical properties of composite chitosan films is presented in Table 2. Chitosan film showed the best tensile strength, elongation at break, puncture strength and puncture deformation in our study. The tensile strength and % elongation were significantly (P < 0.05) decreased with the increase in the proportion of 𝜖-polylysine. At an 𝜖-polylysine and chitosan ratio of 1:5, a reduction of 43% and 55% for tensile strength and % elongation at break was obtained, respectively. These results agreed with those of Park et al.,12 who found that tensile strength and % elongation value decreased with the increase in lysozyme concentration. Chitosan has excellent film-forming properties, while 𝜖-polylysine is a positively charged polypeptide with less film-forming capacity. The reductions in both tensile strength and % elongation at break indicated that the incorporation of 𝜖-polylysine weakened the film structure and integrity, and this impact also improved solubility. 𝜖-Polylysine in film-forming solution could possibly
Table 1. The effect of Maillard reaction time (at 70 ∘ C) on the physical properties of 𝜖-polylysine/chitosan films in comparison with the control film (chitosan) before reaction Sample Chitosan 𝜖-Polylysine/chitosan (1/5) 0h 5h 10 h 𝜖-Polylysine/chitosan (1/10) 0h 5h 10 h 𝜖-Polylysine/chitosan (1/15) 0h 5h 10 h
Thickness (μm)
MC (g kg−1 )
33 ± 2e
181 ± 3a
26.11 ± 0.59g
5.24 ± 0.45c
38 ± 2a 36 ± 2ab 37 ± 2a
181 ± 4a 175 ± 4ab 174 ± 1ab
74.16 ± 2.97a 68.77 ± 4.05b 65.20 ± 1.14b
5.16 ± 0.31c 5.92 ± 0.59b 7.10 ± 0.55a
36 ± 3bc 34 ± 4de 34 ± 4cd
176 ± 2ab 176 ± 3ab 167 ± 4c
52.84 ± 1.70c 44.40 ± 0.17d 36.07 ± 1.56e
5.11 ± 0.24c 5.84 ± 0.47b 5.82 ± 0.58b
36 ± 3ab 34 ± 3de 33 ± 2de
179 ± 4ab 176 ± 3ab 172 ± 5bc
31.92 ± 0.47f 31.83 ± 0.03f 32.21 ± 0.99ef
4.60 ± 0.65d 5.24 ± 0.10c 5.47 ± 0.33bc
S (%)
ΔE
a–g
Mean ± standard deviation. Means in same column with different superscript letters are significantly different (P < 0.05). MC, moisture content; S, solubility in water; ΔE, total colour difference.
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(A)
(B)
(C)
(D)
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Figure 1. Cross-sectional morphology of the films. (A) Pure chitosan film; (B) the film formed by the mixture of 1:5 𝜖-polylysine (PL) and chitosan (CH); (C) the film formed by the Maillard reaction products (MRPs) of 1:5 PL and CH with a reaction time 5 h; (D) the film formed by the MRPs of 1:5 PL and CH with a reaction time 10 h.
disrupt the uniform structure formation during the drying process and weaken inter-molecular hydrogen bonding among chitosan molecules. However, according to the report from Abugoch et al.28 the presence of quinoa protein extract (6.7% w/v) in the film increased the extensibility up to four times compared to films only from chitosan. Similar mechanical properties were also reported in the literature for chitosan film incorporated with nisin (1020 IU mg−1 ).10 In the range of nisin concentrations investigated, the highest amount of nisin incorporated (204 IU mg−1 ) into chitosan film obviously increased the % of elongation at break from 3.45% to 30.72%, whereas the tensile strength decreased sharply from 37.0 MPa to 13.6 MPa. The puncture test is a measure of the resistance of the film to perforation. Table 2 shows that the puncture strength significantly (P < 0.05) decreased, but puncture deformation appeared to increase with the increase in proportion of 𝜖-polylysine. This result indicted that the incorporation of more 𝜖-polylysine into the chitosan–𝜖-polylysine network could reduce the inter-molecular interactions and promote mobility of the polymer chains in the direction of the deformation. In addition, the effect of MRPs on % elongation at break, puncture strength and puncture deformation were limited for all the films; only tensile strength showed differences, at 𝜖-polylysine–chitosan ratios of 1:10 and 1:15, the tensile strength values were increased slightly from 71.1 ± 2.8 MPa, 73.6 ± 2.4 MPa at 0 h to 77.1 ± 0.3 MPa, 80.5 ± 2.5 MPa at 5 h, respectively. In previous research, the cross-linking effect of
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the Maillard reaction could contribute to an improvement of mechanical properties.33 This difference might be due to chitosan composition and suppliers, incorporated components and film preparation.34
Film microstruture There was no plasticiser included in the film formulation and all films were easily peeled from the Plexiglas plate. The cross-sectional morphologies of films formed by chitosan or conjugates between chitosan and 𝜖-polylysine (reaction time of 10 h) were observed by scanning electron microscopy and the results showed that they were homogeneous without any pores or cracks (Fig. 1A and D). The chitosan film was smooth and compact (Fig. 1A). Nevertherless, composite films simply formed by the mixture showed multilayer and irregular structure (Fig. 1B). Such a multilayer structure was also observed in composite films based on chitosan and methylcellulose.35 However, composite films prepared from the conjugates had a more compact and dense structure (Fig. 1C and D) than that by the mixture (Fig. 1B). When the reaction time was longer, the structure was more uniform (Fig. 1D). The more compact films might be responsible for the lower solubility in water. This phenomenon could be attributed to cross-linking via covalent bonding between 𝜖-polylysine and chitosan; a higher chitosan concentration increased the probability of chemical interaction, which might be associated with the greater inter-molecular
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J Sci Food Agric (2014)
The structure and properties of edible films formed by the conjugate of 𝜖-polylysine and chitosan
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Table 2. The effect of Maillard reaction time (at 70 ∘ C) on mechanical properties of 𝜖-polylysine/chitosan films in comparison with the control film (chitosan) before reaction Sample Chitosan 𝜖-Polylysine/chitosan (1/5) 0h 5h 10 h 𝜖-Polylysine/chitosan (1/10) 0h 5h 10 h 𝜖-Polylysine/chitosan (1/15) 0h 5h 10 h
PS (N mm−1 )
PD (mm)
43.80 ± 3.62a
575.6 ± 34.2a
5.19 ± 0.21ab
60.93 ± 1.58f 64.30 ± 3.77f 66.05 ± 3.24ef
19.79 ± 4.69d 22.76 ± 4.31cd 24.43 ± 2.86bcd
410.7 ± 6.8e 424.3 ± 19.2e 413.8 ± 15.7e
4.98 ± 0.17b 5.13 ± 0.21ab 5.40 ± 0.34a
71.11 ± 2.78de 77.11 ± 0.34bc 78.44 ± 3.35bc
22.23 ± 3.22cd 21.46 ± 1.21d 21.78 ± 1.38d
495.6 ± 25.8d 539.4 ± 9.3bc 522.4 ± 13.9cd
4.27 ± 0.22c 4.57 ± 0.24c 4.51 ± 0.24c
73.63 ± 2.40cd 80.49 ± 2.49b 78.51 ± 1.31bc
27.58 ± 1.42cd 29.74 ± 5.58b 40.80 ± 3.67a
553.7 ± 25.8abc 555.2 ± 20.9ab 543.8 ± 19.7abc
4.53 ± 0.13c 4.53 ± 0.10c 4.54 ± 0.17c
TS (MPa)
E (%)
106.06 ± 9.80a
a – f Mean ± standard deviation (n = 5). Means in the same column with different superscript letters are significantly different (P < 0.05). TS, tensile strength; E, % elongation at break; PS, puncture strength; PD, puncture deformation.
10 Antibacterial zone (cm2)
aggregation in the three-dimensional network.36 A similar structure was found in chitosan film incorporating cinnamon essential oils (1% v/v). It revealed sheets stacked in compact layers due to the cross-linking function of cinnamon essential oils.15 According to the result reported by Su et al.,18 a continuous matrix was not formed in carboxymethyl cellulose–soy protein isolate blend films, even though they were partly covalently cross-linked through the Maillard reaction. However, films with the addition of plasticiser had more compact structures, because the plasticiser increased an association with the long-chain polymers.18 These results indicated that the effect of the MRPs on the film structure was limited and related to the reaction parameters.
E. coli S. aureus
8 6 4 2 0
Evaluation of antimicrobial activity of the films The antimicrobial activities of films produced by 𝜖-polylysine and chitosan MRPs against E. coli and S. aureus by the agar diffusion method were investigated. The pure chitosan film did not exhibit any antimicrobial activity against E. coli and S. aureus; even the contact area under film discs on agar surface was not clear (data not shown). This observation was in agreement with that from Pranoto et al.10 and Li et al.11 For 𝜖-polylysine–chitosan ratios of 1:15 and 1:10 there was no markedly inhibitory zone surrounding film discs and the growth of tested microorganisms could not be found on the contact area under the film discs on the agar surface (data not shown). Increasing the level of 𝜖-polylysine to a higher ratio (1:5) showed the formation of a clear inhibition zone of 4.7 ± 0.6 cm2 and 6.1 ± 0.5 cm2 , respectively, against E. coli and S. aureus (Fig. 2), in which no tested microorganisms existed. This result indicated that incorporating 𝜖-polylysine could significantly (P < 0.05) generate antimicrobial activity of chitosan films. Compared with films produced by the mixture of 𝜖-polylysine and chitosan, no visible changes were observed for the MRPs with heating time at 5 h (as shown in Fig. 2). However, the composite films exhibited significantly (P < 0.05) reducing zone area against both E. coli and S. aureus with the extension of heating time to 10 h. The loss of antimicrobial activity was attributed to the cross-linking of chitosan with 𝜖-polylysine and the structure of films from the MRPs (Fig. 1D). Chemical interaction between groups of chitosan and 𝜖-polylysine hindered the release of 𝜖-polylysine to J Sci Food Agric (2014)
0
5 Reaction time (h)
10
Figure 2. The effect of Maillard reaction time on the antimicrobial activity of the films formed by conjugation of 𝜖-polylysine and chitosan at a ratio of 1:5. Vertical bars represent standard deviations.
inhibit pathogen surrounding film discs during agar diffusion process. Similar results were also reported by Pranoto et al.10 with chitosan-based films incorporated with potassium sorbate. The reason for the inhibitory activity of 𝜖-polylysine towards microbial growth was that its electrostatic adsorption onto the cell surface of microorganisms by its polycationic amino groups led to the disruption of the outer membrane and abnormal distribution of cytoplasm.37 Therefore, the chemical modification of amino groups of 𝜖-polylysine lowered its antimicrobial activity.19
CONCLUSION An expected reduction of tensile strength, % of elongation at break and puncture strength was observed for composite chitosan films with the increase in the proportion of 𝜖-polylysine compared with pure chitosan film. The moisture content of composite chitosan films was not significantly affected by the Maillard reaction, whereas the solubility in water of the composite films was markedly decreased compared with those of blend
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activity of chitosan films 𝜖-polylysine into chitosan, ensuring food quality and perishable foods such as
ACKNOWLEDGEMENTS This research was funded by the National Natural Science Foundation of China (NSFC31071609).
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18 Su JF, Huang Z, Yuan XY, Wang XY and Li M, Structure and properties of carboxymethyl cellulose/soy protein isolate blend edible films crosslinked by Maillard reactions. Carbohydr Polym 79:145–153 (2010). 19 Yoshida T and Nagasawa T, Epsilon-poly-L-lysine: Microbial production, biodegradation and application potential. Appl Microbiol Biotechnol 62:21–26 (2003). ˘ A, Antimi20 Üialan ˙IU, Uçar KDA, Arcan ˙I, Korel F and Yemenicioglu crobial potential of polylysine in edible films. Food Sci Technol Res 17:375–380 (2011). 21 Babiker EE, Effect of chitosan conjugation on the functional properties and bactericidal activity of gluten peptides. Food Chem 79:367–372 (2002). 22 Song Y, Babiker EE, Usui M, Saito A and Kato A, Emulsifying properties and bactericidal action of chitosan–lysozyme conjugates. Food Res Int 35:459–466 (2002). 23 Xu D, Wang X, Jiang J, Yuan F and Gao Y, Impact of whey protein–beet pectin conjugation on the physicochemical stability of 𝛽-carotene emulsions. Food Hydrocolloid 28:258–266 (2012). 24 Oseacutes J, Fabregat-Vaacutezquez M, Pedroza-Islas R, Tomaacutes SA, Cruz-Orea A and Mateacute JI, Development and characterization of composite edible films based on whey protein isolate and mesquite gum. J Food Eng 92:56–62 (2009). 25 Gontard N, Guilbert S and Cuq JL, Edible wheat gluten films – influence of the main process variables on film properties using response-surface methodology. J Food Sci 57:190–199 (1992). 26 American Society for Testing and Material (ASTM), Standard test methods for tensile properties of thin plastic sheeting, D882-91. Annual Book of ASTM. ASTM, Philadelphia, PA (1996). 27 Seydim AC and Sarikus G, Antimicrobial activity of whey protein based edible films incorporated with oregano, rosemary and garlic essential oils. Food Res Int 39:639–644 (2006). 28 Abugoch LE, Tapia C, Villaman MC, Yazdani-Pedram M and Diaz-Dosque M, Characterization of quinoa protein–chitosan blend edible films. Food Hydrocoll 25:879–886 (2011). 29 Di Pierro P, Chico B, Villalonga R, Mariniello L, Damiao AE, Masi P, et al., Chitosan–whey protein edible films produced in the absence or presence of transglutaminase: Analysis of their mechanical and barrier properties. Biomacromolecules 7:744–749 (2006). 30 Sebti I, Chollet E, Degraeve P, Noel C and Peyrol E, Water sensitivity, antimicrobial, and physicochemical analyses of edible films based on HPMC and/or chitosan. J Agric Food Chem 55:693–699 (2007). 31 Liu WG and Yao KD, Chitosan and its derivatives A promising non-viral vector for gene transfection. J Control Release 83:1–11 (2002). 32 Pillai CKS, Paul W and Sharma CP, Chitin and chitosan polymers: chemistry, solubility and fiber formation. Prog Polym Sci 34:641–678 (2009). 33 Nagahama H, Kashiki T, Nwe N, Jayakumar R, Furuike T and Tamura H, Preparation of biodegradable chitin/gelatin membranes with GlcNAc for tissue engineering applications. Carbohydr Polym 73:456–463 (2008). 34 Ojagh SM, Rezaei M, Razavi SH and Hosseini SMH, Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water. Food Chem 122:161–166 (2010). 35 Pinotti A, Garcia MA, Martino MN and Zaritzky NE, Study on microstructure and physical properties of composite films based on chitosan and methylcellulose. Food Hydrocolloid 21:66–72 (2007). 36 Nie W, Yuan X, Zhao J, Zhou Y and Bao H, Rapidly in situ forming chitosan/𝜖-polylysine hydrogels for adhesive sealants and hemostatic materials. Carbohydr Polym 96:342–348 (2013). 37 Shima S, Matsuoka H, Iwamoto T and Sakai H, Antimicrobial action of epsilon-poly-L-lysine. J Antibiot 37:1449–1455 (1984).
© 2014 Society of Chemical Industry
J Sci Food Agric (2014)
Revised: 24 February 2014
Accepted article published: 10 March 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6644
Effect of Maillard reaction products on the physical and antimicrobial properties of edible films based on 𝝐-polylysine and chitosan Yingying Wang, Fuguo Liu, Chunxuan Liang, Fang Yuan and Yanxiang Gao* Abstract BACKGROUND: Edible films based on Maillard reaction products (MRPs) of 𝝐-polylysine and chitosan, without the use of any plasticiser, were prepared by solution casting. The effect of Maillard reaction parameters (reaction time and the ratio of polylysine/chitosan) of 𝝐-polylysine and chitosan on the structure, moisture content, water solubility, total colour difference and mechanical properties of edible films formed by MRPs were systematically evaluated. RESULTS: Scanning electron microscopy confirmed that edible films prepared by the MRPs of 𝝐-polylysine and chitosan through the Maillard reaction exhibited a more compact and dense structure than those from the mixture of biopolymers without the presence of MRPs. The tensile strength and % elongation values of films from the mixture were decreased significantly with the rise of 𝝐-polylysine (P < 0.05). The moisture content of the films was not significantly affected by Maillard reaction, whereas water solubility was decreased and total colour difference was increased significantly (P < 0.05) with the extension of Maillard reaction time. In addition, antimicrobial activity of chitosan films against E. coli and S. aureus. could be achieved by incorporating 𝝐-polylysine into chitosan. CONCLUSION: These films can ensure food quality and safety, especially for coating highly perishable foods, such as meat products. © 2014 Society of Chemical Industry Keywords: 𝜖-polylysine; chitosan; Maillard reaction; structure and functional properties; edible film
INTRODUCTION Chitosan, a linear cationic polysaccharide, has been proved to be non-toxic, biodegradable, biofunctional, biocompatible1 – 3 and has also been well known for its good film-forming and antimicrobial properties.3 – 6 One of the reasons for the inhibitory microbial activity of chitosan was that the polycationic amino groups interacted with negatively charged microbial cell menbranes and changed the membrane permeability, leading to the leakage of intracellular constituents of the microorganisms.7,8 The other reason was the formation of a film over the surface of the cell membrane, hindering nutrients from passing into the cell.9 However, only chitosan film could never display any antimicrobial effect against Escherichia coli and Staphlococcus aureus, and even the contact area under film discs on an agar surface was not clear.10,11 The development of improving functional property of edible films is an active research field. Antimicrobials could be possibly enhanced by chitosan-based films and released in a controlled manner.12 Antimicrobial agents, such as potassium sorbate, nisin, garlic oil, thyme essential oil and so on, have been added to chitosan films.10,13 The incorporation of those additives other than cross-linking agents generally lowered tensile strength,14 increased water vapour transmission15 and even changed the appearance of films. Umemura and Kawai,16,17 who investigated the Maillard reaction between chitosan and cellulose or hemicellulose model compounds, found that better tensile properties of J Sci Food Agric (2014)
chitosan films were obtained when the compounds were added at 20 wt%. Su et al.18 studied the structure and properties of carboxymethyl cellulose–soy protein isolate blend edible films cross-linked by Maillard reactions. These authors found that the crystallinity of the soy protein isolate was greatly reduced by Maillard reactions, the water sensitivity of the films was decreased and the mechanical properties were improved with increasing carboxymethyl cellulose content. 𝜖-Polylysine, which is a homo-poly-amino acid characterised by the peptide bond between carboxyl and 𝜖-amino groups of L-lysine, is water soluble, biodegradable, edible and non-toxic and shows a wide spectrum of antimicrobial activity.19 Chitosan films lacking polylysine were ineffective against E. coli, but the incorporation of polylysine into chitosan films could give fully formed zones against E. coli.20 However, much less understanding has been obtained in terms of the effect of Maillard reaction between 𝜖-polylysine and chitosan on the functional property of edible films, such as antimicrobial property, physical characteristics and mechanical properties. To the best of our knowledge, no
∗
Correspondence to: Yanxiang Gao, P. O. Box 112, No.17 Qinghua East Road, Haidian District, Beijing 100083, China. E-mail: [email protected] Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China
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www.soci.org information is available concerning the influence of the Maillard reaction between 𝜖-polylysine and chitosan onthe antimicrobial property of edible films. Therefore, the objective of this study was to enhance the antimicrobial property of edible films based on chitosan by incorporating 𝜖-polylysine. Physical characteristics and mechanical properties of the edible films were evaluated. Furthermore, film microstructures were characterised by scanning electron microscopy and the effect of Maillard reaction products on film structure is also discussed.
EXPERIMENTAL Materials 𝜖-Polylysine (purity of 97.0%) was purchased from Silver Elephant Bio-engineering Co., Ltd (Zhejiang, China). Chitosan (YK111018213) with a deacetylation degree of 91.3%, moisture and ash contents of 70.7 g kg−1 and 8.4 g kg−1 , respectively, was purchased from Golden-Shell Biochemical Co. Ltd, (Zhejiang, China). All other chemicals used were of analytical grade, unless otherwise stated.
Bacterial strains and maintenance The bacterial strains used in this study were Escherichia coli CICC 10302 and Staphylococcus aureus CICC 10786. These bacteria were obtained from the China Center of Industrial Culture Collection (CICC, Beijing, China) and kept at 4 ∘ C on nutrient agar slants. Overnight cultures of bacterial strains were grown and agitated at 140–150 rpm in an incubator shaker for 24 h in brain–heart infusion (Merck Co., Darmstadt, Germany) at 37 ∘ C.
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Moisture content. Moisture content was determined by drying small film-strips in an oven at 105 ∘ C until constant weight (dry sample weight). Moisture content was calculated by dividing weight loss by the original weight. Samples were measured at least in triplicate.24 Film solubility in water. Film solubility (S) was measured using the modified method proposed by Gontard et al.25 Films were cut into pieces (3 × 4 cm) and dried at 105 ∘ C until constant weight (W 0 ). Each film sample was immersed in 100 mL of distilled water for 24 h at 25 ± 2 ∘ C. After that, the remaining pieces of the film were dried at 105 ∘ C until constant weight (W 1 ). Triplicate measurements were performed for each type of the film and the average was taken as the result. The solubility (%) in water was calculated as follows: S=
W0 − W 1 × 100 W0
(1)
Surface colour Film colour was determined by using a colorimeter (DC-P3; Beijing Xingguang Color Measurement Instruments Co., Beijing, China) with the method by Hosseini et al.15 Measurements were taken as an average of at least eight points of each sample by placing the film sample over the standard white plate. Total colour difference (ΔE) was calculated as follows: √ (2) ΔE = (L∗ − L)2 + (a∗ − a)2 + (b∗ − b)2
where L*, a* and b* are values of the white plate, and L, a and b are values of the samples measured. Methods Film preparation A reported method21,22 with minor modifications was applied to prepare film samples. Some mixtures of 𝜖-polylysine and chitosan in the weight ratios of 1:5, 1:10 and 1:15 were dissolved in distilled de-ionised water under moderate stirring. The solution was first frozen hard at −18 ∘ C, and then lyophilised at a shelf temperature of −25 ∘ C, and a pressure of 0.2 mBar for 35 h in a freeze-dryer (Alpha 1–2 LD Plus Martin Christ Company, Osterode am Harz, Germany). The frozen dried mixture of 𝜖-polylysine and chitosan was incubated at 70 ∘ C under 79% relative humidity in a desiccator containing saturated KBr solution23 at the bottom for given times (0, 5 and 10 h). After the incubation, the reaction product was dissolved in 1% (v/v) acetic acid at a concentration of 10 g kg−1 , followed by vacuum filtration to eliminate insolubles and bubbles. After that, 20 g of each film-forming solution was poured on Plexiglas plates (8.0 × 8.0 cm). After being dried at room temperature for 24 h, all films were stored at 25 ∘ C and 60% relative humidity in a constant temperature and humidity chamber (Yi-heng Scientific Apparatus Co., Shanghai, China). These films were equilibrated for at least 48 h before being subsequently removed from the casting surface for further evaluation.
Evaluation of film characteristics Thickness. Film thickness was determined using a digital micrometer (Chengdu Chengliang Co., Chengdu, China). For each film, the values presented were an average of at least 15 random measurements.
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Mechanical properties Tensile tests were performed using a texture analyser (TMS-Pro; Food Technology Corporation, Sterling, VA, USA) according to ASTM26 with a head speed of 1 mm s−1 . At least five samples (6 × 40 mm) of each film were tested. Tensile strength (in MPa) was calculated by dividing maximum force by film cross-section (thickness × width). The % of elongation at break was calculated as (length extended/original length) × 100. Puncture tests were conducted with a cylindrical probe (2 mm in diameter) at speed of 1 mm s−1 . The puncture strength (in N mm−1 ) was calculated as puncture force/thickness of the film. Puncture deformation (in mm) was the fall distance of the probe when the film was punctured. Scanning electron microscopy The cross-sections of the films were examined with scanning electron microscopy (JSM-6360; JEOL Techniques, Tokyo, Japan). The specimens were mounted on bronze stubs using double-sided tape and then coated with a layer of gold. All samples were observed at an acceleration voltage of 10 kV. Antimicrobial assay Antimicrobial activity tests of edible films were performed using the agar diffusion method.27 The edible films were cut into 21-mm diameter discs and placed on Mueller–Hinton agar (Merck) plates and the plates were then incubated at 37 ∘ C for 24 h. Agar plates had been previously seeded with 0.1 mL of inoculum
© 2014 Society of Chemical Industry
J Sci Food Agric (2014)
The structure and properties of edible films formed by the conjugate of 𝜖-polylysine and chitosan containing the indicator microorganisms in the range of 105 to 106 CFU mL−1 . The diameter of inhibitory zones surrounding film discs was then measured with a sliding caliper in triplicate. The antibacterial zone was the area of inhibition surrounding film discs, which was calculated as (whole zone area − film disc area). Statistical analysis The data were analysed using SPSS 16.0. Duncan’s multiple range test (P < 0.05) was used to detect the differences among the mean values of the film properties.
RESULTS AND DISCUSSION Physical properties of films The thickness, moisture content, solubility in water (S) and total colour difference (ΔE) of individual and composite films are shown in Table 1. Film thickness depended on the nature and composition of films.28 Compared with the pure chitosan film, the thickness of composite films was significantly (P < 0.05) increased with an 𝜖-polylysine–chitosan ratio of 1:5. This result was in agreement with those reported by Abugoch et al.,28 Di Pierro et al.29 and Sebti et al.30 Moisture content of composite films was similar to that of pure chitosan film (Table 1). However, the thickness and moisture content of 𝜖-polylysine–chitosan (1/10) samples were decreased with the extension of Maillard reaction time. This could be explained by the formation of covalent bonds by 𝜖-polylysine and chitosan, leading to a compactness of the film network. Chitosan film had a low solubility in water (S) at room temperature after 24 h of dipping in water. However, the incorporation of 𝜖-polylysine into chitosan film formulations increased its water solubility significantly (P < 0.05), from 26.1 ± 0.6% to 74.1 ± 3.0%. This could be explained by considering that chitosan exhibited a hydrophobic nature for the presence of N-acetyl groups on the chitosan backbone,31 and when hydrophilic 𝜖-polylysine was presented, the film network disintegrated, resulting in faster diffusion of water into films. The solubility of chitosan in acidified polyol was substantially high. Polyols such as polyethylene glycol and glycerol-2-phosphate have been reported to aid the formation
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of water-soluble chitosan at neutral pH.32 The solubility was also related to film-forming polymer content and nature (Table 1). The solubility was decreased with the extension of reaction time except for the 𝜖-polylysine and chitosan ratio of 1:15. After 10 h of Maillard reaction, the solubilities of the MRPs with 𝜖-polylysine and chitosan ratios of 1:5 and 1:10 were reduced to 65.2 ± 1.1% and 36.0 ± 1.6% from 74.1 ± 3.0% and 52.8 ± 1.7%, respectively. The more compact and dense structures (Fig. 1D) of composite films were unfavourable for the diffusion of water into films and resulted in a decrease in the solubility.15 Generally, the formation of MRPs is accompanied by a colour change during the Maillard reaction. The total colour difference (ΔE) of films is shown in Table 1. Pure chitosan film was more transparent and had a slightly yellow appearance, and its colour difference was 5.2 ± 0.5. Visually, the transparency of composite films was reduced continuously with the progress of Maillard reaction. From Table 1, it could be found that the ΔE value at the reaction time of 10 h was significantly (P < 0.05) higher than that at 0 h. Therefore, the solubility and total colour difference of composite films depended on the extent of the Maillard reaction. Mechanical properties of the films The effect of incorporating 𝜖-polylysine and the MRPs on the mechanical properties of composite chitosan films is presented in Table 2. Chitosan film showed the best tensile strength, elongation at break, puncture strength and puncture deformation in our study. The tensile strength and % elongation were significantly (P < 0.05) decreased with the increase in the proportion of 𝜖-polylysine. At an 𝜖-polylysine and chitosan ratio of 1:5, a reduction of 43% and 55% for tensile strength and % elongation at break was obtained, respectively. These results agreed with those of Park et al.,12 who found that tensile strength and % elongation value decreased with the increase in lysozyme concentration. Chitosan has excellent film-forming properties, while 𝜖-polylysine is a positively charged polypeptide with less film-forming capacity. The reductions in both tensile strength and % elongation at break indicated that the incorporation of 𝜖-polylysine weakened the film structure and integrity, and this impact also improved solubility. 𝜖-Polylysine in film-forming solution could possibly
Table 1. The effect of Maillard reaction time (at 70 ∘ C) on the physical properties of 𝜖-polylysine/chitosan films in comparison with the control film (chitosan) before reaction Sample Chitosan 𝜖-Polylysine/chitosan (1/5) 0h 5h 10 h 𝜖-Polylysine/chitosan (1/10) 0h 5h 10 h 𝜖-Polylysine/chitosan (1/15) 0h 5h 10 h
Thickness (μm)
MC (g kg−1 )
33 ± 2e
181 ± 3a
26.11 ± 0.59g
5.24 ± 0.45c
38 ± 2a 36 ± 2ab 37 ± 2a
181 ± 4a 175 ± 4ab 174 ± 1ab
74.16 ± 2.97a 68.77 ± 4.05b 65.20 ± 1.14b
5.16 ± 0.31c 5.92 ± 0.59b 7.10 ± 0.55a
36 ± 3bc 34 ± 4de 34 ± 4cd
176 ± 2ab 176 ± 3ab 167 ± 4c
52.84 ± 1.70c 44.40 ± 0.17d 36.07 ± 1.56e
5.11 ± 0.24c 5.84 ± 0.47b 5.82 ± 0.58b
36 ± 3ab 34 ± 3de 33 ± 2de
179 ± 4ab 176 ± 3ab 172 ± 5bc
31.92 ± 0.47f 31.83 ± 0.03f 32.21 ± 0.99ef
4.60 ± 0.65d 5.24 ± 0.10c 5.47 ± 0.33bc
S (%)
ΔE
a–g
Mean ± standard deviation. Means in same column with different superscript letters are significantly different (P < 0.05). MC, moisture content; S, solubility in water; ΔE, total colour difference.
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(A)
(B)
(C)
(D)
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Figure 1. Cross-sectional morphology of the films. (A) Pure chitosan film; (B) the film formed by the mixture of 1:5 𝜖-polylysine (PL) and chitosan (CH); (C) the film formed by the Maillard reaction products (MRPs) of 1:5 PL and CH with a reaction time 5 h; (D) the film formed by the MRPs of 1:5 PL and CH with a reaction time 10 h.
disrupt the uniform structure formation during the drying process and weaken inter-molecular hydrogen bonding among chitosan molecules. However, according to the report from Abugoch et al.28 the presence of quinoa protein extract (6.7% w/v) in the film increased the extensibility up to four times compared to films only from chitosan. Similar mechanical properties were also reported in the literature for chitosan film incorporated with nisin (1020 IU mg−1 ).10 In the range of nisin concentrations investigated, the highest amount of nisin incorporated (204 IU mg−1 ) into chitosan film obviously increased the % of elongation at break from 3.45% to 30.72%, whereas the tensile strength decreased sharply from 37.0 MPa to 13.6 MPa. The puncture test is a measure of the resistance of the film to perforation. Table 2 shows that the puncture strength significantly (P < 0.05) decreased, but puncture deformation appeared to increase with the increase in proportion of 𝜖-polylysine. This result indicted that the incorporation of more 𝜖-polylysine into the chitosan–𝜖-polylysine network could reduce the inter-molecular interactions and promote mobility of the polymer chains in the direction of the deformation. In addition, the effect of MRPs on % elongation at break, puncture strength and puncture deformation were limited for all the films; only tensile strength showed differences, at 𝜖-polylysine–chitosan ratios of 1:10 and 1:15, the tensile strength values were increased slightly from 71.1 ± 2.8 MPa, 73.6 ± 2.4 MPa at 0 h to 77.1 ± 0.3 MPa, 80.5 ± 2.5 MPa at 5 h, respectively. In previous research, the cross-linking effect of
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the Maillard reaction could contribute to an improvement of mechanical properties.33 This difference might be due to chitosan composition and suppliers, incorporated components and film preparation.34
Film microstruture There was no plasticiser included in the film formulation and all films were easily peeled from the Plexiglas plate. The cross-sectional morphologies of films formed by chitosan or conjugates between chitosan and 𝜖-polylysine (reaction time of 10 h) were observed by scanning electron microscopy and the results showed that they were homogeneous without any pores or cracks (Fig. 1A and D). The chitosan film was smooth and compact (Fig. 1A). Nevertherless, composite films simply formed by the mixture showed multilayer and irregular structure (Fig. 1B). Such a multilayer structure was also observed in composite films based on chitosan and methylcellulose.35 However, composite films prepared from the conjugates had a more compact and dense structure (Fig. 1C and D) than that by the mixture (Fig. 1B). When the reaction time was longer, the structure was more uniform (Fig. 1D). The more compact films might be responsible for the lower solubility in water. This phenomenon could be attributed to cross-linking via covalent bonding between 𝜖-polylysine and chitosan; a higher chitosan concentration increased the probability of chemical interaction, which might be associated with the greater inter-molecular
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J Sci Food Agric (2014)
The structure and properties of edible films formed by the conjugate of 𝜖-polylysine and chitosan
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Table 2. The effect of Maillard reaction time (at 70 ∘ C) on mechanical properties of 𝜖-polylysine/chitosan films in comparison with the control film (chitosan) before reaction Sample Chitosan 𝜖-Polylysine/chitosan (1/5) 0h 5h 10 h 𝜖-Polylysine/chitosan (1/10) 0h 5h 10 h 𝜖-Polylysine/chitosan (1/15) 0h 5h 10 h
PS (N mm−1 )
PD (mm)
43.80 ± 3.62a
575.6 ± 34.2a
5.19 ± 0.21ab
60.93 ± 1.58f 64.30 ± 3.77f 66.05 ± 3.24ef
19.79 ± 4.69d 22.76 ± 4.31cd 24.43 ± 2.86bcd
410.7 ± 6.8e 424.3 ± 19.2e 413.8 ± 15.7e
4.98 ± 0.17b 5.13 ± 0.21ab 5.40 ± 0.34a
71.11 ± 2.78de 77.11 ± 0.34bc 78.44 ± 3.35bc
22.23 ± 3.22cd 21.46 ± 1.21d 21.78 ± 1.38d
495.6 ± 25.8d 539.4 ± 9.3bc 522.4 ± 13.9cd
4.27 ± 0.22c 4.57 ± 0.24c 4.51 ± 0.24c
73.63 ± 2.40cd 80.49 ± 2.49b 78.51 ± 1.31bc
27.58 ± 1.42cd 29.74 ± 5.58b 40.80 ± 3.67a
553.7 ± 25.8abc 555.2 ± 20.9ab 543.8 ± 19.7abc
4.53 ± 0.13c 4.53 ± 0.10c 4.54 ± 0.17c
TS (MPa)
E (%)
106.06 ± 9.80a
a – f Mean ± standard deviation (n = 5). Means in the same column with different superscript letters are significantly different (P < 0.05). TS, tensile strength; E, % elongation at break; PS, puncture strength; PD, puncture deformation.
10 Antibacterial zone (cm2)
aggregation in the three-dimensional network.36 A similar structure was found in chitosan film incorporating cinnamon essential oils (1% v/v). It revealed sheets stacked in compact layers due to the cross-linking function of cinnamon essential oils.15 According to the result reported by Su et al.,18 a continuous matrix was not formed in carboxymethyl cellulose–soy protein isolate blend films, even though they were partly covalently cross-linked through the Maillard reaction. However, films with the addition of plasticiser had more compact structures, because the plasticiser increased an association with the long-chain polymers.18 These results indicated that the effect of the MRPs on the film structure was limited and related to the reaction parameters.
E. coli S. aureus
8 6 4 2 0
Evaluation of antimicrobial activity of the films The antimicrobial activities of films produced by 𝜖-polylysine and chitosan MRPs against E. coli and S. aureus by the agar diffusion method were investigated. The pure chitosan film did not exhibit any antimicrobial activity against E. coli and S. aureus; even the contact area under film discs on agar surface was not clear (data not shown). This observation was in agreement with that from Pranoto et al.10 and Li et al.11 For 𝜖-polylysine–chitosan ratios of 1:15 and 1:10 there was no markedly inhibitory zone surrounding film discs and the growth of tested microorganisms could not be found on the contact area under the film discs on the agar surface (data not shown). Increasing the level of 𝜖-polylysine to a higher ratio (1:5) showed the formation of a clear inhibition zone of 4.7 ± 0.6 cm2 and 6.1 ± 0.5 cm2 , respectively, against E. coli and S. aureus (Fig. 2), in which no tested microorganisms existed. This result indicated that incorporating 𝜖-polylysine could significantly (P < 0.05) generate antimicrobial activity of chitosan films. Compared with films produced by the mixture of 𝜖-polylysine and chitosan, no visible changes were observed for the MRPs with heating time at 5 h (as shown in Fig. 2). However, the composite films exhibited significantly (P < 0.05) reducing zone area against both E. coli and S. aureus with the extension of heating time to 10 h. The loss of antimicrobial activity was attributed to the cross-linking of chitosan with 𝜖-polylysine and the structure of films from the MRPs (Fig. 1D). Chemical interaction between groups of chitosan and 𝜖-polylysine hindered the release of 𝜖-polylysine to J Sci Food Agric (2014)
0
5 Reaction time (h)
10
Figure 2. The effect of Maillard reaction time on the antimicrobial activity of the films formed by conjugation of 𝜖-polylysine and chitosan at a ratio of 1:5. Vertical bars represent standard deviations.
inhibit pathogen surrounding film discs during agar diffusion process. Similar results were also reported by Pranoto et al.10 with chitosan-based films incorporated with potassium sorbate. The reason for the inhibitory activity of 𝜖-polylysine towards microbial growth was that its electrostatic adsorption onto the cell surface of microorganisms by its polycationic amino groups led to the disruption of the outer membrane and abnormal distribution of cytoplasm.37 Therefore, the chemical modification of amino groups of 𝜖-polylysine lowered its antimicrobial activity.19
CONCLUSION An expected reduction of tensile strength, % of elongation at break and puncture strength was observed for composite chitosan films with the increase in the proportion of 𝜖-polylysine compared with pure chitosan film. The moisture content of composite chitosan films was not significantly affected by the Maillard reaction, whereas the solubility in water of the composite films was markedly decreased compared with those of blend
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www.soci.org films. In addition, the antimicrobial could be generated by incorporating which expanded their applications in safety, especially for coating highly meat products.
activity of chitosan films 𝜖-polylysine into chitosan, ensuring food quality and perishable foods such as
ACKNOWLEDGEMENTS This research was funded by the National Natural Science Foundation of China (NSFC31071609).
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