- Email: [email protected]
Preparation and properties of chitosan–soybean trypsin inhibitor blend film with anti-Aspergillus flavus activity
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/indcrop
Preparation and properties of chitosan–soybean trypsin inhibitor blend film with anti-Aspergillus flavus activity Bin Zhang, Dong-Feng Wang ∗ , Hai-Yan Li, Ying Xu, Li Zhang Department of Food Chemistry and Nutrition, College of Food Science and Technology, Ocean University of China, Yu Shan Road 5, Qingdao 266003, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
The chitosan-based blend films were prepared from chitosan, soybean trypsin inhibitor
Received 11 August 2008
extract (STI)/wild soybean trypsin inhibitor extract (WTI) and glycerol (Gly) solutions, the
Received in revised form
properties of which were also investigated, including thickness, mechanical property, water
30 October 2008
vapor transmission, optical transmittance, and solubility. In addition, the resulting films
Accepted 31 October 2008
were characterized by scanning electron microscope (SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The result of SEM images showed the surface and cross-section of chitosan–STI/WTI–Gly blend film had more smooth and dense mor-
Keywords:
phology than pure chitosan film, which suggested there was a better compatibility among
Chitosan
the three components. XRD and FTIR spectra indicated that the possible interaction force
Trypsin inhibitor
among the components might be the hydrogen bonds of N H· · ·O C and O H· · ·O C. Fur-
Blend film
thermore, the antifungal activity against A. flavus by the prepared blend films had been
A. flavus
investigated. The facts that the germination and growth of A. flavus were strongly inhibited by chitosan–STI/WTI–Gly film indicated the blend films might be useful as potential bio-control packaging against A. flavus during the peanuts and other cereals storage. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Aspergillus flavus is one of the major spoilage moulds of intermediate moisture foods that are widely distributed in tropical and subtropical zones around the globe. A. flavus can infect several agricultural crops such as peanuts, maize, tree nuts, and rice, resulting in the production of one of the most toxic and potent carcinogenic metabolites known as aflatoxin (Binder et al., 2007). Therefore, a variety of methods such as physical methods (aeration, temperature and moisture control, modified atmospheres, etc.) (Cleveland et al., 2003; Magan and Aldred, 2007) or by fungistats of which the potassium sorbate and sodium benzoate are the commonly used (LópezMalo et al., 2005), were adopted for inhibition on the fungal
growth and subsequent mycotoxin production. While, more attention has been particularly given to the natural antifungal peptide (Kim et al., 2006; Wang and Ng, 2006a; Wang and Ng, 2007), some of which have been demonstrated important antiA. flavus properties (Fakhoury and Woloshuk, 2001; B. Zhang et al., 2007; T. Zhang et al., 2007). Soybean whey is a byproduct during the preparation of soybean protein, and approximately 300 million ton of the whey is wasted and abandoned every year in China (Wang and Ying, 2007). Trypsin inhibitor (TI), large quantities in the whey, possesses various physiology functions used in the biological pesticide and biomedical fields (Zhang et al., 2006). What is more, TIs of different size and amino acid sequence have been isolated from various cereals including corn kernels, and
Abbreviations: TI, Trypsin inhibitor; STI, Soybean (Glycine max) trypsin inhibitor extract; WTI, Wild soybean (Glycine soya) trypsin inhibitor extract; Gly, Glycerol; A. flavus, Aspergillus flavus. ∗ Corresponding author. Tel.: +86 532 82031575; fax: +86 532 82032468. E-mail address: [email protected] (D.-F. Wang). 0926-6690/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2008.10.007
542
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
identified the activity of anti-A. flavus (Chen et al., 1999a,b; Kim et al., 2006; Mellon et al., 2007). However, there are rarely questioned about cultivated and wild soybean TI anti-A. flavus activity (Ye et al., 2001; Wang and Ng, 2006b; Yang et al., 2006) in the reports. Chitosan obtained by the deacetylation of chitin is one of the most abundant natural aminopolysaccharides. Owing to its good biodegradability, biocompatibility and non-toxicity, it has been extensively used over a wide range of applications, including the biomedical area and edible coating or films. Currently, chitosan and its films have been documented for its anti-fungal (Sébastien et al., 2006), anti-microbial (Fu et al., 2005; Sunzuki et al., 2007) and anti-viral (Neamnark et al., 2007) properties. Therefore, many active biopolymers including proteins (Tanabe et al., 2002), polysaccharides (Li et al., 2006), lipids (Pranoto et al., 2005) or their combinations have been incorporated in the edible chitosan films, to enhance its excellent oxygen, carbon dioxide barrier and interesting antimicrobial properties. Nevertheless, the chitosan-based blend materials, incorporated in anti-viral peptides, such as protein inhibitors, to be employed as packaging, particularly as an edible packaging film for resistance to A. flavus infection, have not been reported so far. Therefore, the objective of this study is to prepare blend films by making from soybean TI extract/wild soybean TI extract incorporated in chitosan and glycerol solution, study the intrinsic properties and investigate the possible molecular miscibility among the components, further to study the activity against A. flavus germination and growth on peanuts.
2.
Materials and methods
2.1.
Materials
TI extract was prepared from the cultivated soybean (Glycine max) (STI) from a local supermarket and wild soybean (Glycine soya) (WTI) which was manually collected from the plants grown in Qingdao city (36◦ 38 N, 120◦ 45 E), Shandong province, China. The cultivated/wild soybean seed flour with distilled water (1:6, w/v) was stirring for 12 h at 4 ◦ C, with subsequent centrifugation at 3000 × g for 15 min. The sediment obtained was extracted once more. The obtained supernatants were mixed and adjusted to pH 4.5 (B. Zhang et al., 2007; T. Zhang et al., 2007) before being centrifuged at 5000 × g for 20 min. The concentrated supernatant obtained (soybean whey) after freeze drying was referred as crude STI/WTI extract used in the preparation of chitosan–STI/WTI blend film. And the crude STI/WTI extract was further purified using the hydrophobic interaction chromatography following the previous report (Yeboah et al., 1996). Glycerol (Gly) and acetic acid were obtained from Sigma Chemical Co. Chitosan (degree of deacetylation was 90%) was prepared following the method of Jia and Li (2002). All the chemicals and reagents were of analytical grade.
2.2.
Film preparation
The chitosan–STI/WTI–Gly blend film was fabricated by a casting/solvent evaporation (Cao et al., 2007) of acetic acid chi-
tosan, STI/WTI and glycerol solutions. Initially, STI/WTI (0.2%, w/v) was dispersed in an aqueous solution of acetic acid (1.0%, v/v). Then chitosan and glycerol were added to the solution to get a final concentration of 1.8% (w/v) and 1.2% (w/v), until the homogenous solution was obtained. The solution was deaerated under vacuum (0.09 MPa) at room temperature for 2 h prior to casting film, to prevent pinhole formation in the film when the casting solvent evaporated. Then the blend solution was cast onto the rectangular polyvinyl chloride mold (bottom; 8.0 cm × 12.0 cm) and dried at 45 ◦ C for 12 h. Film thickness was controlled by casting the same amount (10.0 mL) of forming solution per mold. Finally, the resulting film was manually peeled off, stored in plastic bags and held in desiccators for further barrier, mechanical properties and biological activity testing. Above the added concentrations of chitosan, STI/WTI, glycerol and other operating conditions had been optimized by Box-Behnken design experiment, with anti-A. flavus activity as evaluation indexes (data was not showed).
2.3.
Measurement of film properties
Thickness of the films was determined using a precision digital coating thickness gauge Elcometer A 300 FNP 23 (England). The measurements were taken at 15 different points of each specimen and the mean values were taken. Mechanical properties were measured by using a Texture Analyser (TMS-PRO, FTC, USA). Sample films were cut into 20.0 mm wide and 80.0 mm long strips. Grip separation was set at 40.0 mm, with a cross-head speed of 50.0 mm/min. Tensile strength (TS) and percentage of elongation at break (EB) were evaluated in eight samples from each type of film and the mean values were taken, according to the standard testing method (ASTM, 1995). Film transparency was determined according to the following method. The films were cut into rectangular shapes (15.0 mm × 40.0 mm) and placed on the internal side of a spectrophotometer cell. Transparency of films was measured using a spectrophotometer (UV 2102 PC, UNICO, USA) at 560 nm. Three replicates of each film were tested. A modified method from Pierro et al. (2007) was used to measure film solubility. The solubility studies of the films were carried out by incubating the samples at 25 ◦ C in deionized water. From each film pieces of 40.0 mm × 40.0 mm were cut and stored in desiccators with silica gel for 7 days. Sample films were weighed (Wdry ) to the nearest 0.0001 g and placed into test beakers with 80.0 mL deionized water at room temperature until the film reached to constant weight. After that, the remaining pieces of film were collected by filtration, dried again in an oven at 50 ◦ C to constant weight (Wfinal dry ). The percentage of total soluble matter (solubility) was calculated as follows: solubility (%) = 100 × (Wdry − Wfinal dry )/Wdry . The measurement was repeated five times for each type of film, and an average was taken as the result. The water vapor transmission (WVP) of the films was determined by gravimetric test according to modified ASTM method (Gontard et al., 1994) as previously described. Three grams of anhydrous calcium chloride were introduced in each weighing bottle (25.0 mm in diameter). The film sample having diameter of about 40.0 mm was placed on top of the cup and sealed by melted paraffin, which area exposed to vapor transmission was about 3.46 cm2 . The cups were then weighed and placed
543
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
Table 1 – Properties of chitosan and chitosan–STI/WTI–Gly films. Film type
Thickness (×10−2 mm)
TS (Pa)
EB (%)
WVP (×10−7 g mm/cm2 h mmHg)
Transparency (%)
Solubility (%)
Chitosan film Chitosan–STI–Gly film Chitosan–WTI–Gly film
2.47 ± 0.12a 2.92 ± 0.17b 2.84 ± 0.11b
3731.1 ± 75.5a 2158.9 ± 20.9b 2059.4 ± 38.0b
29.6 ± 1.7a 69.0 ± 2.9b 73.5 ± 2.3b
2.57 ± 0.35a 2.45 ± 0.52b 2.43 ± 0.42b
88.7 ± 0.2a 82.8 ± 0.3b 83.2 ± 0.1b
98.1 ± 1.2a 34.6 ± 2.0b 31.7 ± 1.6b
Data represent the means ± S.D. of measurement for there different samples. Student–Newman–Keuls (SNK) test is used to determine the significance, and the means with different letters in the same column are significantly different at P < 0.05. TS is tensile strength; EB is elongation at break; WVP is water vapor permeability.
in desiccators in which provided a 100% relative humidity, stored in a thermostat incubator at 25.0 ◦ C. The water vapor transferred through the films at different time intervals were determined from the weight gain of the cups. The WVP was calculated as follows: WVP = [Wd]/[S t(p2 − p1 )], where W was the weight of water absorbed in the cups, d was the thickness of the film, S was the area of the exposed film, t was the time for weight change, p2 − p1 was the vapor pressure differential across the film.
distilled water (Control), chitosan and chitosan–STI/WTI–Gly blend film solution and dried at 45 ◦ C for 12 h. The prepared carriers of the films (dried paper disks) were deposited on the surface of Czapek culture medium followed by 15.0 L of A. flavus spores (102 /mL) inoculated on the surface of the paper disks. After incubation at 28 ◦ C for 48 h until the growth of A. flavus was evaluated macroscopically, the diameters (mm) of growth inhibition was determined. Above each petri dish was conducted in triplicate.
2.4.
2.6.
Characterization analyses
The surface and cross-section of the chitosan and chitosan–STI/WTI–Gly blend films were coated with the fine gold palladium film and examined by a scanning electron microscope (SEM) (JSM-6390LV, JEOL Ltd., Japan). Fourier transform infrared (FTIR) spectrometry was used to characterize the functional groups of the films in order to determine the possible interaction mode among chitosan, STI/WTI and glycerol in the composite system. Samples were analyzed between 400 and 4000 cm−1 with a resolution of 4 cm−1 in a PerkinElmer spectrum 2000 (USA). X-ray diffraction patterns of chitosan, STI/WTI, chitosan film and chitosan–STI/WTI–Gly blend film were analyzed using an X-ray diffractometer (XPERT, Philips, Netherlands) in the angular range of 5–40 (2) with Nickel-filtered Cu K␣ radiation at a voltage of 40 kV and current of 30 mA, to investigate the crystalline property.
2.5.
Anti-A. flavus activity assay
The purified STI/WTI was added at a final concentration of 0.0, 0.6, 1.2, 1.8 and 2.4 mol/L to the conical flasks, which contained 10.0 mL of 10% potato dextrose broth (PDB)/Czapeks liquid medium and 0.1 mL of freshly harvested A. flavus conidia (104 /mL). The conidia were incubated for up to 48 h at 28 ◦ C. Negative controls were 10 mmol/L phosphate buffer or heat-inactivated purified STI/WTI. Morphological changes of hyphae were examined periodically during the incubation using light microscopy. Above each conical flask was conducted in triplicate for one concentration, according to the previous report (Chen et al., 1999a). Antifungal activity of prepared chitosan film and chitosan–STI/WTI–Gly blend film against A. flavus was carried out in 100 mm × 15 mm petri dish containing Czapek culture medium according to the previous report (Ouattara et al., 2000) with a further modification. The sterile blank paper disks (7.0 mm in diameter) were immersed in the
Inhibition of A. flavus growth on peanuts
Peanuts were surface sterilized with 1.0% NaClO solution for 60 s and rinsed in sterile distilled water before coating with chitosan and chitosan–STI/WTI–Gly solution. The peanuts coating with the film were dried at 45 ◦ C for 12 h and put into conical flasks. Then each flask was inoculated with 0.1 mL, 102 /mL of A. flavus spore suspension. After incubation at 28 ◦ C for 5 days with vigorous shaking for 1 h once a day, the growth of A. flavus was evaluated macroscopically. Above the flask was conducted in triplicate for each coating film.
2.7.
Statistical analysis
Measurements of each property of the films were conducted at least three times in a randomized design and all measurements were carried out at least twice. Statistical analysis was conducted using the SPSS package (SPSS 13.0 for windows, SPSS Inc., Chicago, IL). And the Student’s–Newman–Keuls (SNK) test was used to determine the significance different of the properties of the films at P < 0.05.
3.
Results and discussion
3.1.
Properties of film
The properties of chitosan and chitosan–STI/WTI–Gly films were displayed in Table 1. As can be seen from the results, the chitosan–STI/WTI–Gly blend film (2.92/2.84 mm) had a significant increase in thickness as the level of STI/WTI and glycerol incorporated, compared with the pure chitosan film (2.47 mm). The edible films must withstand the normal stress encountered during its application and subsequent shipping and handling of the food. High tensile strength (TS) is generally required, but deformation values must be adjusted according to the intended application of the films. TS of the chitosan–STI/WTI–Gly film (2158/2059 Pa) decreased signifi-
544
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
Fig. 1 – SEM micrographs of chitosan and chitosan–STI/WTI–Gly films. Surface and cross-section (A1 and A2) of chitosan film; surface and cross-section (B1 and B2) of chitosan–STI–Gly film; surface and cross-section (C1 and C2) of chitosan–WTI–Gly film.
cantly, compared with simple chitosan film (3731 Pa), mainly affected by the incorporation of STI/WTI protein and glycerol. Particularly, the protein could exert negative effects on both coating operation and tensile property, due to their intrinsic brittleness (Lee et al., 2008). And also the TS values were influenced by the negative influence of the interaction between chitosan and glycerol (Chillo et al., 2008). Elongation at the break (EB) is an indication of the film’s flexibility and extensibility. The average EB values of the blend films behaved inversely to the TS values, increasing from 29.6% for chitosan film to 69.0%/73.5% for chitosan–STI/WTI–Gly film, which mainly effected by the added glycerol distributing uniformly and improving flexibility of protein incorporated films (Gounga et al., 2007). Water vapor permeability (WVP) values of the blend films decreased significantly, since STI/WTI was hydrophilic materials and could be well incorporated into the chitosan network structure. And the addition of STI/WTI and glycerol might cause hydrogen bond formation, thus reinforce the compactness of the blend films (Prodpran et al., 2007). Since a main function of the edible film was to impede moisture transfer between food and the surrounding atmosphere, or between two components of a heterogeneous food product, WVP should be as low as possible (Bourtoom and Chinnan, 2008). Solubility in water is also an important property, which governs potential applications of these materials to food preservation. Generally, films with low water solubility are necessary for the protection of foodstuffs with high or intermediate water activity (aw). Pure chitosan film was almost soluble (98.1%) or very difficult to recover after the water immersion process. The addition of STI/WTI and glycerol decreased the solubility to 34.6%/31.7% significantly. Color attributes are of prime importance because they directly influence consumer acceptability. The pure chitosan film was nearly transparent (88.7%) at a wavelength of 560 nm. The percent transmittance value of the chitosan–STI/WTI–Gly
film decreased significantly (82.8%/83.2%), caused by the dispersion of STI/WTI and glycerol in the chitosan matrix.
3.2.
Surface and cross-section structure (SEM) analysis
The surface and cross-section morphology of chitosan and chitosan–STI/WTI–Gly films (Fig. 1) were analyzed by scanning electron microscopy (SEM). The surface and cross-section of chitosan film appeared to be certain discontinuous zones and small pores. However, the chitosan–STI/WTI–Gly blend film exhibited a dense and uniform microstructure, which might be due to the reorientation of polar functional groups and change of networking structure introduced by protein and glycerol. The smooth and dense morphology of the surface and cross-section also suggested that there was a better compatibility among chitosan, STI/WTI and glycerol, perhaps the result of strong intermolecular interaction caused by the produced hydrogen bonding (Pierro et al., 2007).
3.3.
FTIR analysis spectroscopy
The infrared spectrophometry of STI, WTI, chitosan film, chitosan–STI/WTI–Gly blend film were shown in Fig. 2. The signal of the chitosan film could be significantly recognized from the intensity of peaks at spectral line A. The characteristic peak of STI (B1) was almost in concordance with infrared absorption of WTI (B2). As the spectral peak of chitosan–STI/WTI–Gly blend film (C1/C2) displayed, the absorb band at 3379 cm−1 /3357 cm−1 , associated with the stretch vibration absorption peak of O–H, had strong absorption and a significantly downward shift compared with the spectral of chitosan film (A), which perhaps proved that more hydrogen bonds had generated because of the incorporation of protein and glycerol. Obviously, the absorb band in the blend film (C1 and C2) at about 1647 cm−1 had been blue shift to 1561 cm−1 , compared with line B1 (1645 cm−1 ), B2 (1647 cm−1 )
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
545
Fig. 2 – FTIR spectra: (A) chitosan film; (B1) STI; (B2) WTI; (C1) chitosan–STI–Gly film; (C2) chitosan–WTI–Gly film.
and A (1647 cm-1 ) associated with amide I absorption, mainly resulted from the formation of hydrogen bonds between –NH2 , –COOH, –OH of STI/WTI and –C O of chitosan (Schmidt et al., 2005). Therefore, the absorb band in the line C1 and C2 at 1561 cm−1 , had greatly strengthened compared with line A. Except these, the absorb band in the line C1 and C2 at about 1250 cm−1 , associated with bend vibration absorption peak of O–H, had reduced considerably compared with line B1 and B2. All the above, it is likely that the hydrogen bonds of N H· · ·O C and O H· · ·O C had formed which perhaps was the main major interaction force in the blend films (Wang et al., 2006; Ma and Liu, 2008).
3.4.
X-ray diffraction analysis
The X-ray diffractograms of chitosan powder, chitosan film and chitosan–STI/WTI–Gly blend film were displayed in Fig. 3. As observed, the chitosan powder (A) was in a crystalline state because two main diffraction peaks (2 = 11.5◦ and 20.25◦ ) were observed in its X-ray diffraction pattern, agreed with the finding of previous report (Nunthanid et al., 2001). The diffractogram of chitosan film (B) showed the crystalline peaks at 8.3◦ , 11.4◦ and 22.4◦ , which were similar to the previous work (Sashiwa et al., 2003; Wan et al., 2003). The small peak at
approximately 16.1◦ was attributed to the anhydrous crystal of chitosan whereas the diffraction peak at around 22◦ was observed in chitosan film prepared from dissolving chitosan in acetic acid solution (Bangyekan et al., 2006). The X-ray diffraction pattern of STI (C1) was similar to WTI (C2), and there was no obvious X-ray diffraction peak in the diffraction pattern. The intensity of diffraction peak of the blend films (D1 and D2) at 18.1◦ and 22.4◦ , compared with chitosan film, became more flat and broad. It illustrated that the addition of protein and glycerol decreased the crystallines of chitosan film. This phenomenon was perhaps due to the significant hydrogen bonding interaction (Wang et al., 2007) among chitosan molecule, protein and glycerol, which was consistent with the results of FTIR and no reappearance of chitosan powder diffractogram peaks suggested that there was not a phase separation among the components of the composite films. In other words, the addition of protein and glycerol improved the compatibility among these components.
3.5.
Anti-A. flavus activity assay
The results of anti-A. flavus (Fig. 4) showed that the hyphal growth in the control containing heat-inactivated TI was similar to that in the phosphate buffer control. However, it was
Fig. 3 – XRD patterns: (A) chitosan powder; (B) chitosan film; (C1) STI; (C2) WTI; (D1) chitosan–STI–Gly film; (D2) chitosan–WTI–Gly film.
546
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
Fig. 4 – Growth of A. flavus conidia (0.1 mL, 104 /mL) after 48 h at 25 ◦ C in 10 mL of 10% potato dextrose broth (PDB) (left picture)/Czapeks liquid medium (right picture). (A1/A2) 0.6 mol/L purified STI/WTI; (B1/B2) 1.2 mol/L purified STI/WTI; (C1/C2) 1.8 mol/L STI/WTI; (D1/D2) 2.4 mol/L purified STI/WTI.
notable that both the hyphal growth and mycelium mass of A. flavus were inhibited by purified STI and WTI. Strong reduction in hyphal growth was observed in the purified STI/WTI range of 0–1.8 mol/L. And with a concentration of 1.8 mol/L, the growth was almost inhibited completely. The IC50 values of purified STI and WTI activity against A. flavus were 1.6 mol/L and 1.0 mol/L (data was not showed) which were stronger than some of the antifungal peptide in the previous reports (Shivaraj and Pattabiraman, 1981; Wong and Ng, 2003). In order to evaluate the anti- A. flavus activity by chitosan and chitosan–STI/WTI–Gly films, the further experimental approach was tested. The results (Table 2) showed that A. flavus conidia inoculated on the surface of the paper disk immersed in simple chitosan solution could germinate and growth normally. The diameter of A. flavus growth was about 14.3 mm after 48 h at 28 ◦ C, without significantly different compared with the control (14.6 mm), which suggested the simple chitosan film were not responsible for inhibition on the germination and growth of A. flavus. On the opposite, the chitosan–STI/WTI–Gly blend film gave an obvious inhibition activity on which the A. flavus conidia were completely inhibited without germination and growth.
Table 2 – Diameters of A. flavus growth inhibition by chitosan and chitosan–STI/WTI–Gly films. Film type Control Chitosan solution Chitosan–STI–Gly film Chitosan–WTI–Gly film
Diameters of growth inhibition (mm) 14.6 ± 2.5 14.3 ± 2.0 -
Incubation periods of petri dishes at 28 ◦ C were 48 h. Data represent the means ± S.D. of measurement for there different samples. The transverse lines represent that the A. flavus conidia were inhibited completely without germination and growth.
The germination and growth of A. flavus conidia on peanuts coating with chitosan film and chitosan–STI/WTI–Gly blend film beforehand was observed after peanuts incubated at 28 ◦ C for 5 days (Fig. 5). Compared to the blank control (Fig. 5A), the chitosan–STI/WTI–Gly blend film could inhibit the A. flavus conidia germination and growth completely (C and D), however no obvious visible inhibition on A. flavus conidia germination and growth on peanuts by simple chitosan film was observed (B). Taken together, the inhibition on A. flavus infec-
Fig. 5 – Inhibition of A. flavus growth on peanuts coating with chitosan and chitosan–STI/WTI–Gly films. (A) Negative control; (B) coating with chitosan film; (C) coating with chitosan–STI–Gly film; (D) coating with chitosan–WTI–Gly film.
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
tion was mainly due to the activity of STI/WTI incorporated in the blend film. Alpha amylase secreted by A. flavus is an important hydrolase activity for breaking down the plant storage polysaccharide starch to produce low molecular weight glucose, maltose and maltotriose, which are known to induce aflatoxin B1 biosynthesis (Shivaraj and Pattabiraman, 1981). It had been demonstrated that constitutive levels of a 14kDa TI in corn kernels were associated with resistance to A. flavus infection and aflatoxin production. And the 14-kDa TI resisted to A. flavus by inhibition the activity of secreted ␣amylase (Shivaraj and Pattabiraman, 1981; Chen et al., 1999b; Fakhoury and Woloshuk, 1999). Except these, some TIs had been reported to inhibit both trypsin and ␣-amylase (Chen et al., 1992; Richardson, 1991). Therefore, similar to the reported inhibitors from the corn and other plant tissues (Mellon et al., 2007), STI/WTI might also work on the availability of simple sugars for A. flavus growth by limiting the hydrolase. In other word, it was likely that STI/WTI exerted its anti-A. flavus activity by the inhibition of ␣-amylase activity required for conidia germination, and little was known about the modes of action of STI/WTI as A. flavus growth inhibitor, which needed a further study. In addition, chitosan film itself showed some antimicrobial effect even though it did not reveal inhibitory zone in any microorganisms tested (Ouattara et al., 2000; Pranoto et al., 2005). And the dense structure of the blend films could control the moisture and oxygen transmission of grain acting as a physical barrier, away from the contents favorable to the growth and mycotoxin contamination of A. flavus. Accordingly, characterization of the mode of action of blend films could constitute a first step leading to the development of anti-A. flavus infection on peanut or other cereals.
4.
Conclusion
The smooth and dense morphology of chitosan–STI/WTI–Gly blend films were prepared by a casting/solvent evaporation method. In particular, the blend films had increasing elongation at break significantly and decreasing water vapor transmission rate. X-ray diffraction and FTIR spectroscopy were used to evaluate the interaction mode among chitosan, STI/WTI and glycerol molecules, the results of which suggested that the addition of protein and glycerol improved the compatibility of the components, and the hydrogen bonds of N H· · ·O C and O H· · ·O C had formed as the main major interaction force in the blend films. Furthermore, the prepared blend films could inhibit the growth of A. flavus strongly, which indicated that the blend films might be useful as potential biocontrol packaging on peanuts or other cereals against A. flavus under storage conditions.
Acknowledgements We thank the key project from Shandong province (2007GG10009005) and the marine project for public benefits from oceanic bureau of China (200805038). Special thanks to Dr. Peter Bucheli from Nestlé R&D Centre Shanghai Ltd.
547
references
ASTM, 1995. Standard test methods for tensile properties of thin plastic sheeting. In: Annual Book of American Standard Testing Methods. American Society for Testing Materials, Philadelphia. Bangyekan, C., Aht-Ong, D., Srikulkit, K., 2006. Preparation and properties evaluation of chitosan-coated cassava starch films. Carbohydr. Polym. 63, 61–71. Binder, E.M., Tan, L.M., Chin, L.J., Handl, J., Richard, J., 2007. World wide occurrence of mycotoxins in commodities, feeds and feed ingredients. Anim. Feed Sci. Technol. 137, 265–282. Bourtoom, T., Chinnan, M.S., 2008. Preparation and properties of rice starchechitosan blend biodegradable film, Available online at www.sciencedirect.com. Cao, N., Fu, Y.H., He, J.H., 2007. Preparation and physical properties of soy protein isolate and gelatin composite films. Food Hydrocolloid 21, 1153–1162. Chen, M.S., Feng, G., Zen, K.C., Richardson, M., Valdes-Rodriguez, S., Reeck, G.R., Kramer, K.J., 1992. ␣-Amylases from three species of stored grain Coleoptera and their inhibition by wheat and corn proteinaceous inhibitors. Insect Biochem. Mol. Biol. 22, 261–268. Chen, Z.Y., Brown, R.L., Lax, A.R., Cleveland, T.E., Russin, J.S., 1999a. Inhibition of plant-pathogenic fungi by a corn trypsin inhibitor overexpressed in Escherichia coli. Appl. Environ. Microbiol. 65, 1320–1324. Chen, Z.Y., Brown, R.L., Russin, J.S., Lax, A.R., Cleveland, T.E., 1999b. A corn trypsin inhibitor with antifungal activity inhibits Aspergillus flavus ␣-amylase. Biochem. Cell Biol. 89, 902–907. Chillo, S., Flores, S., Mastromatteo, M., Conte, A., Gerschenson, L., Nobile, M.A., 2008. Influence of glycerol and chitosan on tapioca starch-based edible film properties. J. Food Eng., Available online at www.sciencedirect.com. Cleveland, T.E., Dowd, P.F., Desjardin, A.E., Bhatnagar, D., Cotty, P.J., 2003. United States Department of Agriculture-Agricultural Research Service research on pre-harvest prevention of mycotoxins and mycotoxigenic fungi in US crops. Pest Manage. Sci. 59, 629–642. Fakhoury, A.M., Woloshuk, C.P., 1999. Amyl, the ␣-amylase gene of Aspergillus flavus: involvement in aflatoxin biosynthesis in maize kernels. Phytopathology 89, 908–914. Fakhoury, A.M., Woloshuk, C.P., 2001. Inhibition of growth of Aspergillus flavus and fungal ␣-amylases by a lectin-like protein from Lablab purpureus. Am. Phytopathol. Soc. 14, 955–961. Fu, J.H., Ji, J., Yuan, W.Y., Shen, J.C., 2005. Construction of anti-adhesive and antibacterial multilayer films via layer-by-layer assembly of heparin and chitosan. Biomaterials 26, 6684–6692. Gontard, N., Duchez, C., Cuq, J.L., Guilbert, S., 1994. Edible composite films of wheat gluten and lipids: water vapour permeability and other physical properties. Int. J. Food Sci. Technol. 29, 39–43. Gounga, M.E., Xu, S.Y., Wang, Z., 2007. Whey protein isolate-based edible films as affected by protein concentration, glycerol ratio and pullulan addition in film formation. J. Food Eng. 83, 521–530. Jia, Z.S., Li, X.L., 2002. Determination of the deacetylation degree of chitosan by acid base conductometric titration. Chin. J. Anal. Chem. 7, 846–848. Kim, M.H., Park, S.C., Kim, J.Y., Lee, S.Y., Lim, H.T., Cheong, H., Hahm, K.S., Park, Y., 2006. Purification and characterization of a heat-stable serine protease inhibitor from the tubers of new potato variety “Golden Valley”. Biochem. Biophys. Res. Commun. 346, 681–686.
548
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
Lee, J.W., Son, S.M., Hong, S.I., 2008. Characterization of protein-coated polypropylene films as a novel composite structure for active food packaging application. J. Food Eng. 86, 484–493. Li, B., Kennedy, J.F., Peng, J.L., Yie, X., Xie, B.J., 2006. Preparation and performance evaluation of glucomannan–chitosan–nisin ternary antimicrobial blend film. Carbohydr. Polym. 65, 488–494. López-Malo, A., Alzamora, S.M., Palou, E., 2005. Aspergillus flavus growth in the presence of chemical preservatives and naturally occurring antimicrobial compounds. Int. J. Food Microbiol. 99, 119–128. Ma, J.H., Liu, W.S., 2008. PP non-woven specialties via chemical degradation. Polym. Mater. Sci. Eng. 24, 147–150. Magan, N., Aldred, D., 2007. Post-harvest control strategies: minimizing mycotoxins in the food chain. Int. J. Food Microbiol. 119, 131–139. Mellon, J.E., Cotty, P.J., Dowd, M.K., 2007. Aspergillus flavus hydrolases: their roles in pathogenesis and substrate utilization. Appl. Microbiol. Biotechnol. 77, 497–504. Nunthanid, J., Puttipipatkhachorn, S., Yamamoto, K., Peck, G.E., 2001. Physical properties and molecular behavior of chitosan films. Drug Dev. Ind. Pharm. 27, 143–157. Neamnark, A., Sanchavanakit, N., Pavasant, P., Bunaprasert, T., Supaphol, P., Rujiravanit, R., 2007. In vitro biocompatibility evaluations of hexanoyl chitosan film. Carbohydr. Polym. 68, 166–172. Ouattara, B., Simard, R.E., Piette, G., Bégin, A., Holley, R.A., 2000. Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan. International Journal of Food Microbiology 62, 139–148. Pierro, P.D., Chico, B., Villalonga, R., Mariniello, L., Masi, P., Porta, R., 2007. Transglutaminase-catalyzed preparation of chitosan–ovalbumin films. Enzyme Microb. Technol. 40, 437–441. Prodpran, T., Benjakul, S., Artharn, A., 2007. Properties and microstructure of protein-based film from round scad (Decapterus maruadsi) muscle as affected by palm oil and chitosan incorporation. Int. J. Biol. Macromol. 41, 605–614. Pranoto, Y., Rakshit, S.K., Salokhe, V.M., 2005. Enhancing antimicrobial activity of chitosan films by incorporating garlic oil, potassium sorbate and nisin. Food Sci. Technol. 38, 859–865. Richardson, M., 1991. Seed storage proteins: the enzyme inhibitors. Methods Plant Biochem. 5, 259–305. Sashiwa, H., Fujishima, S., Yamano, N., Kawasaki, N., Nakayama, A., Muraki, E., Sukwttanasinitt, M., Pichyangkura, R., Aiba, S.I., 2003. Enzymatic production of N-acetyl-glucosamine from chitin. Degradation study of N-acetylchitooligosaccharide and the effect of mixing of crude enzymes. Carbohydr. Polym. 51, 391–395. Schmidt, V., Giacomelli, C., Soldi, V., 2005. Thermal stability of films formed by soy protein isolate–sodium dodecyl sulfate. Polym. Degrad. Stab. 87, 25–31. Sébastien, F., Stéphane, G., Copinet, A., Coma, V., 2006. Novel biodegradable films made from chitosan and poly (lactic acid) with antifungal properties against mycotoxinogen strains. Carbohydr. Polym. 65, 185–193.
Shivaraj, B., Pattabiraman, T.N., 1981. Characterization of an unusual ␣-amylase/trypsin inhibitor from ragi (Eleusine coracana Gearta.). Biochem. J. 193, 29–36. Sunzuki, S., Ying, B., Yamane, H., Tachi, H., Shimahashi, K., Ogawa, K., Kitamura, S., 2007. Surface structure of chitosan and hybrid chitosan–amylose films—restoration of the antibacterial properties of chitosan in the amylose film. Carbohyd. Res. 342, 2490–2493. Tanabe, T., Okitsu, N., Tachibana, A., Yamauchi, K., 2002. Preparation and characterization of keratin–chitosan composite film. Biomaterials 23, 817–825. Wan, Y., Creber, K.A.M., Preppley, B., Bui, V.T., 2003. Ionic conductivity of chitosan membranes. Polymer 44, 1057–1065. Wang, B., Wang, K.Y., Dan, W.H., Zhang, T.Y., Ye, Y., 2006. Konjac glucomannan–collagen–chitosan blend films. J. Biomed. Eng. 23, 102–106. Wang, H.X., Ng, T.B., 2006a. Ganodermin, an antifungal protein from fruiting bodies of the medicinal mushroom Ganoderma lucidum. Peptides 27, 27–30. Wang, H.X., Ng, T.B., 2006b. Concurrent isolation of a Kunitz-type trypsin inhibitor with antifungal activity and a novel lectin from Pseudostellaria heterophylla roots. Biochem. Biophys. Res. Commun. 342, 349–353. Wang, H.X., Ng, T.B., 2007. Isolation and characterization of an antifungal peptide with antiproliferative activity from seeds of Phaseolus vulgaris cv. ‘Spotted Bean’. Appl. Microbiol. Biotechnol. 74, 125–130. Wang, Q.S., Ying, T.J., 2007. Research progress on waste water utilization in soybean processing. Food Sci. 28, 594–598. Wang, X.Y., Du, Y.M., Luo, J.W., Lin, B.F., Kennedy, J.F., 2007. Chitosan/organic rectorite nanocomposite films: structure, characteristic and drug delivery behaviour. Carbohydr. Polym. 69, 41–49. Wong, J.H., Ng, T.B., 2003. Gymnin, a potent defensin-like antifungal peptide from the Yunnan bean (Gymnocladus chinensis Baill). Peptides 24, 963–968. Yang, X.Y., Li, J., Wang, X.W., Fang, W.G., Michael, J.B., She, R., Xiao, Y.H., Pei, Y., 2006. Psc-AFP, an antifungal protein with trypsin inhibitor activity from Psoralea corylifolia seeds. Peptides 27, 1726–1731. Ye, X.Y., Ng, T.B., Rao, P.F., 2001. A Bowman–Birk-type trypsin–chymotrypsin inhibitor from broad beans. Biochem. Biophys. Res. Commun. 289, 91–96. Yeboah, N.A., Arahira, M., Udaka, K., Fukazawa, C., 1996. A rapid purification method for soybean Bowman–Birk protease inhibitor using hydrophobic interaction chromatography. Protein Expr. Purif. 7, 309–314. Zhang, B., Zhang, L., Yu, L.N., Wang, D.F., 2007. Preparation of immobilized trypsin chitosan resin and its absorption property for soybean trypsin inhibitor. Sci. Technol. Food Ind. 7, 65–68. Zhang, L., Wang, D.F., Zhang, B., Sun, L.P., 2006. Advances in the study of protease inhibitors from leguminous plants. Soybean Sci. 3, 314–319. Zhang, T., Shi, Z.Q., Hu, L.B., Cheng, L.G., Wang, F., 2007. Antifungal compounds from Bacillus subtilis B-FS06 inhibiting the growth of Aspergillus flavus. World J. Microbiol. Biotechnol. 24, 783–788.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/indcrop
Preparation and properties of chitosan–soybean trypsin inhibitor blend film with anti-Aspergillus flavus activity Bin Zhang, Dong-Feng Wang ∗ , Hai-Yan Li, Ying Xu, Li Zhang Department of Food Chemistry and Nutrition, College of Food Science and Technology, Ocean University of China, Yu Shan Road 5, Qingdao 266003, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
The chitosan-based blend films were prepared from chitosan, soybean trypsin inhibitor
Received 11 August 2008
extract (STI)/wild soybean trypsin inhibitor extract (WTI) and glycerol (Gly) solutions, the
Received in revised form
properties of which were also investigated, including thickness, mechanical property, water
30 October 2008
vapor transmission, optical transmittance, and solubility. In addition, the resulting films
Accepted 31 October 2008
were characterized by scanning electron microscope (SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The result of SEM images showed the surface and cross-section of chitosan–STI/WTI–Gly blend film had more smooth and dense mor-
Keywords:
phology than pure chitosan film, which suggested there was a better compatibility among
Chitosan
the three components. XRD and FTIR spectra indicated that the possible interaction force
Trypsin inhibitor
among the components might be the hydrogen bonds of N H· · ·O C and O H· · ·O C. Fur-
Blend film
thermore, the antifungal activity against A. flavus by the prepared blend films had been
A. flavus
investigated. The facts that the germination and growth of A. flavus were strongly inhibited by chitosan–STI/WTI–Gly film indicated the blend films might be useful as potential bio-control packaging against A. flavus during the peanuts and other cereals storage. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Aspergillus flavus is one of the major spoilage moulds of intermediate moisture foods that are widely distributed in tropical and subtropical zones around the globe. A. flavus can infect several agricultural crops such as peanuts, maize, tree nuts, and rice, resulting in the production of one of the most toxic and potent carcinogenic metabolites known as aflatoxin (Binder et al., 2007). Therefore, a variety of methods such as physical methods (aeration, temperature and moisture control, modified atmospheres, etc.) (Cleveland et al., 2003; Magan and Aldred, 2007) or by fungistats of which the potassium sorbate and sodium benzoate are the commonly used (LópezMalo et al., 2005), were adopted for inhibition on the fungal
growth and subsequent mycotoxin production. While, more attention has been particularly given to the natural antifungal peptide (Kim et al., 2006; Wang and Ng, 2006a; Wang and Ng, 2007), some of which have been demonstrated important antiA. flavus properties (Fakhoury and Woloshuk, 2001; B. Zhang et al., 2007; T. Zhang et al., 2007). Soybean whey is a byproduct during the preparation of soybean protein, and approximately 300 million ton of the whey is wasted and abandoned every year in China (Wang and Ying, 2007). Trypsin inhibitor (TI), large quantities in the whey, possesses various physiology functions used in the biological pesticide and biomedical fields (Zhang et al., 2006). What is more, TIs of different size and amino acid sequence have been isolated from various cereals including corn kernels, and
Abbreviations: TI, Trypsin inhibitor; STI, Soybean (Glycine max) trypsin inhibitor extract; WTI, Wild soybean (Glycine soya) trypsin inhibitor extract; Gly, Glycerol; A. flavus, Aspergillus flavus. ∗ Corresponding author. Tel.: +86 532 82031575; fax: +86 532 82032468. E-mail address: [email protected] (D.-F. Wang). 0926-6690/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2008.10.007
542
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
identified the activity of anti-A. flavus (Chen et al., 1999a,b; Kim et al., 2006; Mellon et al., 2007). However, there are rarely questioned about cultivated and wild soybean TI anti-A. flavus activity (Ye et al., 2001; Wang and Ng, 2006b; Yang et al., 2006) in the reports. Chitosan obtained by the deacetylation of chitin is one of the most abundant natural aminopolysaccharides. Owing to its good biodegradability, biocompatibility and non-toxicity, it has been extensively used over a wide range of applications, including the biomedical area and edible coating or films. Currently, chitosan and its films have been documented for its anti-fungal (Sébastien et al., 2006), anti-microbial (Fu et al., 2005; Sunzuki et al., 2007) and anti-viral (Neamnark et al., 2007) properties. Therefore, many active biopolymers including proteins (Tanabe et al., 2002), polysaccharides (Li et al., 2006), lipids (Pranoto et al., 2005) or their combinations have been incorporated in the edible chitosan films, to enhance its excellent oxygen, carbon dioxide barrier and interesting antimicrobial properties. Nevertheless, the chitosan-based blend materials, incorporated in anti-viral peptides, such as protein inhibitors, to be employed as packaging, particularly as an edible packaging film for resistance to A. flavus infection, have not been reported so far. Therefore, the objective of this study is to prepare blend films by making from soybean TI extract/wild soybean TI extract incorporated in chitosan and glycerol solution, study the intrinsic properties and investigate the possible molecular miscibility among the components, further to study the activity against A. flavus germination and growth on peanuts.
2.
Materials and methods
2.1.
Materials
TI extract was prepared from the cultivated soybean (Glycine max) (STI) from a local supermarket and wild soybean (Glycine soya) (WTI) which was manually collected from the plants grown in Qingdao city (36◦ 38 N, 120◦ 45 E), Shandong province, China. The cultivated/wild soybean seed flour with distilled water (1:6, w/v) was stirring for 12 h at 4 ◦ C, with subsequent centrifugation at 3000 × g for 15 min. The sediment obtained was extracted once more. The obtained supernatants were mixed and adjusted to pH 4.5 (B. Zhang et al., 2007; T. Zhang et al., 2007) before being centrifuged at 5000 × g for 20 min. The concentrated supernatant obtained (soybean whey) after freeze drying was referred as crude STI/WTI extract used in the preparation of chitosan–STI/WTI blend film. And the crude STI/WTI extract was further purified using the hydrophobic interaction chromatography following the previous report (Yeboah et al., 1996). Glycerol (Gly) and acetic acid were obtained from Sigma Chemical Co. Chitosan (degree of deacetylation was 90%) was prepared following the method of Jia and Li (2002). All the chemicals and reagents were of analytical grade.
2.2.
Film preparation
The chitosan–STI/WTI–Gly blend film was fabricated by a casting/solvent evaporation (Cao et al., 2007) of acetic acid chi-
tosan, STI/WTI and glycerol solutions. Initially, STI/WTI (0.2%, w/v) was dispersed in an aqueous solution of acetic acid (1.0%, v/v). Then chitosan and glycerol were added to the solution to get a final concentration of 1.8% (w/v) and 1.2% (w/v), until the homogenous solution was obtained. The solution was deaerated under vacuum (0.09 MPa) at room temperature for 2 h prior to casting film, to prevent pinhole formation in the film when the casting solvent evaporated. Then the blend solution was cast onto the rectangular polyvinyl chloride mold (bottom; 8.0 cm × 12.0 cm) and dried at 45 ◦ C for 12 h. Film thickness was controlled by casting the same amount (10.0 mL) of forming solution per mold. Finally, the resulting film was manually peeled off, stored in plastic bags and held in desiccators for further barrier, mechanical properties and biological activity testing. Above the added concentrations of chitosan, STI/WTI, glycerol and other operating conditions had been optimized by Box-Behnken design experiment, with anti-A. flavus activity as evaluation indexes (data was not showed).
2.3.
Measurement of film properties
Thickness of the films was determined using a precision digital coating thickness gauge Elcometer A 300 FNP 23 (England). The measurements were taken at 15 different points of each specimen and the mean values were taken. Mechanical properties were measured by using a Texture Analyser (TMS-PRO, FTC, USA). Sample films were cut into 20.0 mm wide and 80.0 mm long strips. Grip separation was set at 40.0 mm, with a cross-head speed of 50.0 mm/min. Tensile strength (TS) and percentage of elongation at break (EB) were evaluated in eight samples from each type of film and the mean values were taken, according to the standard testing method (ASTM, 1995). Film transparency was determined according to the following method. The films were cut into rectangular shapes (15.0 mm × 40.0 mm) and placed on the internal side of a spectrophotometer cell. Transparency of films was measured using a spectrophotometer (UV 2102 PC, UNICO, USA) at 560 nm. Three replicates of each film were tested. A modified method from Pierro et al. (2007) was used to measure film solubility. The solubility studies of the films were carried out by incubating the samples at 25 ◦ C in deionized water. From each film pieces of 40.0 mm × 40.0 mm were cut and stored in desiccators with silica gel for 7 days. Sample films were weighed (Wdry ) to the nearest 0.0001 g and placed into test beakers with 80.0 mL deionized water at room temperature until the film reached to constant weight. After that, the remaining pieces of film were collected by filtration, dried again in an oven at 50 ◦ C to constant weight (Wfinal dry ). The percentage of total soluble matter (solubility) was calculated as follows: solubility (%) = 100 × (Wdry − Wfinal dry )/Wdry . The measurement was repeated five times for each type of film, and an average was taken as the result. The water vapor transmission (WVP) of the films was determined by gravimetric test according to modified ASTM method (Gontard et al., 1994) as previously described. Three grams of anhydrous calcium chloride were introduced in each weighing bottle (25.0 mm in diameter). The film sample having diameter of about 40.0 mm was placed on top of the cup and sealed by melted paraffin, which area exposed to vapor transmission was about 3.46 cm2 . The cups were then weighed and placed
543
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
Table 1 – Properties of chitosan and chitosan–STI/WTI–Gly films. Film type
Thickness (×10−2 mm)
TS (Pa)
EB (%)
WVP (×10−7 g mm/cm2 h mmHg)
Transparency (%)
Solubility (%)
Chitosan film Chitosan–STI–Gly film Chitosan–WTI–Gly film
2.47 ± 0.12a 2.92 ± 0.17b 2.84 ± 0.11b
3731.1 ± 75.5a 2158.9 ± 20.9b 2059.4 ± 38.0b
29.6 ± 1.7a 69.0 ± 2.9b 73.5 ± 2.3b
2.57 ± 0.35a 2.45 ± 0.52b 2.43 ± 0.42b
88.7 ± 0.2a 82.8 ± 0.3b 83.2 ± 0.1b
98.1 ± 1.2a 34.6 ± 2.0b 31.7 ± 1.6b
Data represent the means ± S.D. of measurement for there different samples. Student–Newman–Keuls (SNK) test is used to determine the significance, and the means with different letters in the same column are significantly different at P < 0.05. TS is tensile strength; EB is elongation at break; WVP is water vapor permeability.
in desiccators in which provided a 100% relative humidity, stored in a thermostat incubator at 25.0 ◦ C. The water vapor transferred through the films at different time intervals were determined from the weight gain of the cups. The WVP was calculated as follows: WVP = [Wd]/[S t(p2 − p1 )], where W was the weight of water absorbed in the cups, d was the thickness of the film, S was the area of the exposed film, t was the time for weight change, p2 − p1 was the vapor pressure differential across the film.
distilled water (Control), chitosan and chitosan–STI/WTI–Gly blend film solution and dried at 45 ◦ C for 12 h. The prepared carriers of the films (dried paper disks) were deposited on the surface of Czapek culture medium followed by 15.0 L of A. flavus spores (102 /mL) inoculated on the surface of the paper disks. After incubation at 28 ◦ C for 48 h until the growth of A. flavus was evaluated macroscopically, the diameters (mm) of growth inhibition was determined. Above each petri dish was conducted in triplicate.
2.4.
2.6.
Characterization analyses
The surface and cross-section of the chitosan and chitosan–STI/WTI–Gly blend films were coated with the fine gold palladium film and examined by a scanning electron microscope (SEM) (JSM-6390LV, JEOL Ltd., Japan). Fourier transform infrared (FTIR) spectrometry was used to characterize the functional groups of the films in order to determine the possible interaction mode among chitosan, STI/WTI and glycerol in the composite system. Samples were analyzed between 400 and 4000 cm−1 with a resolution of 4 cm−1 in a PerkinElmer spectrum 2000 (USA). X-ray diffraction patterns of chitosan, STI/WTI, chitosan film and chitosan–STI/WTI–Gly blend film were analyzed using an X-ray diffractometer (XPERT, Philips, Netherlands) in the angular range of 5–40 (2) with Nickel-filtered Cu K␣ radiation at a voltage of 40 kV and current of 30 mA, to investigate the crystalline property.
2.5.
Anti-A. flavus activity assay
The purified STI/WTI was added at a final concentration of 0.0, 0.6, 1.2, 1.8 and 2.4 mol/L to the conical flasks, which contained 10.0 mL of 10% potato dextrose broth (PDB)/Czapeks liquid medium and 0.1 mL of freshly harvested A. flavus conidia (104 /mL). The conidia were incubated for up to 48 h at 28 ◦ C. Negative controls were 10 mmol/L phosphate buffer or heat-inactivated purified STI/WTI. Morphological changes of hyphae were examined periodically during the incubation using light microscopy. Above each conical flask was conducted in triplicate for one concentration, according to the previous report (Chen et al., 1999a). Antifungal activity of prepared chitosan film and chitosan–STI/WTI–Gly blend film against A. flavus was carried out in 100 mm × 15 mm petri dish containing Czapek culture medium according to the previous report (Ouattara et al., 2000) with a further modification. The sterile blank paper disks (7.0 mm in diameter) were immersed in the
Inhibition of A. flavus growth on peanuts
Peanuts were surface sterilized with 1.0% NaClO solution for 60 s and rinsed in sterile distilled water before coating with chitosan and chitosan–STI/WTI–Gly solution. The peanuts coating with the film were dried at 45 ◦ C for 12 h and put into conical flasks. Then each flask was inoculated with 0.1 mL, 102 /mL of A. flavus spore suspension. After incubation at 28 ◦ C for 5 days with vigorous shaking for 1 h once a day, the growth of A. flavus was evaluated macroscopically. Above the flask was conducted in triplicate for each coating film.
2.7.
Statistical analysis
Measurements of each property of the films were conducted at least three times in a randomized design and all measurements were carried out at least twice. Statistical analysis was conducted using the SPSS package (SPSS 13.0 for windows, SPSS Inc., Chicago, IL). And the Student’s–Newman–Keuls (SNK) test was used to determine the significance different of the properties of the films at P < 0.05.
3.
Results and discussion
3.1.
Properties of film
The properties of chitosan and chitosan–STI/WTI–Gly films were displayed in Table 1. As can be seen from the results, the chitosan–STI/WTI–Gly blend film (2.92/2.84 mm) had a significant increase in thickness as the level of STI/WTI and glycerol incorporated, compared with the pure chitosan film (2.47 mm). The edible films must withstand the normal stress encountered during its application and subsequent shipping and handling of the food. High tensile strength (TS) is generally required, but deformation values must be adjusted according to the intended application of the films. TS of the chitosan–STI/WTI–Gly film (2158/2059 Pa) decreased signifi-
544
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
Fig. 1 – SEM micrographs of chitosan and chitosan–STI/WTI–Gly films. Surface and cross-section (A1 and A2) of chitosan film; surface and cross-section (B1 and B2) of chitosan–STI–Gly film; surface and cross-section (C1 and C2) of chitosan–WTI–Gly film.
cantly, compared with simple chitosan film (3731 Pa), mainly affected by the incorporation of STI/WTI protein and glycerol. Particularly, the protein could exert negative effects on both coating operation and tensile property, due to their intrinsic brittleness (Lee et al., 2008). And also the TS values were influenced by the negative influence of the interaction between chitosan and glycerol (Chillo et al., 2008). Elongation at the break (EB) is an indication of the film’s flexibility and extensibility. The average EB values of the blend films behaved inversely to the TS values, increasing from 29.6% for chitosan film to 69.0%/73.5% for chitosan–STI/WTI–Gly film, which mainly effected by the added glycerol distributing uniformly and improving flexibility of protein incorporated films (Gounga et al., 2007). Water vapor permeability (WVP) values of the blend films decreased significantly, since STI/WTI was hydrophilic materials and could be well incorporated into the chitosan network structure. And the addition of STI/WTI and glycerol might cause hydrogen bond formation, thus reinforce the compactness of the blend films (Prodpran et al., 2007). Since a main function of the edible film was to impede moisture transfer between food and the surrounding atmosphere, or between two components of a heterogeneous food product, WVP should be as low as possible (Bourtoom and Chinnan, 2008). Solubility in water is also an important property, which governs potential applications of these materials to food preservation. Generally, films with low water solubility are necessary for the protection of foodstuffs with high or intermediate water activity (aw). Pure chitosan film was almost soluble (98.1%) or very difficult to recover after the water immersion process. The addition of STI/WTI and glycerol decreased the solubility to 34.6%/31.7% significantly. Color attributes are of prime importance because they directly influence consumer acceptability. The pure chitosan film was nearly transparent (88.7%) at a wavelength of 560 nm. The percent transmittance value of the chitosan–STI/WTI–Gly
film decreased significantly (82.8%/83.2%), caused by the dispersion of STI/WTI and glycerol in the chitosan matrix.
3.2.
Surface and cross-section structure (SEM) analysis
The surface and cross-section morphology of chitosan and chitosan–STI/WTI–Gly films (Fig. 1) were analyzed by scanning electron microscopy (SEM). The surface and cross-section of chitosan film appeared to be certain discontinuous zones and small pores. However, the chitosan–STI/WTI–Gly blend film exhibited a dense and uniform microstructure, which might be due to the reorientation of polar functional groups and change of networking structure introduced by protein and glycerol. The smooth and dense morphology of the surface and cross-section also suggested that there was a better compatibility among chitosan, STI/WTI and glycerol, perhaps the result of strong intermolecular interaction caused by the produced hydrogen bonding (Pierro et al., 2007).
3.3.
FTIR analysis spectroscopy
The infrared spectrophometry of STI, WTI, chitosan film, chitosan–STI/WTI–Gly blend film were shown in Fig. 2. The signal of the chitosan film could be significantly recognized from the intensity of peaks at spectral line A. The characteristic peak of STI (B1) was almost in concordance with infrared absorption of WTI (B2). As the spectral peak of chitosan–STI/WTI–Gly blend film (C1/C2) displayed, the absorb band at 3379 cm−1 /3357 cm−1 , associated with the stretch vibration absorption peak of O–H, had strong absorption and a significantly downward shift compared with the spectral of chitosan film (A), which perhaps proved that more hydrogen bonds had generated because of the incorporation of protein and glycerol. Obviously, the absorb band in the blend film (C1 and C2) at about 1647 cm−1 had been blue shift to 1561 cm−1 , compared with line B1 (1645 cm−1 ), B2 (1647 cm−1 )
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
545
Fig. 2 – FTIR spectra: (A) chitosan film; (B1) STI; (B2) WTI; (C1) chitosan–STI–Gly film; (C2) chitosan–WTI–Gly film.
and A (1647 cm-1 ) associated with amide I absorption, mainly resulted from the formation of hydrogen bonds between –NH2 , –COOH, –OH of STI/WTI and –C O of chitosan (Schmidt et al., 2005). Therefore, the absorb band in the line C1 and C2 at 1561 cm−1 , had greatly strengthened compared with line A. Except these, the absorb band in the line C1 and C2 at about 1250 cm−1 , associated with bend vibration absorption peak of O–H, had reduced considerably compared with line B1 and B2. All the above, it is likely that the hydrogen bonds of N H· · ·O C and O H· · ·O C had formed which perhaps was the main major interaction force in the blend films (Wang et al., 2006; Ma and Liu, 2008).
3.4.
X-ray diffraction analysis
The X-ray diffractograms of chitosan powder, chitosan film and chitosan–STI/WTI–Gly blend film were displayed in Fig. 3. As observed, the chitosan powder (A) was in a crystalline state because two main diffraction peaks (2 = 11.5◦ and 20.25◦ ) were observed in its X-ray diffraction pattern, agreed with the finding of previous report (Nunthanid et al., 2001). The diffractogram of chitosan film (B) showed the crystalline peaks at 8.3◦ , 11.4◦ and 22.4◦ , which were similar to the previous work (Sashiwa et al., 2003; Wan et al., 2003). The small peak at
approximately 16.1◦ was attributed to the anhydrous crystal of chitosan whereas the diffraction peak at around 22◦ was observed in chitosan film prepared from dissolving chitosan in acetic acid solution (Bangyekan et al., 2006). The X-ray diffraction pattern of STI (C1) was similar to WTI (C2), and there was no obvious X-ray diffraction peak in the diffraction pattern. The intensity of diffraction peak of the blend films (D1 and D2) at 18.1◦ and 22.4◦ , compared with chitosan film, became more flat and broad. It illustrated that the addition of protein and glycerol decreased the crystallines of chitosan film. This phenomenon was perhaps due to the significant hydrogen bonding interaction (Wang et al., 2007) among chitosan molecule, protein and glycerol, which was consistent with the results of FTIR and no reappearance of chitosan powder diffractogram peaks suggested that there was not a phase separation among the components of the composite films. In other words, the addition of protein and glycerol improved the compatibility among these components.
3.5.
Anti-A. flavus activity assay
The results of anti-A. flavus (Fig. 4) showed that the hyphal growth in the control containing heat-inactivated TI was similar to that in the phosphate buffer control. However, it was
Fig. 3 – XRD patterns: (A) chitosan powder; (B) chitosan film; (C1) STI; (C2) WTI; (D1) chitosan–STI–Gly film; (D2) chitosan–WTI–Gly film.
546
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
Fig. 4 – Growth of A. flavus conidia (0.1 mL, 104 /mL) after 48 h at 25 ◦ C in 10 mL of 10% potato dextrose broth (PDB) (left picture)/Czapeks liquid medium (right picture). (A1/A2) 0.6 mol/L purified STI/WTI; (B1/B2) 1.2 mol/L purified STI/WTI; (C1/C2) 1.8 mol/L STI/WTI; (D1/D2) 2.4 mol/L purified STI/WTI.
notable that both the hyphal growth and mycelium mass of A. flavus were inhibited by purified STI and WTI. Strong reduction in hyphal growth was observed in the purified STI/WTI range of 0–1.8 mol/L. And with a concentration of 1.8 mol/L, the growth was almost inhibited completely. The IC50 values of purified STI and WTI activity against A. flavus were 1.6 mol/L and 1.0 mol/L (data was not showed) which were stronger than some of the antifungal peptide in the previous reports (Shivaraj and Pattabiraman, 1981; Wong and Ng, 2003). In order to evaluate the anti- A. flavus activity by chitosan and chitosan–STI/WTI–Gly films, the further experimental approach was tested. The results (Table 2) showed that A. flavus conidia inoculated on the surface of the paper disk immersed in simple chitosan solution could germinate and growth normally. The diameter of A. flavus growth was about 14.3 mm after 48 h at 28 ◦ C, without significantly different compared with the control (14.6 mm), which suggested the simple chitosan film were not responsible for inhibition on the germination and growth of A. flavus. On the opposite, the chitosan–STI/WTI–Gly blend film gave an obvious inhibition activity on which the A. flavus conidia were completely inhibited without germination and growth.
Table 2 – Diameters of A. flavus growth inhibition by chitosan and chitosan–STI/WTI–Gly films. Film type Control Chitosan solution Chitosan–STI–Gly film Chitosan–WTI–Gly film
Diameters of growth inhibition (mm) 14.6 ± 2.5 14.3 ± 2.0 -
Incubation periods of petri dishes at 28 ◦ C were 48 h. Data represent the means ± S.D. of measurement for there different samples. The transverse lines represent that the A. flavus conidia were inhibited completely without germination and growth.
The germination and growth of A. flavus conidia on peanuts coating with chitosan film and chitosan–STI/WTI–Gly blend film beforehand was observed after peanuts incubated at 28 ◦ C for 5 days (Fig. 5). Compared to the blank control (Fig. 5A), the chitosan–STI/WTI–Gly blend film could inhibit the A. flavus conidia germination and growth completely (C and D), however no obvious visible inhibition on A. flavus conidia germination and growth on peanuts by simple chitosan film was observed (B). Taken together, the inhibition on A. flavus infec-
Fig. 5 – Inhibition of A. flavus growth on peanuts coating with chitosan and chitosan–STI/WTI–Gly films. (A) Negative control; (B) coating with chitosan film; (C) coating with chitosan–STI–Gly film; (D) coating with chitosan–WTI–Gly film.
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
tion was mainly due to the activity of STI/WTI incorporated in the blend film. Alpha amylase secreted by A. flavus is an important hydrolase activity for breaking down the plant storage polysaccharide starch to produce low molecular weight glucose, maltose and maltotriose, which are known to induce aflatoxin B1 biosynthesis (Shivaraj and Pattabiraman, 1981). It had been demonstrated that constitutive levels of a 14kDa TI in corn kernels were associated with resistance to A. flavus infection and aflatoxin production. And the 14-kDa TI resisted to A. flavus by inhibition the activity of secreted ␣amylase (Shivaraj and Pattabiraman, 1981; Chen et al., 1999b; Fakhoury and Woloshuk, 1999). Except these, some TIs had been reported to inhibit both trypsin and ␣-amylase (Chen et al., 1992; Richardson, 1991). Therefore, similar to the reported inhibitors from the corn and other plant tissues (Mellon et al., 2007), STI/WTI might also work on the availability of simple sugars for A. flavus growth by limiting the hydrolase. In other word, it was likely that STI/WTI exerted its anti-A. flavus activity by the inhibition of ␣-amylase activity required for conidia germination, and little was known about the modes of action of STI/WTI as A. flavus growth inhibitor, which needed a further study. In addition, chitosan film itself showed some antimicrobial effect even though it did not reveal inhibitory zone in any microorganisms tested (Ouattara et al., 2000; Pranoto et al., 2005). And the dense structure of the blend films could control the moisture and oxygen transmission of grain acting as a physical barrier, away from the contents favorable to the growth and mycotoxin contamination of A. flavus. Accordingly, characterization of the mode of action of blend films could constitute a first step leading to the development of anti-A. flavus infection on peanut or other cereals.
4.
Conclusion
The smooth and dense morphology of chitosan–STI/WTI–Gly blend films were prepared by a casting/solvent evaporation method. In particular, the blend films had increasing elongation at break significantly and decreasing water vapor transmission rate. X-ray diffraction and FTIR spectroscopy were used to evaluate the interaction mode among chitosan, STI/WTI and glycerol molecules, the results of which suggested that the addition of protein and glycerol improved the compatibility of the components, and the hydrogen bonds of N H· · ·O C and O H· · ·O C had formed as the main major interaction force in the blend films. Furthermore, the prepared blend films could inhibit the growth of A. flavus strongly, which indicated that the blend films might be useful as potential biocontrol packaging on peanuts or other cereals against A. flavus under storage conditions.
Acknowledgements We thank the key project from Shandong province (2007GG10009005) and the marine project for public benefits from oceanic bureau of China (200805038). Special thanks to Dr. Peter Bucheli from Nestlé R&D Centre Shanghai Ltd.
547
references
ASTM, 1995. Standard test methods for tensile properties of thin plastic sheeting. In: Annual Book of American Standard Testing Methods. American Society for Testing Materials, Philadelphia. Bangyekan, C., Aht-Ong, D., Srikulkit, K., 2006. Preparation and properties evaluation of chitosan-coated cassava starch films. Carbohydr. Polym. 63, 61–71. Binder, E.M., Tan, L.M., Chin, L.J., Handl, J., Richard, J., 2007. World wide occurrence of mycotoxins in commodities, feeds and feed ingredients. Anim. Feed Sci. Technol. 137, 265–282. Bourtoom, T., Chinnan, M.S., 2008. Preparation and properties of rice starchechitosan blend biodegradable film, Available online at www.sciencedirect.com. Cao, N., Fu, Y.H., He, J.H., 2007. Preparation and physical properties of soy protein isolate and gelatin composite films. Food Hydrocolloid 21, 1153–1162. Chen, M.S., Feng, G., Zen, K.C., Richardson, M., Valdes-Rodriguez, S., Reeck, G.R., Kramer, K.J., 1992. ␣-Amylases from three species of stored grain Coleoptera and their inhibition by wheat and corn proteinaceous inhibitors. Insect Biochem. Mol. Biol. 22, 261–268. Chen, Z.Y., Brown, R.L., Lax, A.R., Cleveland, T.E., Russin, J.S., 1999a. Inhibition of plant-pathogenic fungi by a corn trypsin inhibitor overexpressed in Escherichia coli. Appl. Environ. Microbiol. 65, 1320–1324. Chen, Z.Y., Brown, R.L., Russin, J.S., Lax, A.R., Cleveland, T.E., 1999b. A corn trypsin inhibitor with antifungal activity inhibits Aspergillus flavus ␣-amylase. Biochem. Cell Biol. 89, 902–907. Chillo, S., Flores, S., Mastromatteo, M., Conte, A., Gerschenson, L., Nobile, M.A., 2008. Influence of glycerol and chitosan on tapioca starch-based edible film properties. J. Food Eng., Available online at www.sciencedirect.com. Cleveland, T.E., Dowd, P.F., Desjardin, A.E., Bhatnagar, D., Cotty, P.J., 2003. United States Department of Agriculture-Agricultural Research Service research on pre-harvest prevention of mycotoxins and mycotoxigenic fungi in US crops. Pest Manage. Sci. 59, 629–642. Fakhoury, A.M., Woloshuk, C.P., 1999. Amyl, the ␣-amylase gene of Aspergillus flavus: involvement in aflatoxin biosynthesis in maize kernels. Phytopathology 89, 908–914. Fakhoury, A.M., Woloshuk, C.P., 2001. Inhibition of growth of Aspergillus flavus and fungal ␣-amylases by a lectin-like protein from Lablab purpureus. Am. Phytopathol. Soc. 14, 955–961. Fu, J.H., Ji, J., Yuan, W.Y., Shen, J.C., 2005. Construction of anti-adhesive and antibacterial multilayer films via layer-by-layer assembly of heparin and chitosan. Biomaterials 26, 6684–6692. Gontard, N., Duchez, C., Cuq, J.L., Guilbert, S., 1994. Edible composite films of wheat gluten and lipids: water vapour permeability and other physical properties. Int. J. Food Sci. Technol. 29, 39–43. Gounga, M.E., Xu, S.Y., Wang, Z., 2007. Whey protein isolate-based edible films as affected by protein concentration, glycerol ratio and pullulan addition in film formation. J. Food Eng. 83, 521–530. Jia, Z.S., Li, X.L., 2002. Determination of the deacetylation degree of chitosan by acid base conductometric titration. Chin. J. Anal. Chem. 7, 846–848. Kim, M.H., Park, S.C., Kim, J.Y., Lee, S.Y., Lim, H.T., Cheong, H., Hahm, K.S., Park, Y., 2006. Purification and characterization of a heat-stable serine protease inhibitor from the tubers of new potato variety “Golden Valley”. Biochem. Biophys. Res. Commun. 346, 681–686.
548
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 541–548
Lee, J.W., Son, S.M., Hong, S.I., 2008. Characterization of protein-coated polypropylene films as a novel composite structure for active food packaging application. J. Food Eng. 86, 484–493. Li, B., Kennedy, J.F., Peng, J.L., Yie, X., Xie, B.J., 2006. Preparation and performance evaluation of glucomannan–chitosan–nisin ternary antimicrobial blend film. Carbohydr. Polym. 65, 488–494. López-Malo, A., Alzamora, S.M., Palou, E., 2005. Aspergillus flavus growth in the presence of chemical preservatives and naturally occurring antimicrobial compounds. Int. J. Food Microbiol. 99, 119–128. Ma, J.H., Liu, W.S., 2008. PP non-woven specialties via chemical degradation. Polym. Mater. Sci. Eng. 24, 147–150. Magan, N., Aldred, D., 2007. Post-harvest control strategies: minimizing mycotoxins in the food chain. Int. J. Food Microbiol. 119, 131–139. Mellon, J.E., Cotty, P.J., Dowd, M.K., 2007. Aspergillus flavus hydrolases: their roles in pathogenesis and substrate utilization. Appl. Microbiol. Biotechnol. 77, 497–504. Nunthanid, J., Puttipipatkhachorn, S., Yamamoto, K., Peck, G.E., 2001. Physical properties and molecular behavior of chitosan films. Drug Dev. Ind. Pharm. 27, 143–157. Neamnark, A., Sanchavanakit, N., Pavasant, P., Bunaprasert, T., Supaphol, P., Rujiravanit, R., 2007. In vitro biocompatibility evaluations of hexanoyl chitosan film. Carbohydr. Polym. 68, 166–172. Ouattara, B., Simard, R.E., Piette, G., Bégin, A., Holley, R.A., 2000. Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan. International Journal of Food Microbiology 62, 139–148. Pierro, P.D., Chico, B., Villalonga, R., Mariniello, L., Masi, P., Porta, R., 2007. Transglutaminase-catalyzed preparation of chitosan–ovalbumin films. Enzyme Microb. Technol. 40, 437–441. Prodpran, T., Benjakul, S., Artharn, A., 2007. Properties and microstructure of protein-based film from round scad (Decapterus maruadsi) muscle as affected by palm oil and chitosan incorporation. Int. J. Biol. Macromol. 41, 605–614. Pranoto, Y., Rakshit, S.K., Salokhe, V.M., 2005. Enhancing antimicrobial activity of chitosan films by incorporating garlic oil, potassium sorbate and nisin. Food Sci. Technol. 38, 859–865. Richardson, M., 1991. Seed storage proteins: the enzyme inhibitors. Methods Plant Biochem. 5, 259–305. Sashiwa, H., Fujishima, S., Yamano, N., Kawasaki, N., Nakayama, A., Muraki, E., Sukwttanasinitt, M., Pichyangkura, R., Aiba, S.I., 2003. Enzymatic production of N-acetyl-glucosamine from chitin. Degradation study of N-acetylchitooligosaccharide and the effect of mixing of crude enzymes. Carbohydr. Polym. 51, 391–395. Schmidt, V., Giacomelli, C., Soldi, V., 2005. Thermal stability of films formed by soy protein isolate–sodium dodecyl sulfate. Polym. Degrad. Stab. 87, 25–31. Sébastien, F., Stéphane, G., Copinet, A., Coma, V., 2006. Novel biodegradable films made from chitosan and poly (lactic acid) with antifungal properties against mycotoxinogen strains. Carbohydr. Polym. 65, 185–193.
Shivaraj, B., Pattabiraman, T.N., 1981. Characterization of an unusual ␣-amylase/trypsin inhibitor from ragi (Eleusine coracana Gearta.). Biochem. J. 193, 29–36. Sunzuki, S., Ying, B., Yamane, H., Tachi, H., Shimahashi, K., Ogawa, K., Kitamura, S., 2007. Surface structure of chitosan and hybrid chitosan–amylose films—restoration of the antibacterial properties of chitosan in the amylose film. Carbohyd. Res. 342, 2490–2493. Tanabe, T., Okitsu, N., Tachibana, A., Yamauchi, K., 2002. Preparation and characterization of keratin–chitosan composite film. Biomaterials 23, 817–825. Wan, Y., Creber, K.A.M., Preppley, B., Bui, V.T., 2003. Ionic conductivity of chitosan membranes. Polymer 44, 1057–1065. Wang, B., Wang, K.Y., Dan, W.H., Zhang, T.Y., Ye, Y., 2006. Konjac glucomannan–collagen–chitosan blend films. J. Biomed. Eng. 23, 102–106. Wang, H.X., Ng, T.B., 2006a. Ganodermin, an antifungal protein from fruiting bodies of the medicinal mushroom Ganoderma lucidum. Peptides 27, 27–30. Wang, H.X., Ng, T.B., 2006b. Concurrent isolation of a Kunitz-type trypsin inhibitor with antifungal activity and a novel lectin from Pseudostellaria heterophylla roots. Biochem. Biophys. Res. Commun. 342, 349–353. Wang, H.X., Ng, T.B., 2007. Isolation and characterization of an antifungal peptide with antiproliferative activity from seeds of Phaseolus vulgaris cv. ‘Spotted Bean’. Appl. Microbiol. Biotechnol. 74, 125–130. Wang, Q.S., Ying, T.J., 2007. Research progress on waste water utilization in soybean processing. Food Sci. 28, 594–598. Wang, X.Y., Du, Y.M., Luo, J.W., Lin, B.F., Kennedy, J.F., 2007. Chitosan/organic rectorite nanocomposite films: structure, characteristic and drug delivery behaviour. Carbohydr. Polym. 69, 41–49. Wong, J.H., Ng, T.B., 2003. Gymnin, a potent defensin-like antifungal peptide from the Yunnan bean (Gymnocladus chinensis Baill). Peptides 24, 963–968. Yang, X.Y., Li, J., Wang, X.W., Fang, W.G., Michael, J.B., She, R., Xiao, Y.H., Pei, Y., 2006. Psc-AFP, an antifungal protein with trypsin inhibitor activity from Psoralea corylifolia seeds. Peptides 27, 1726–1731. Ye, X.Y., Ng, T.B., Rao, P.F., 2001. A Bowman–Birk-type trypsin–chymotrypsin inhibitor from broad beans. Biochem. Biophys. Res. Commun. 289, 91–96. Yeboah, N.A., Arahira, M., Udaka, K., Fukazawa, C., 1996. A rapid purification method for soybean Bowman–Birk protease inhibitor using hydrophobic interaction chromatography. Protein Expr. Purif. 7, 309–314. Zhang, B., Zhang, L., Yu, L.N., Wang, D.F., 2007. Preparation of immobilized trypsin chitosan resin and its absorption property for soybean trypsin inhibitor. Sci. Technol. Food Ind. 7, 65–68. Zhang, L., Wang, D.F., Zhang, B., Sun, L.P., 2006. Advances in the study of protease inhibitors from leguminous plants. Soybean Sci. 3, 314–319. Zhang, T., Shi, Z.Q., Hu, L.B., Cheng, L.G., Wang, F., 2007. Antifungal compounds from Bacillus subtilis B-FS06 inhibiting the growth of Aspergillus flavus. World J. Microbiol. Biotechnol. 24, 783–788.