Development of new antioxidant active packaging films based on ethylene vinyl alcohol copolymer (EVOH) and green tea extract

Ethylene vinyl alcohol copolymer (EVOH) films containing green tea extract were successfully produced by extrusion. The films were brown and translucent, and the addition of the extract increased the water and oxygen barrier at low relative humidity but increased the water sensitivity, the glass transition temperature, and the crystallinity of the films and improved their thermal resistance. An analysis by HPLC revealed that the antioxidant components of the extract suffered partial degradation during extrusion, reducing the content of catechin gallates and increasing the concentration of free gallic acid. Exposure of the films to various food simulants showed that the liquid simulants increased their capacity to reduce DPPH(?) and ABTS(?+) radicals. The release of green tea extract components into the simulant monitored by HPLC showed that all compounds present in the green tea extract were partially released, although the extent and kinetics of release were dependent on the type of food. In aqueous food simulants, gallic acid was the main antioxidant component released with partition coefficient values ca. 200. In 95% ethanol (fatty food simulant) the K value for gallic acid decreased to 8 and there was a substantial contribution of catechins (K in the 1000 range) to a greatly increased antioxidant efficiency. Kinetically, gallic acid was released more quickly than catechins, owing to its faster diffusivity in the polymer matrix as a consequence of its smaller molecular size, although the most relevant effect is the plasticization of the matrix by alcohol, increasing the diffusion coefficient >10-fold. Therefore, the materials here developed with the combination of antioxidant substances that constitute the green tea extract could be used in the design of antioxidant active packaging for all type of foods, from aqueous to fatty products, the compounds responsible for the protection being those with the higher compatibility with the packaged product.     

Characterization of the interaction between two food aroma components, alpha-pinene and ethyl butyrate, and ethylene-vinyl alcohol copolymer (EVOH) packaging films as a function of environmental humidity

The ethylene-vinyl alcohol copolymers (EVOHs) are well-known high oxygen barrier materials that are being used successfully in the design of packaging structures for oxygen-sensitive food or pharmaceutical products. Recently, there has been increasing interest in using EVOH materials to provide a high barrier to organic compounds as a means to reduce food aroma scalping. However, the barrier function of this family of materials diminishes significantly in humid environments, and it is supposed that so does the organic vapor barrier. In this work, a new sorption-based method to characterize the interaction between food aroma and polymer films for packaging as a function of relative humidity is presented and is used to determine the barrier to ethyl butyrate and alpha-pinene of EVOH at 23 degrees C. The results show that although EVOH is an excellent barrier to food aroma when dry, a property that even improves at low relative humidity (RH), the solubility and diffusivity of the compounds tested increase dramatically with humidity at medium to high water activities. However, even in the worst case (100% RH), EVOH outperforms low-density polyethylene (LDPE) as a barrier to organic vapors at least 500,000-fold.     

Preparation and characterization of chitosan-based nanocomposite films with antimicrobial activity

Four different types of chitosan-based nanocomposite films were prepared using a solvent-casting method by incorporation with four types of nanoparticles, that is, an unmodified montmorillonite (Na-MMT), an organically modified montmorillonite (Cloisite 30B), a Nano-silver, and a Ag-zeolite (Ag-Ion). X-ray diffraction patterns of the nanocomposite films indicated that a certain degree of intercalation was formed in the nanocomposite films, with the highest intercalation in the Na-MMT-incorporated films followed by films with Cloisite 30B and Ag-Ion. Scanning electron micrographs showed that in all of the nanocomposite films, except the Nano-silver-incorporated one, nanoparticles were dispersed homogeneously throughout the chitosan polymer matrix. Consequently, mechanical and barrier properties of chitosan films were affected through intercalation of nanoparticles, that is, tensile strength increased by 7-16%, whereas water vapor permeability decreased by 25-30% depending on the nanoparticle material tested. In addition, chitosan-based nanocomposite films, especially silver-containing ones, showed a promising range of antimicrobial activity.     

Development of flexible antimicrobial films using essential oils as active agents

The antimicrobial activity in the vapor-phase of laboratory-made flexible films of polypropylene (PP) and polyethylene/ethylene vinyl alcohol copolymer (PE/EVOH) incorporating essential oil of cinnamon ( Cinnamomum zeylanicum), oregano ( Origanum vulgare), clove ( Syzygium aromaticum), or cinnamon fortified with cinnamaldehyde was evaluated against a wide range of microorganisms: the Gram-negative bacteria Escherichia coli, Yersinia enterocolitica, Pseudomonas aeruginosa, and Salmonella choleraesuis; the Gram-positive bacteria Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and Enterococcus faecalis; the molds Penicillium islandicum, Penicillium roqueforti, Penicillium nalgiovense, Eurotium repens, and A spergillus flavus and the yeasts Candida albicans, Debaryomyces hansenii, and Zigosaccharomyces rouxii. Films with a nominal concentration of 4% (w/w) of fortified cinnamon or oregano essential oil completely inhibited the growth of the fungi; higher concentrations were required to inhibit the Gram-positive bacteria (8 and 10%, respectively), and higher concentrations still were necessary to inhibit the Gram-negative bacteria. PP films were more effective than PE/EVOH films. The atmospheres generated by the antimicrobial films inside Petri dishes were quantitatively analyzed using headspace-single drop microextraction (HS-SDME) in combination with gas chromatography-mass spectrometry (GC-MS). The analyses showed that the oregano-fortified PP films released higher levels of carvacrol and thymol, and the cinnamon-fortified PP films released higher levels of cinnamaldehyde, during the first 3-6 h of incubation, than the corresponding PE/EVOH films. Shelf-life tests were also performed, demonstrating that the antifungal activities of the films persisted for more than two months after their manufacture. In addition, migration tests (overall and specific) were performed, using both aqueous and fatty simulants, to ensure that the films meet EU regulations regarding food contact materials. Following contact with the tested films, the substances that had migrated into the aqueous simulants were recovered by direct immersion-single drop extraction (DI-SDME) and then analyzed by GC-MS. The fatty stimulant (isooctane) was directly injected into the chromatographic system.     

Development and characterization of films based on chemically cross-linked gliadins

The aim of the present work has been to study the possibility of obtaining modified gliadin films with improved water resistance and mechanical properties by means of promoting intermolecular covalent bonds between polypeptide chains. Prior to casting films, formaldehyde, glutaraldehyde, and glyoxal were used to cross-link proteins at concentrations ranging from 1% to 4% (grams per 100 g of protein). Mechanical properties (tensile strength and elongation at break), water vapor permeability, moisture sorption isotherms, and optical properties of the films produced were evaluated as a function of the cross-linker used. Experimental results showed that some properties of gliadin films were considerably modified. Cross-linking improved the water resistance of films, avoiding their disintegration. Their water barrier properties were also enhanced, but their moisture sorption properties remained unchanged. Formaldehyde imparted greater mechanical strength to films than glutaraldehyde or glyoxal, increasing tensile strength values 10-fold. Addition of the cross-linkers at concentrations in excess of 2.5% did not further improve the mechanical or barrier properties. However, modification with glutaraldehyde or glyoxal imparted an increasingly yellowish tint to the films.     

Physicochemical and mechanical properties of experimental coextruded food-packaging films containing a buried layer of recycled low-density polyethylene

Migrational, permeation, and tensile properties of experimental five- and eight-layer coextruded and laminated films containing a middle buried layer of recycled low-density polyethylene (LDPE) comprising 40-50% bw of the multilayer structure were determined. Respective films containing 100% virgin LDPE as the buried layer were taken as controls. Results showed that the percentage of recycled LDPE in the multilayer structure did not affect overall migration values to distilled water, 3% acetic acid, and iso-octane. In all cases, overall migration values were lower than the upper acceptable limit (10 mg/dm(2)) set by the European Union. Transmission rate values to O(2), CO(2), and water vapor were also not affected by the percentage of recycled LDPE in the multilayer structure. On the basis of O(2) transmission rates, low-barrier, barrier, and high-barrier multilayer structures were produced. Likewise, tensile properties (tensile strength, percent elongation at break, and Young's modulus) were not affected by the percentage of recycled material in the multilayer structure. Finally, all experimental films produced no adverse effects in taste or odor of the food-contacting phase tested. The above findings are discussed in relation to the high quality of the primary LDPE scrap used throughout this work in combination with the functional barrier hypothesis. On the basis of the present results it is proposed that primary LDPE scrap may be used as a middle layer comprising 40-50% bw of multilayer food-packaging films without any compromise in migrational, barrier, mechanical, and organoleptic properties.     

Bioautography and chemical characterization of antimicrobial compound(s) in commercial water-soluble annatto extracts

Annatto preparations based on extracts of the seed of tropical bush Bixa orellana L consist of carotenoid-type pigments. Previous reports indicate that commercial annatto extracts have biological activities against microorganisms of significance to food fermentation, preservation, and safety. The objective of this study was to separate and identify the compound(s) responsible for the antimicrobial activity of annatto preparations. Commercial water-soluble annatto extracts were screened by thin-layer chromatography and bioautography followed by liquid chromatography/photodiode array/mass spectrometry (LC/PDA/MS) analysis of active fractions. Bioautography revealed two fractions with antimicrobial activity against Staphylococcus aureus. LC/PDA/MS analysis of both fractions revealed 9'-cis-norbixin (UV(max) 460 and 489 nm) and all-trans-norbixin (UV(max) 287, 470, and 494 nm) as the major components. Structure confirmation was achieved by (1)H NMR spectroscopy. Results indicate that 9'-cis-norbixin and all-trans-norbixin are responsible for the antimicrobial properties of annatto.     

Characterization, antimicrobial activity, and mechanism of a high-performance (-)-epigallocatechin-3-gallate (EGCG)-CuII/polyvinyl alcohol (PVA) nanofibrous membrane

(-)-Epigallocatechin-3-gallate (EGCG) exhibited strong antimicrobial activity. However, the easy oxidation of EGCG has limited its application. To increase the antimicrobial activity and stability of EGCG, the EGCG-Cu(II) complex was formed by chelating copper ions and then electronspun into polyvinyl alcohol (PVA) nanofibers. Electronspun nanofibrous membranes were investigated by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM), which showed that the average fiber diameter was 210 nm. Minimal inhibitory concentrations (MICs) of EGCG-Cu(II)/PVA membranes were tested against the tested strains. The bactericidal activity of EGCG-Cu(II) was suppressed by ethylenediaminetetraacetic acid (EDTA). Cell killing was accompanied by the leakage of intracellular proteins, indicating that the cytoplasmic membrane was badly damaged after exposure to the EGCG-Cu(II)/PVA membrane. We observed the process of cell damage by SEM. On the basis of experimental evidence and theoretical analyses, the mechanism proposed that copper ions played a cooperative role in the bactericidal process of EGCG. To evaluate the antimicrobial activity of the EGCG-Cu(II)/PVA membrane, we developed a rapid detection method by labeling cells with water-soluble CdTe quantum dots.     

Immobilization of subtilisin on polycaprolactam for antimicrobial food packaging applications

Subtilisin was immobilized on polycaprolactam and used for food packaging applications to reduce the transference of microorganisms from the packaging material to the packaged food material. The optimized conditions for subtilisin immobilization was as follows: pH, 8; temperature, 4 °C; glutaraldehyde, 0.5%; incubation time, 25 h; and subtilisin concentration, 600 μL. The formation of -CH═N- at 1576 cm(-1) in the Fourier transform infrared (FTIR) spectrum confirmed the immobilization. Subtilisin-immobilized polycaprolactam (SIP) exhibited the highest residual activity of 106.67 ± 4.41% and 104.67 ± 0.88% at 40 °C and pH 8 and retained residual activity of 94% at the end of 56 days when compared to 21.33 ± 4.10% in the case of free subtilisin. SIP significantly (p < 0.05) lowered the colony forming units (CFU), dry weight, and protein and carbohydrate contents in bacterial and fungal biofilm. Practical application of the SIP on ham steaks at 4 and 20 °C showed a 2-3 times reduction of Staphylococcus aureus as well as Escherichia coli cells in the range of p < 0.05.     

Development and characterization of biodegradable films made from wheat gluten protein fractions

Gliadins and glutenins were extracted from commercial wheat gluten on the basis of their extractability in ethanol and used to produce film-forming solutions. Films cast using these gliadin- and glutenin-rich solutions were characterized. Glycerol was used as a plasticizer, and its effect on the films was also studied. Films obtained from the glutenin fraction presented higher tensile strength values and lower elongation at break and water vapor permeability values than gliadin films. Gliadin films disintegrated when immersed in water. The GAB isotherm model was used to describe the equilibrium moisture sorption of the films. The glycerol concentration largely modified mechanical and water vapor barrier properties of both film types.     

Development of flexible antimicrobial packaging materials against Campylobacter jejuni by incorporation of gallic acid into zein-based films

In this study, antimicrobial films were developed against Campylobacter jejuni by incorporation of gallic acid (GA) into zein-based films. The zein and zein-wax composite films containing GA between 2.5 and 10 mg/cm(2) were effective on different C. jejuni strains in a concentration-dependent manner. Zein and zein-wax composite films showed different release profiles in distilled water but quite similar release profiles at solid agar medium. Depending on incorporated GA concentration, 60-80% of GA released from the films, while the remaining GA was bound or trapped by film matrix. The GA at 2.5 and 5 mg/cm(2) caused a considerable increase in elongation (57-280%) of all zein films and eliminated their classical flexibility problems. The zein-wax composite films were less flexible than zein films, but the films showed similar tensile strengths and Young's modulus. Scanning electron microscopy indicated different morphologies of zein and zein-wax composite films. This study clearly showed the good potential of zein and GA to develop flexible antimicrobial films against C. jejuni.     

Preparation and characterization of films from pea protein

The conditions for protein film preparation from an alkaline dispersion of a pea protein isolate were investigated in the presence of polyols as plasticizers. Mechanical and barrier properties of resulting films were studied as a function of protein dispersion conditions, protein and plasticizer concentrations and ratios, chain length of the plasticizer, and pH and composition of the alkaline medium. Neither the mode of protein hydration nor the pea isolate origin had a significant effect on the mechanical properties of pea protein films. However, increasing the plasticizer chain length induced slightly higher surface hydrophobicity but poor mechanical properties. Addition of monoglycerides to film-forming solution allowed a significant improvement of the films during aging. Both tensile strength and surface hydrophobicity increased when ammonium hydroxide was used as protein dispersing agent instead of sodium hydroxide.     

Isolation and characterization of an extracellular antimicrobial protein from Aspergillus oryzae

A 17 kDa antimicrobial protein was isolated from growth medium containing the filamentous fungus Aspergillus oryzae by extracting the supernatants from the culture media, ion exchange chromatography on CM-sepharose, and C18 reverse-phase high-performance liquid chromatography. This antimicrobial protein, which we considered to be an extracellular antimicrobial protein from A. oryzae (exAP-AO17), possessed antimicrobial activity but lacked hemolytic activity. The exAP-AO17 protein strongly inhibited pathogenic microbial strains, including pathogenic fungi, Fusarium moniliform var. subglutinans and Colletotrichum coccodes, and showed antibacterial activity against bacteria, including E. coli O157 and Staphylococcus aureus. To confirm that the protein acts as a regulation factor for extracellular secretion, we examined growth under varying conditions of N sources, C sources, ions, ambient pH, and stress. Various culture conditions were found to induce characteristic changes in the expression of protein synthesis as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Highly basic polypeptides were regulated by suppressing the ambient pH under acidic conditions and strongly induced under alkaline conditions, thus confirming that pH regulation is physiologically relevant. The expression of exAP-AO17 was upregulated by heat shock upon growth in the presence of NaCl. Automated Edman degradation showed that the N-terminal sequence of exAP-AO17 was NH 2-GLPGPAGAVGFAGKDQNM-. ExAP-AO17 showed partial sequence homology with a collagen belonging to the animal source. These results suggest that exAP-AO17 is an excellent candidate as a lead compound for the development of novel oral or other types of anti-infective agents.     

Production and characterization of films from cotton stalk xylan

Composite film production based on cotton stalk xylan was studied, and the mechanical and physical properties of the films formed were investigated. Xylan and lignin were separated from cellulose by alkali extraction and, then, lignin was removed using ethanol washing. Self-supporting continuous films could not be produced using pure cotton stalk xylan. However, film formation was achieved using 8-14% (w/w) xylan without complete removal of lignin during xylan isolation. Keeping about 1% lignin in xylan (w/w) was determined to be sufficient for film formation. Films were produced by casting the film-forming solutions, followed by solvent evaporation in a temperature (20 degrees C) and relative humidity (40%) controlled environment. The elastic modulus and hypothetical coating strength of the films obtained by using 8% xylan were significantly different from the ones containing 10-14% xylan. The water vapor transfer rates (WVTR) decreased with increasing xylan concentration, which made the films thicker. The glycerol addition as an additional plasticizer resulting in more stretchable films having higher WVTR and lower water solubility values. As a result, film production was successfully achieved from xylan, which was extracted from an agricultural waste (cotton stalk), and the film-forming effect of lignin on pure xylan has been demonstrated.     

Mechanical, barrier, and antimicrobial properties of apple puree edible films containing plant essential oils

Edible films, as carriers of antimicrobial compounds, constitute an approach for incorporating plant essential oils (EOs) onto fresh-cut fruit surfaces. The effect against Escherichia coli O157:H7 of oregano, cinnamon, and lemongrass oils in apple puree film-forming solution (APFFS) and in an edible film made from the apple puree solution (APEF) was investigated along with the mechanical and physical properties of the films. Bactericidal activities of APFFS, expressed as BA50 values (BA50 values are defined as the percentage of antimicrobial that killed 50% of the bacteria under the test conditions) ranged from 0.019% for oregano oil to 0.094% for cinnamon oil. Oregano oil in the apple puree and in the film was highly effective against E. coli O157:H7. The data show that (a) the order of antimicrobial activities was oregano oil > lemongrass oil > cinnamon oil and (b) addition of the essential oils into film-forming solution decreased water vapor permeability and increased oxygen permeability, but did not significantly alter the tensile properties of the films. These results show that plant-derived essential oils can be used to prepare apple-based antimicrobial edible films for various food applications.     

Development of an iron chelating polyethylene film for active packaging applications

Metal-promoted oxidation reactions are a major cause of food quality deterioration. Active packaging offers novel approaches to controlling such oxidation for the purpose of extending shelf life. Herein, we report modification of the surface of polyethylene (PE) films to possess metal chelating activity. Metal chelating carboxylic acids were introduced to the film surface using cross-linking agents [polyethylenimine (PEI) or ethylenediamine (ED)] to increase the number of available carboxylic acids. ATR-FTIR, contact angle, dye assay, and iron chelating assay were used to characterize changes in surface chemistry after each functionalization step. The chelator poly(acrylic acid) (PAA) was attached to the surface at a density of 9.12 ± 0.71 nmol carboxyl groups/cm2, and exhibited an iron chelating activity. The results indicate that PAA-modified PE films might have a higher affinity to Fe3? than Fe2? with the optimum binding pH at 5.0. Such inexpensive active packaging materials are promising in food industry for the preservation of liquid and semiliquid food products and have application in heavy metal chelation therapy for biomedical materials as well.     

Structure and physical properties of starch/poly vinyl alcohol/laponite RD nanocomposite films

Nanocomposites of starch, poly vinyl alcohol (PVOH), and laponite RD (LRD) were produced by solution mixing and cast into films. In general, an increase in LRD content (0-20%) enhanced tensile strength and decreased water vapor permeability, irrespective of the relative humidity (50% and 75% RH). Tensile strength (TS) of starch/PVOH/LRD films ranged from 6.51 to 13.3 MPa. At 75% RH, TS was up to 65% higher as compared to films with sodium montmorillonite as filler. The most striking results were obtained with respect to elongation at break (E%), which ranged from 144% to 312%. Contrary to other polymer/clay nanocomposites, E% increased on addition of 5-20% LRD and was up to 175% higher than the control without clay. Nanocomposite structure and interactions were investigated using X-ray diffraction, transmission electron microscopy, and differential scanning calorimetry. Results indicated that LRD was a compatibilizer and cross-linking agent between polymers, and has the potential for use in biodegradable packaging films with good mechanical performance even in high humidity conditions.     

Isolation and characterization of aminopeptidase (Jc-peptidase) from Japanese cedar pollen (Cryptomeria japonica)

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.     

Structure and physical properties of starch/poly vinyl alcohol/sodium montmorillonite nanocomposite films

Nanocomposites of starch, poly vinyl alcohol (PVOH), and sodium montmorillonite (Na(+)MMT) were produced by solution mixing and cast into films. Tensile strength (TS) and elongation at the break (E%) of the films ranged from 11.60 to 22.35 MPa and 28.93-211.40%, respectively, while water vapor permeability (WVP) ranged from 0.718 to 1.430 g·mm/kPa·h·m(2). In general, an increase in Na(+)MMT content (0-20%) enhanced TS and decreased E% and WVP. Use of higher molecular weight PVOH increased both TS and E% and also decreased WVP. Mechanical properties were negatively affected, but water vapor barrier properties improved with increasing starch content (0-80%). X-ray diffraction and transmission electron microscopy were used to analyze the nanostructure, and molecular conformations and interactions in the multicomponent nanocomposites were inferred from glass transition behavior. Interactions between starch and PVOH were strongest, followed by polymer/clay interactions. On the basis of this insight, a conceptual model was presented to explain the phenomena of intercalation and exfoliation in the starch/PVOH/Na(+)MMT nanocomposites.