Improvement in water stability and other related functional properties of thin cast kafirin protein films

Improvement in the water stability and other related functional properties of thin (<50 μm) kafirin protein films was investigated. Thin conventional kafirin films and kafirin microparticle films were prepared by casting in acetic acid solution. Thin kafirin films cast from microparticles were more stable in water than conventional cast kafirin films. Treatment of kafirin microparticles with heat and transglutaminase resulted in slightly thicker films with reduced tensile strength. In contrast, glutaraldehyde treatment resulted in up to a 43% increase in film tensile strength. The films prepared from microparticles treated with glutaraldehyde were quite stable in ambient temperature water, despite the loss of plasticizer. This was probably due to the formation of covalent cross-linking between free amino groups of the kafirin polypeptides and carbonyl groups of the aldehyde. Thus, such thin glutaraldehyde-treated kafirin microparticle films appear to have good potential for use as biomaterials in aqueous applications.     

Effect of preparation conditions on protein secondary structure and biofilm formation of kafirin

Various extraction and drying conditions for the isolation of kafirin from dry-milled, whole grain sorghum have been investigated, with a view to optimizing extraction of the protein for commercial food coatings and packaging films. The addition of sodium hydroxide to an aqueous ethanol extractant increased the yield and solubility of kafirin. Subsequent heat drying at 40 degrees C was shown to cause the kafirin to aggregate as indicated by an increase in intermolecular beta-sheets. Extraction of the flour using ethanol (70%, w/w) with 0.5% (w/w) sodium metabisulfite and 0.35% (w/w) sodium hydroxide at 70 degrees C followed by freeze-drying of the protein was found to produce a yield of 54% kafirin with good film-forming properties. The kafirin films were assessed for their sensory properties, tensile strength, strain, and water vapor permeability. Fourier transform infrared spectroscopy was used to study the secondary structure of the extracted kafirins. The best films were made with kafirin containing a large proportion of nativelike alpha-helical structures with little intermolecular beta-sheet content as indicated by the Fourier transform infrared reflectance peak intensity ratios associated with these secondary structures. The principal factor affecting the secondary structure of the protein appeared to be the temperature at which the protein was dried. Heat drying resulted in a greater proportion of intermolecular beta-sheets. Any industrial-scale extraction must therefore minimize protein aggregation and maximize native alpha-helical structures to achieve optimal film quality.     

Effect of Microwave Heating of Kafirin on the Functional Properties of Kafirin Films

To improve the functional properties of cast kafirin films, dry kafirin, extracted with an aqueous ethanol‐based solvent at 70°C, was microwave‐heated. No effect on film tensile properties was found. Two strategies were employed to improve the effect of microwaving: extraction of kafirin using an aqueous tert‐butanol‐based solvent at ambient temperature to minimize temperature‐induced denaturation and wetting the kafirin to increase its dielectric properties. Microwave heating this kafirin to 90 or 96°C and holding for 1–2 min more than doubled maximum tensile strength and Young's modulus, and decreased strain by about one‐third compared with films made from nonmicrowaved kafirin. Film water vapor permeability was reduced by at least one‐third. Digestibility of microwaved kafirin and films was also substantially decreased, and film biodegradability was slowed slightly. Microwave heating gave a film microstructure with fewer and smaller size pores. SDS‐PAGE showed microwave‐induced intermolecular cross‐linking of the kafirin monomers, which was possibly responsible for the modification of film properties. Microwave heating of kafirin can be used to modify kafirin film properties, but the kafirin must be microwaved wet and be as close as possible to its native state.     

Physical,Mechanical, and Barrier Properties of Kafirin Films from Red and White Sorghum Milling Fractions

Fractions from the sorghum dry milling industry, including bran, are a potential source of kafirin. Free‐standing plasticized cast films were prepared from defatted kafirin preparations from red and white sorghum flour and bran fractions, and from commercial zein. All the kafirin preparations were able to form films. However, there were differences in film thickness, clarity, flexibility, surface texture, odor, and color between the different kafirin films. Bran kafirin films were highly colored, less flexible with a less smooth surface texture compared with films from flour, probably due to higher levels of contaminants in the bran kafirins. The strong color of the bran films could limit their use in certain coating applications. The kafirin films had much higher tensile strength and lower extensibility than zein film, probably because of the presence of β‐ and γ‐kafirins in the kafirin, giving high levels of disulfide cross‐linking in the kafirin films. The kafirin films had poorer water barrier properties than zein film, possibly due to greater thickness or to poorer flexibility, which may have caused microcracks.     

Properties of chemically and physically treated wheat gluten films

Chemical (vapors of formaldehyde), physical (temperature, UV and gamma radiation), and aging treatments were applied to wheat gluten films. Changes in film mechanical properties, water vapor permeability, solubility, and color coordinates were investigated. An aging of 360 h led to a 75 and 314% increase in tensile strength and Young's modulus, respectively, and a 36% decrease in elongation. Severe thermal (above 110 degrees C, 15 min) and formaldehyde treatments highly improved the mechanical resistance of the films. Under these conditions, up to 376 and 654% increase in tensile strength and Young's modulus and up to 66% decrease in elongation have been observed. Water solubility was only slightly modified, whereas water vapor permeability was not affected. Color coordinates of films heated above 95 degrees C changed to a great extent. An almost total insolubilization of proteins in sodium dodecyl sulfate occurred for heat- and formaldehyde-treated films, due to the modification of protein network leading to changes in properties of the films.     

丁香酚对高粱醇溶蛋白可食性膜结构及性能的影响

为开发天然的可降解、可食性包装材料,以高粱醇溶蛋白为原料,采用溶液共混的方法制备可食性丁香酚/高粱醇溶蛋白复合膜,分析不同浓度丁香酚对可食性高粱醇溶蛋白膜物理性能及微观结构的影响并探讨其变化机理。结果表明,添加4%丁香酚可优化蛋白膜的机械性能,提升膜的拉伸强度(TS)和断裂伸长率(EAB);添加丁香酚不影响蛋白膜的水蒸气透过系数(WVP),但略微提高了蛋白膜的溶解度;添加4%丁香酚可增加蛋白膜对紫外光和可见光的吸光度值,即增强膜的光阻隔性能。DSC测量显示,添加丁香酚后降低了高粱醇溶蛋白的玻璃态转变温度(Tg),表明丁香酚提高了丁香酚/高粱醇溶蛋白复合膜的延展性;FTIR分析结果表明,添加丁香酚后使得高粱醇溶蛋白二级结构中的α-螺旋、无规则卷曲转变为β-折叠、β-转角,表明丁香酚有助于提高丁香酚/高粱醇溶蛋白复合膜的机械性能;SEM结果显示,4%丁香酚与高粱醇溶蛋白的相容性良好,制备的复合膜截面光滑紧致。本研究结果为可降解、可食性膜新材料的研究及应用推广提供了理论参考。     

Development of biodegradable films from whey proteins by cross-linking and entrapment in cellulose

When cross-linked by heating or by gamma-irradiation and entrapped in cellulose, whey proteins can generate insoluble biofilms with good mechanical properties and high resistance to attack by proteolytic enzymes. Interchain cross-linking of proteins generated an increase in the puncture strength, and a decrease in water vapor permeability. Gelatin was added in film formulation as a stabilizer to improve the puncture strength and film appearance. The structure of the biofilms was also analyzed. SDS-PAGE revealed that heating and gamma-irradiation produce an increase of the molecular weight of the cross-linked protein. Size exclusion chromatography showed a molecular mass of 40 kDa for un-cross-linked whey proteins, whereas for the soluble fractions of the cross-linked proteins, molecular distributions were between 600 and 3800 kDa for the heated proteins and between 1000 and 2000 kDa for gamma-irradiated proteins. No major alteration of the structural conformation of the proteins was observed by FTIR for biofilms obtained after heat treatment, whereas gamma-irradiation induced some modifications in the protein structure. X-ray diffraction analysis suggests that cross-linking by gamma-irradiation seems to modify the conformation of proteins, which became more ordered and more stable.     

Transgenic sorghum with altered kafirin synthesis: kafirin solubility, polymerization, and protein digestion

Transgenic sorghum (TG) lines with altered kafirin synthesis, particularly suppression of γ-kafirin synthesis, and improved protein quality have been developed. The proportion of kafirin extracted with 60% tert-butyl alcohol alone was greatly increased in the TG lines. However, the total amount of kafirin remained unchanged. Further, in the TG lines, the kafirin was much less polymerized by disulfide bonding. There was also evidence of compensatory synthesis of other kafirin proteins. Cooked protein digestibility was increased in the TG form, even after removal of interfering starch. The TG protein bodies were intermediate in appearance between the normal type and the invaginated high digestibility mutants. Hence, the increased protein digestibility of these TG lines is probably related to their lower levels of disulfide-bonded kafirin polymerization, allowing better access of proteases. This work appears to confirm that disulfide bond formation in kafirin is responsible for the reduced protein digestibility of cooked sorghum.     

Gamma-irradiation influence on the structure and properties of calcium caseinate-whey protein isolate based films. Part 1. Radiation effect on the structure of proteins gels and films

Brookfield viscosimetry, Fourier transform infrared spectroscopy, transmission electron microscopy (TEM), and measurements of the texture strength of gels formed with CaCl2 and the mechanical and barrier properties of the film were applied in studies of gel formation and structural and mechanical properties of gels and films prepared using calcium caseinate (CC)-whey protein isolate (WPI)-glycerol (1:1:1), control, and irradiated with 60Co gamma rays using a 32 kGy dose. The irradiated gels have appeared to be more "fine-stranded" as compared to the more "particulate" control gels and lead to the formation of more rigid films with improved mechanical strength and barrier properties. This results from cross-linking and the modification of protein conformations were induced by irradiation, in particular the increase in the beta-sheet and beta-strand contents. Structural modifications taking place in CC-WPI composition are related to modifications taking place separately in CC and WPI. Improvement of the properties of the films after irradiation corresponds to the increased density of the cross-linked material because no change in the porosity of the films was observed by TEM.     

Physicochemical properties and bioactivity of nisin-containing cross-linked hydroxypropylmethylcellulose films

Cross-linked hydroxypropylmethylcellulose (HPMC) cast films with citric acid as polycarboxylic cross-linker were elaborated to study the effect of cross-linking level on various properties. Increased amounts of cross-linking agent were not connected to statistically different tensile strength and Young's modulus. Whatever the cross-linking level of the film was, the ultimate elongation parameter decreased by approximately 60% compared to the HMPC control film. Moisture sorption isotherms and water contact angle meter showed that the effect of cross-linking degree tends to reduce the hygroscopic and hydrophilic characteristics of films. In addition, to control bacteria growth on food surfaces, the antimicrobial activity of both 98% cross-linked HPMC-nisin and control HPMC-nisin films was tested on Micrococcus luteus. Despite the incorporation of a significant content of nisin, cross-linked HPMC-nisin films were completely inactive on the microbial strain compared to the HPMC-nisin control films. Cross-linking conditions likely either denatured the nisin or irreversibly bound nisin to the cross-linked HPMC. However, nisin adsorbed into films made from previously cross-linked HPMC maintained its activity.     

Carbon respiration and nitrogen dynamics in Corsican pine litter amended with aluminium and tannins

We investigated the carbon (C) mineralisation and nitrogen (N) dynamics in litter from a Corsican pine forest in response to individual and combined additions of aluminium (Al), condensed tannin (extracted from fresh Corsican pine needles) and hydrolysable tannin (commercial tannic acid). Production rates of CO₂, NH₄⁺ and NO₃⁻ concentrations, tannin concentrations and Al speciation were determined at various time intervals during a 28-day incubation experiment. The addition of Al decreased CO₂ production and shut down nitrification. Exchangeable NH₄⁺ strongly increased in the Al-amended litter, likely due to (i) decreased microbial uptake of NH₄⁺, (ii) the inhibition of nitrification and (iii) competition for soil organic matter (SOM) binding sites by Al. Both tannin species affected C mineralisation and/or N dynamics, be it in different ways. Addition of tannic acid led to a strong increase of the C mineralisation rate and microbial uptake of N, caused by rapid degradation of this labile tannin and subsequent increased microbial nutrient demand. Net immobilisation of N occurred as long as one week after addition. Condensed tannin was not consumed but probably strongly bound to (nitrogenous) SOM compounds, forming recalcitrant complexes and decreasing net N mineralisation. Complexation of Al by tannins in solution before addition to the litter mitigated the Al-induced release of exchangeable NH₄⁺. In the case of condensed tannin with complexed Al, this was due to detoxification of Al through complexation. Increased microbial demand for N likely played a major role in decreased NH₄⁺ accumulation in the samples to which tannic acid with complexed Al was added. Nitrification was shut down despite of the complexation of Al by either condensed tannin or tannic acid.     

Physicochemical, antimicrobial, and cytotoxic characteristics of a chitosan film cross-linked by a naturally occurring cross-linking agent, aglycone geniposidic acid

The purpose of this study was to evaluate the characteristics of a chitosan film cross-linked by a naturally occurring compound, aglycone geniposidic acid (aGSA). This newly developed aGSA-cross-linked chitosan film may be used as an edible film. The chitosan film without cross-linking (fresh) and the glutaraldehyde-cross-linked chitosan film were used as controls. The characteristics of test chitosan films evaluated were their degree of cross-linking, swelling ratio, mechanical properties, water vapor permeability, antimicrobial capability, cytotoxicity, and enzymatic degradability. It was found that cross-linking of chitosan films by aGSA (at a concentration up to 0.8 mM) significantly increased its ultimate tensile strength but reduced its strain at fracture and swelling ratio. There was no significant difference in the antimicrobial capability between the cross-linked chitosan films and their fresh counterpart. However, the aGSA-cross-linked chitosan film had a lower cytotoxicity, a slower degradation rate, and a relatively lower water vapor permeability as compared to the glutaraldehyde-cross-linked film. These results suggested that the aGSA-cross-linked chitosan film may be a promising material as an edible film.     

Characterization of kafirin and zein oligomers by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Quantitative and qualitative analysis of uncooked zein and kafirin fractions were performed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic profiles. Kafirins and zeins present the same oligomer and monomer compositions with the exception of a 66 kDa oligomer that is only present in kafirins. The quantitative analysis showed differences between zein and kafirin. The composition of each oligomer was established via preparative SDS-PAGE. Part of the cooked oligomers resists reduction; the presence of those oligomers could be related to the decrease on protein digestibility with the cooking process.     

Evolution of wheat gliadins conformation during film formation: a fourier transform infrared study

The secondary structures of wheat gliadins (a major storage protein fraction from gluten) in film-forming solutions and their evolution during film formation were investigated by Fourier transform infrared spectroscopy. In the film-forming solution, wheat gliadins presented a mixture of different secondary structures, with an important contribution of beta-turns induced by proline residues. The presence of plasticizer did not have any influence on protein secondary structure in the film-forming solution. The evolution of protein conformation was followed during drying; the major feature of this evolution was a clear growing of the infrared band at 1622 cm(-1), characteristic of intermolecular hydrogen-bonded beta-sheets. This revealed the formation of protein aggregates during film drying. The influence of the drying temperature on film properties and gliadin secondary structures was also investigated. Higher drying temperatures induced an increase of both the tensile strength of the films and the amount of beta-sheets aggregates. Although the appearance of heat-induced disulfide bridge cross-links has already been described, there is clear evidence that hydrogen-bonded beta-sheets aggregates are also induced by thermal treatment. It was not possible, however, to determine whether there is a direct relationship between the occurrence of these aggregates and the increase of the tensile strength of the films.     

Antimicrobial and physicochemical properties of chitosan-HPMC-based films

To prepare composite films from biopolymers with anti-listerial activity and moisture barrier properties, the antimicrobial efficiency of chitosan-hydroxy propyl methyl cellulose (HPMC) films, chitosan-HPMC films associated with lipid, and chitosan-HPMC films chemically modified by cross-linking were evaluated. In addition, the physicochemical properties of composite films were evaluated to determine their potential for food applications. The incorporation of stearic acid into the composite chitosan-HPMC film formulation decreased water sensitivity such as initial solubility in water and water drop angle. Thus, cross-linking of composite chitosan-HPMC, using citric acid as the cross-linking agent, led to a 40% reduction in solubility in water. The water vapor transfer rate of HPMC film, approximately 270 g x m(-2) x day(-1) x atm(-1), was improved by incorporating chitosan and was further reduced 40% by the addition of stearic acid and/or cross-linking. Anti-listerial activity of films was determined on solid medium by a numeration technique. Chitosan-HPMC-based films, with and without stearic acid, inhibited the growth of Listeria monocytogenes completely. On the other hand, a loss of antimicrobial activity after chemical cross-linking modification was observed. FTIR and 13C NMR analyses were then conducted in order to study a potential chemical modification of biopolymers such as a chemical reaction with the amino group of chitosan. To complete the study, the mechanical properties of composite films were determined from tensile strength assays.     

Chemistry and physical properties of melt-processed and solution-cross-linked corn zein

Corn zein was cross-linked with glutaraldehyde (GDA) using glacial acetic acid (HAc) as catalyst. The objectives are to evaluate the swelling characteristics of GDA cross-linked zein gels in water, ethanol, and their combinations. Similar formulations, upon solvent evaporation, form films. The mechanical properties of the films are compared to compression molded tensile bars from GDA melt-processed zein as a second objective. Chemistry of the cross-linking reaction was based on the aldehyde binding characteristics defined by use of fluorescence spectroscopy; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to demonstrate the cross-linking reaction; FTIR to observe absorption differences of the cross-linked product; differential scanning calorimetry, dynamic mechanical analysis and thermogravimetric analysis to assess thermal properties; and the use of Instron Universal Testing Machine to evaluate mechanical properties. A reaction mechanism for acid catalyzed GDA cross-linking of zein is proposed. Thermal and mechanical properties of tensile bars cut from either film or formed by compression molding were similar, where both showed increased tensile strengths, ductility and stiffness when compared with unmodified controls. Samples that were reacted with 8% GDA by weight based on weight of zein from either process retained their integrity when tensile bars from each were subjected to boiling water for 10 min or soaking in either water or HAc for 24 h. The melt-processed, cross-linked zein is a more environmentally friendly method that would eliminate the need for HAc recovery.     

Molecular structure, physicochemical characterization, and in vitro degradation of barley protein films

Barley protein films were prepared by thermopressing using glycerol as a plasticizer. The combined effects of heating temperature and amount of plasticizer interacted to determine protein conformation and, subsequently, the properties of the film matrix. The film barrier and mechanical properties were systematically investigated using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), SDS-PAGE, and protein solubility tests. These experiments demonstrated that heat treatment induced barley protein unfolding and then protein aggregation and the formation of covalent disulfide bonds to enhance film strength. Increasing the amount of plasticizer reduced protein denaturation and limited protein interactions, resulting in significantly improved film flexibility at the cost of reduced film moisture barrier property and tensile strength. In vitro degradation experiments demonstrated that barley films were resistant in gastric conditions, yet can still be completely degraded by intestinal enzymes, and they possess low cytotoxicity to Caco-2 cells. The prepared barley films have potential for development as delivery systems for gastric-sensitive bioactive compounds to the intestine for release.     

Influence of gallic acid and tannic acid on the mechanical and barrier properties of wheat gluten films

Vital wheat gluten, a byproduct of wheat starch production, is a highly functional ingredient having a unique viscoelasticity that makes it ideal for the production of edible biodegradable films. However, its functional properties must be modified to ensure sufficient strength and elasticity, in addition to water vapor barrier properties. In this study, vital gluten was modified using tannic and gallic acid. It was found that the addition of tannic acid resulted in stiffer, more resistant, and less resilient and flexible films, having as well decreased water vapor permeability. Tannic acid containing films became reddish brown, whereas gallic acid addition did not have an influence on the film appearance. Films containing gallic acid became more elastic. Gallic acid was found to potentially act like a plasticizer. Scanning electron microscopy was used to investigate the ultrastructure of the produced films.     

Solution and film properties of sodium caseinate/glycerol and sodium caseinate/polyethylene glycol edible coating systems.

The aim of this study is to determine the effects of plasticizer hydrogen bonding capability and chain length on the molecular structure of sodium caseinate (NaCAS), in NaCAS/glycerol and NaCAS/polyethylene glycol 400 (PEG) systems. Both solution and film phases were investigated. Glycerol and PEG reduced the viscosity of aqueous NaCAS, with the latter having a greater effect. This was explained in terms of protein/plasticizer aggregate size and changes to the conformation of the caseinate chain. In the film phase, glycerol caused more pronounced changes to the film tensile strength compared with PEG. However, the effect of glycerol on film water vapor permeability was smaller. These observations are attributed to the differences in plasticizer size and hydrogen bonding strength that controls the protein-plasticizer and protein-protein interactions in the films. Glass transition calculations from the tensile strength data indicate that the distribution of bonding interactions is more homogeneous in NaCAS/PEG films than in NaCAS/glycerol films.     

Properties of deamidated gluten films enzymatically cross-linked

Films were prepared at neutral pH from deamidated gluten by casting with or without enzymatic treatment by transglutaminase in the presence of various concentrations of diamines added to the film-forming solution. Variation in the glycerol/deamidated gluten ratio from 0.2 to 0.5 had a major effect on the film mechanical properties, which is characteristic of a plasticizing effect. A ratio of 0.35, producing a tensile strength of 1.14 +/- 0.12 MPa and an elongation at break of 376 +/- 62%, was chosen for most of the enzymatic modifications. The action of transglutaminase with or without the addition of external diamines induced a simultaneous increase in tensile strength and elongation at break of the films but tended to decrease the contact angle between the film surface and a water droplet. The presence of diamines in the film solution affected the elongation at break more than the tensile strength of the films. These diamines, able to react at their two extremities, probably acted as spacers between gluten proteins. The decrease in solubility was related to the formation of high molecular weight polymers in the film. The film properties were unaffected by the type of diamine added as secondary substrate in the transglutaminase reaction.