Structure and functionality of surface-active amylose-fatty amine salt inclusion complexes.

Novel value-added starch-based materials can be produced by forming amylose inclusion complexes (AIC) with hydrophobic compounds. There is currently little research on AIC use as polymeric emulsifiers, particularly for AIC with fatty amine salt ligands. This work evaluated AIC emulsifiers by studying the structure and functionality of AIC composed of high amylose corn starch and fatty amine salts (10-18 carbons, including a mixture simulating vegetable oil composition) produced via steam jet cooking. X-ray scattering verified successful AIC formation, with peaks located near 7.0°, 12.8° and 19.9° 2θ. AIC were easily dispersed in water (80-85 °C) and remained in suspension at room temperature for weeks, unlike the uncomplexed ligands or starch. AIC were highly effective emulsifying agents, with emulsifying activity indexes of 213-229 m2g-1 at pH 5, and zeta potentials, a measure of electrostatic repulsion, as high as 43.4 mV. AIC dispersions had surface tension ranging from 24 to 41 mN/m and displayed surface-active properties superior to amylose complexes formed from fatty acid salts and competitive with common starch-based emulsifiers. These findings demonstrate that fatty amine salt AIC are effective emulsifiers that can be made from low-cost sources of fatty amine salts, such as vegetable oil derivatives.

Fusarium head blight resistance exacerbates nutritional loss of wheat grain at elevated CO2.

The nutritional integrity of wheat is jeopardized by rapidly rising atmospheric carbon dioxide (CO2) and the associated emergence and enhanced virulence of plant pathogens. To evaluate how disease resistance traits may impact wheat climate resilience, 15 wheat cultivars with varying levels of resistance to Fusarium Head Blight (FHB) were grown at ambient and elevated CO2. Although all wheat cultivars had increased yield when grown at elevated CO2, the nutritional contents of FHB moderately resistant (MR) cultivars were impacted more than susceptible cultivars. At elevated CO2, the MR cultivars had more significant differences in plant growth, grain protein, starch, fructan, and macro and micro-nutrient content compared with susceptible wheat. Furthermore, changes in protein, starch, phosphorus, and magnesium content were correlated with the cultivar FHB resistance rating, with more FHB resistant cultivars having greater changes in nutrient content. This is the first report of a correlation between the degree of plant pathogen resistance and grain nutritional content loss in response to elevated CO2. Our results demonstrate the importance of identifying wheat cultivars that can maintain nutritional integrity and FHB resistance in future atmospheric CO2 conditions.

Incorporation of Plasticizers and Co-proteins in Zein Electrospun Fibers.

As a means to alter the physical properties of electrospun zein fibers, plasticizers (glycerol, lactic acid, and oleic acid) or co-proteins (casein, whey protein, rice protein) were mixed with zein using the solvents acetic acid or aqueous ethanol with or without sodium hydroxide. Incorporating plasticizers or co-proteins had a negligible impact on solution viscosity, solution surface tension, and fiber formation, although electron microscopy of fiber mats showed an increase in bead formation with added co-proteins. Gel electrophoresis identified casein and whey protein in spun mats. Infrared spectra demonstrated the inclusion of plasticizers in fiber mats. Glycerol, lactic acid, and oleic acid reduced the glass transition temperature of bulk fibers. Nanoindentation tests of individual fibers found reduced Young's moduli with added lactic or oleic acids but increased moduli with added casein. Thus, electrospinning zein with food-grade plasticizers or proteins physically modifies fibers, yet incorporating significant protein quantities remains a challenge.

Insecticidal Activity of Commiphora erythraea Essential Oil and Its Emulsions Against Larvae of Three Mosquito Species.

The use of essential oils as ecofriendly tools for vector management is one of the mainstreams for biopesticide research. We evaluated the larvicidal properties of Commiphora erythraea (opoponax) essential oil and its fractions against Culex restuans Theobald, Culex pipiens L., and Aedes aegypti L. The use of bio-based amylose-N-1-hexadecylammonium chloride inclusion complex (Hex-Am) and amylose-sodium palmitate inclusion complex (Na-Palm) as emulsifiers for C. erythraea essential oil was also investigated. Bisabolene was the most abundant chemical constituent in the whole essential oil (33.9%), fraction 2 (62.5%), and fraction 4 (23.8%) while curzerene (32.6%) and α-santalene (30.1%) were the dominant chemical constituents in fractions 1 and 3, respectively. LC50 values for the whole essential oil were 19.05 ppm for Cx. restuans, 22.61 ppm for Cx. pipiens, and 29.83 ppm for Ae. aegypti and differed significantly. None of the four C. erythraea essential oil fractions were active against mosquito larvae. Two CYP450 genes (CYP6M11 and CYP6N12) and one GST gene (GST-2) were significantly upregulated in Ae. aegypti larvae exposed to C. erythraea essential oil suggesting their potential involvement in metabolic pathways for C. erythraea essential oil. Essential oil emulsions produced with Hex-Am were more toxic than the whole essential oil while those produced with Na-Palm had similar toxicity as the whole essential oil. These findings demonstrate that C. erythraea essential oil is a promising source of mosquito larvicide and that the use of Hex-Am as an emulsifier can enhance the insecticidal properties of C. erythraea essential oil.

Corn zein undergoes conformational changes to higher ß-sheet content during its self-assembly in an increasingly hydrophilic solvent.

Viscoelasticity of corn zein is associated with the formation of ß-sheet secondary structures; however, studies of the fundamentals of this conformational change are limited due to zein insolubility and poor analytical resolution. Here, changes in soluble zein conformation were evaluated as the protein self-assembles in increasingly hydrophilic solvents to the concentration just before aggregation and precipitation. Circular dichroism spectra of zein showed that α-helix structures decrease in favor of random coil and ß-sheets with increases in water content in an ethanol-water system, similar to observations of zein when it becomes viscoelastic in dough systems. This was further supported by changes in Thioflavin T fluorescence emission spectra and intrinsic viscosity measurements. Two widely recognized molecular models for α-zein (hairpin and superhelical conformations) were tested at 75 and 45% ethanol concentration using molecular dynamics simulation for agreement with experimental results. Increase in solvent hydrophilicity increased ß-sheets and reduced distance between backbone anomeric carbons only for hairpin model, suggesting it to be the more valid of the two. These findings emphasize the importance of transformation to ß-sheets during zein self-assembly and provide further insight into the mechanisms by which the protein is functionalized into viscoelastic systems.

Biopesticide synergy when combining plant flavonoids and entomopathogenic baculovirus.

Four crop plants known to be hosts for the lepidopteran Trichoplusia ni (soybean, green bean, cotton, and cabbage) were treated with the biopesticide AfMNPV baculovirus in a dosage response assay. Treated soybean had, on average, a 6-fold increase in virus activity compared with the other crops. Leaf trichomes on soybeans were not found to be responsible for the observed increase of insecticidal activity. Three flavonoid compounds (daidzein, genistein, and kaempferol) were uniquely found only in the soybean crop, and were not detected in cotton, cabbage, or green bean plant matter. The individual flavonoid compounds did not cause T ni. mortality in no-virus assays when incorporated into artificial insect diet. The combination of the three flavonoid compounds at leaf level concentrations significantly increased baculovirus activity in diet incorporation assays. When the daidzein, genistein, and kaempferol were added to artificial diet, at 3.5-6.5 × leaf level concentrations, virus activity increased 1.5, 2.3, and 4.2-fold for each respective flavonoid. The soybean flavonoid compounds were found to synergistically improve baculovirus activity against T. ni.

Leptospermum scoparium essential oil is a promising source of mosquito larvicide and its toxicity is enhanced by a biobased emulsifier.

Synthetic pesticides are the cornerstone of vector-borne disease control, but alternatives are urgently needed to tackle the growing problem of insecticide resistance and concerns over environmental safety. Leptospermum scoparium J.R. Forst and G. Forst (manuka) essential oil and its four fractions were analyzed for chemical composition and toxicity against Aedes aegypti larvae. The use of bio-based amylose-N-1-hexadecylammonium chloride inclusion complexes (Hex-Am) as an emulsifier for L. scoparium essential oil was also investigated. Fraction 1 was inactive, fractions 2 (LC50 = 12.24 ppm) and 3 (LC50 = 20.58 ppm) were more toxic than the whole essential oil (LC50 = 47.97 ppm), and fraction 4 (LC50 = 35.87 ppm) had similar toxicity as the whole essential oil. Twenty-one chemical constituents were detected in L. scoparium essential oil compared to 16, 5, 19 and 25 chemical constituents in fractions, 1, 2, 3 and 4 respectively. The two most dominant chemical constituents were calamenene (17.78%) and leptospermone (11.86%) for L. scoparium essential oil, calamenene (37.73%) and ledene (10.37%) for fraction 1, leptospermone (56.6%) and isoleptospermone (19.73) for fraction 2, cubenol (24.30%) and caryophyllene oxide (12.38%) for fraction 3, and γ-gurjunene (21.62%) and isoleptospermone (7.88%) for fraction 4. Alpha-pinene, ledene, and aromandendrene were 2-7 times less toxic than the whole essential suggesting that the toxicity of L. scoparium essential oil was either due to other chemical constituents that were not tested or due synergist interactions among chemical constituents. Leptospermum scoparium essential oil-Hex-Am emulsion (LC50 = 29.62) was more toxic than the whole essential oil. These findings suggest that L. scoparium essential oil is a promising source of mosquito larvicide and that Hex-Am is an excellent emulsifier for L. scoparium essential oil for use as a larvicide.

Antimicrobial properties of amylose-fatty ammonium salt inclusion complexes.

Amylose-fatty (C12-C16) ammonium salt inclusion complexes are effective antimicrobial polymers causing growth inhibition of microbes at concentrations as low as 40 µg/mL of the complex (2 µg/mL active cationic ligand). The complex was more effective at controlling microbes than the uncomplexed ligand. The complexes were found to be particularly effective at inhibiting the growth of fungi, yeast, gram (+) bacteria, and algae; its performance was affected by pH. The complexes were not hemolytic at concentrations up to 2000 µg/mL. These agents were determined to be surface active polymers and their antimicrobial mode of action may involve cell membrane thinning or disruption, causing moderate leakage. Increased ligand concentration provided increased antimicrobial activity. Solutions of amylose complexes were found to be stable, retaining their antimicrobial efficacy after autoclaving, or after storage at room temperature for 6 months. Antimicrobial amylose complexes were produced using readily available inexpensive materials via an easily scalable process.

Amylose Inclusion Complexes as Emulsifiers for Garlic and Asafoetida Essential Oils for Mosquito Control.

Although the insecticidal properties of some plant essential oils are well-documented, their use in integrated pest and vector management is complicated by their high volatility, low thermal stability, high sensitivity to oxidation, and low solubility in water. We investigated the use of bio-based N-1-hexadecylammonium chloride and sodium palmitate amylose inclusion complexes as emulsifiers for two essential oils, garlic and asafoetida, known to be highly toxic to mosquito larvae. Four emulsions of each essential oil based on amylose hexadecylammonium chloride and amylose sodium palmitate inclusion complexes were evaluated for their toxicity against Aedes aegypti L. larvae relative to bulk essential oils. All emulsions were significantly more toxic than the bulk essential oil with the lethal dosage ratios ranging from 1.09-1.30 relative to bulk essential oil. Droplet numbers ranged from 1.11 × 109 to 9.55 × 109 per mL and did not change significantly after a 6-month storage period. These findings demonstrated that amylose inclusion complexes enhanced the toxicity of essential oils and could be used to develop new essential oil based larvicides for use in integrated vector management.

Improved hydroxypropyl methylcellulose (HPMC) films through incorporation of amylose-sodium palmitate inclusion complexes.

Polymer film blends of hydroxypropyl methylcellulose (HPMC) and amylose-sodium palmitate inclusion complexes (Na-Palm) were produced with no plasticizer, and were observed to have improved physical and gas barrier properties as compared with pure HPMC. The crystalline amylose helices incorporating the hydrophobic sodium palmitate ligand decreased the water vapor permeability of a 50/50% blended film of HPMC/Na-Palm by 40% and decreased oxygen permeability by 96%. The incorporation of 25% Na-Palm into HPMC films resulted in improved elongation, Young's modulus and toughness. Addition of the amylose-complexes produced relatively smooth, high clarity films which had reduced solubility in neutral and acidic solutions. Increasing concentrations of Na-Palm increased film thermal resilience and increased storage modulus at high temperatures. The heat deflection temperature of the films also increased with increasing concentrations of amylose-complex; HPMC/Na-Palm film blends with >50% Na-Palm displayed almost no material deformation up to 250 °C.

Physical, Rheological, Functional, and Film Properties of a Novel Emulsifier: Frost Grape Polysaccharide from Vitis riparia Michx.

A novel emulsifier, Frost grape polysaccharide (FGP), isolated from natural exudate of the species Vitis riparia Michx, was physically and rheologically characterized. The determination of the physical, structural, thermodynamic, emulsification, film, and rheological properties of FGP provide essential details for the commercial adoption of this novel plant polysaccharide. FGP is capable of producing exceptionally stable emulsions when compared with the industrially ubiquitous gum arabic (GA). The FGP isolate contained a negligible amount of nitrogen (0.03%), indicating that it does not contain an associated glycoprotein, unlike GA. Solutions of FGP have a high degree of thermostability, displaying no loss in viscosity with temperature cycling and no thermal degradation when held at 90 °C. FGP is an excellent film former, producing high tensile strength films which remain intact at temperatures up to 200 °C. This work identified a number of potential food and pharmaceutical applications where FGP is significantly superior to GA.

Effect of Salt and Ethanol Addition on Zein-Starch Dough and Bread Quality.

Development of viscoelastic doughs from non-wheat proteins allows for a wider range of gluten-free products. Little work has been completed to describe mechanisms of zein functionality in food systems. To identify factors responsible for dough development in zein-starch mixtures and their influence on zein bread quality, a mixture of 20% zein-80% maize starch was mixed with water and various reagents. Salts, NaSCN, NaCl, and Na2 SO4 were evaluated at concentrations from 0 to 2M for their influence on the properties of zein-starch dough systems. NaSCN at low concentrations produced softer dough. Ethanol treatments produced softer more workable dough in the absence of salts. Increasing concentrations of NaCl and Na2 SO4 resulted in coalescing of the proteins and no dough formation. The addition of ß-ME had minimal softening effects on zein-starch dough. Specific volumes of zein-starch bread increased with decreasing NaCl addition in bread formulations. Likewise, including 5% ethanol (v/v) in the bread formula increased bread quality.

Rheological characterization of solutions and thin films made from amylose-hexadecylammonium chloride inclusion complexes and polyvinyl alcohol.

The rheological properties of aqueous solutions and films made from blends of polyvinyl alcohol (PVOH) and amylose-hexadecylammonium chloride inclusion complexes (Hex-Am) were investigated to better understand the polymer interactions and processing parameters. Aqueous solutions of Hex-Am displayed non-Newtonian shear thinning characteristics, becoming highly viscous at 4.2% solids and forming a strong mechanical gel at 10% solids. Cationic Hex-Am was observed to have dramatically different rheological temperature response profiles from anionic amylose-sodium palmitate inclusion complexes, displaying a precipitous increase in viscosity upon cooling from 95°C to 50°C. Aqueous solution blends of 1:1 PVOH/Hex-Am lack this precipitous increase in viscosity, indicating that PVOH reduces amylose-chain entanglement. Films cast from varying blends of Hex-Am and PVOH were thermostable to 200°C, and displayed decreasing storage modulus with increasing concentrations of PVOH in film blends. Films cast from Hex-Am/PVOH absorb water vapor at lower rates than their constitutive polymers.

Effect of spray drying on the properties of amylose-hexadecylammonium chloride inclusion complexes.

Water soluble amylose-hexadecyl ammonium chloride complexes were prepared from high amylose corn starch and hexadecyl ammonium chloride by excess steam jet cooking. Amylose inclusion complexes were spray dried to determine the viability of spray drying as a production method. The variables tested in the spray drying process were the% solids of the amylose-hexadecyl ammonium chloride complex being fed into the spray dryer, feed rate and the spray dryer outlet temperature. The amylose-inclusion complexes remained intact in all spray drying conditions tested as determined by X-ray diffraction. The rheological properties of solutions of the spray dried amylose-complexes remained unchanged when compared with the freeze dried control. Particle density and moisture content decreased with increased outlet temperature while particle size increased. X-ray diffraction and DSC analysis confirmed the formation of type II amylose inclusion complexes. Spray drying is a high throughput, low cost continuous commercial production method, which when coupled with excess steam jet cooking allows for the industrial scale production of cationic amylose-hexadecyl ammonium chloride complexes which may have value as flocculating and filtration enhancing agents and other aspects of paper production.

Extraction, composition, and functional properties of dried alfalfa (Medicago sativa L.) leaf protein.

BACKGROUND: Alfalfa is considered a potential feedstock for biofuels; co-products with value-added uses would enhance process viability. This work evaluated dried alfalfa leaves for protein production and describes the functional properties of the protein. RESULTS: Dried alfalfa leaves contained 260 g kg-1 dry basis (DB) crude protein, with albumins being the major fraction (260 g kg-1 of total protein). Alkali solubilization for 2 h at 50 °C, acid precipitation, dialysis, and freeze-drying produced a protein concentrate (600 g kg-1 DB crude protein). Alfalfa leaf protein concentrate showed moderate solubility (maximum 500 g kg-1 soluble protein from pH 5.5 to 10), excellent emulsifying properties (activity 158-219 m2 g-1 protein, stability 17-49 min) and minimal loss of solubility during heating at pH ≥ 7.0. CONCLUSIONS: It is technically feasible to extract protein with desirable emulsifying and heat stability properties from dried alfalfa leaves; however, the dried form may not be a practical starting material for protein production, given the difficulty of achieving high yields and high-purity protein product. © 2016 Society of Chemical Industry.

Preparation and properties of films cast from mixtures of poly(vinyl alcohol) and submicron particles prepared from amylose-palmitic acid inclusion complexes.

The use of starch in polymer composites for film production has been studied for increasing biodegradability, improving film properties and reducing cost. In this study, submicron particles were prepared from amylose-sodium palmitate complexes both by rapidly cooling jet-cooked starch-palmitic acid mixtures and by acidifying solutions of starch-sodium palmitate complexes. Films were cast containing poly(vinyl alcohol) (PVOH) with up to 50% starch particles. Tensile strength decreased and Young's modulus increased with starch concentration, but percent elongations remained similar to controls regardless of preparation method or starch content. Microscopy showed particulate starch distribution in films made with rapidly cooled starch-palmitic acid particles but smooth, diffuse starch staining with acidified sodium palmitate complexes. The mild effects on tensile properties suggest that submicron starch particles prepared from amylose-palmitic acid complexes provide a useful, commercially viable approach for PVOH film modification.

Role of non-covalent interactions in the production of visco-elastic material from zein.

The role of non-covalent interactions in the formation of visco-elastic material from zein was investigated. Hydrophobic interactions were evaluated through the addition of various salts from the Hofmeister series. Urea, ethanol, and beta mercaptoethanol (ß-ME) were used to evaluate the effects of protein denaturation and disulfide bonds on zein's ability to form a visco-elastic material. The addition of NaI and NaSCN altered the properties of visco-elastic materials made from zein, making them softer and more extensible, as did urea and ethanol. The addition of NaCl and Na2SO4 negatively impacted the ability of zein to from a visco-elastic material and at higher concentrations completely disrupted the formation of visco-elastic material. These results indicate that manipulating non-covalent interactions in zein can alter and in some cases, completely disrupt the formation of a visco-elastic material. Specifically this may be due to disruption of hydrophobic interactions within individual zein proteins or interactions between proteins. The reducing agent ß-ME had little effect on zein's ability to form a visco-elastic material. Therefore, the visco-elastic properties of zein arise as a result of non-covalent interactions.

Structural characterization of alpha-zein.

A variety of published physical measurements, computational algorithms, and structural modeling methods have been used to create a molecular model of 19 kDa alpha-zein (Z19). Zetaeins are water-insoluble storage proteins found in corn protein bodies. Analyses of the protein sequence using probability algorithms, structural studies by circular dichroism, infrared spectroscopy, small-angle X-ray scattering (SAXS), light scattering, proton exchange, NMR, and optical rotatory dispersion experiments suggest that Z19 has approximately 35-60% helical character, made up of nine helical segments of about 20 amino acids with glutamine-rich "turns" or "loops". SAXS and light-scattering experiments suggest that in alcohol/water mixtures alpha-zein exists as an oblong structure with an axial ratio of approximately 6:1. Furthermore, ultracentifugation, birefringence, dielectric, and viscosity studies indicate that alpha-zein behaves as an asymmetric particle with an axial ratio of from 7:1 to 28:1. Published models of alpha-zein to date have not been consistent with the experimental data, and for this reason the structure was re-examined using molecular mechanics and dynamics simulations creating a new three-dimensional (3D) structure for Z19. From the amino acid sequence and probability algorithms this analysis suggested that alpha-zein has coiled-coil tendencies resulting in alpha-helices with about four residues per turn in the central helical sections with the nonpolar residue side chains forming a hydrophobic face inside a triple superhelix. The nine helical segments of the 19 kDa protein were modeled into three sets of three interacting coiled-coil helices with segments positioned end to end. The resulting structure lengthens with the addition of the N- and C-terminal sections, to give an axial ratio of approximately 6 or 7:1 in agreement with recent experiments. The natural carotenoid, lutein, is found to fit into the core of the triple-helical segments and help stabilize the configuration. Molecular dynamics simulations with explicit methanol/water molecules as solvent have been carried out to refine the 3D structure.

Rheological studies utilizing various lots of zein in N,N-dimethylformamide solutions.

Rheological studies were carried out on solutions of zein in N,N-dimethylformamide (DMF), where the specific lot of zein, concentration, time, and temperature were varied. DMF is a good solvent for zein, giving clear, relatively low viscosity solutions. It was found that all of the zein solutions behaved in a non-Newtonian fashion. At high concentration and elevated temperature, zein solutions will increase in viscosity with time. A temperature study on the rate of viscosity rise illustrated that at temperatures above 40 degrees C, the rate of viscosity rise increased in a non-Arrhenius fashion. There can be significant lot to lot variations in commercially obtained zein that gives rise to differences in viscosity and rate of viscosity rises. With the samples studied, viscosity was found to double from one lot of zein to another. Size exclusion chromatography suggests that compositional differences between the lots drive the observed differences in viscosity.