Functional characterization of biodegradable films obtained from whole Paecilomyces variotii biomass.

The indiscriminate use of petroleum-based polymers and plastics for single-use food packaging has led to serious environmental problems due the non-biodegradable characteristics. Thus, much attention has been focused on the research of new biobased and biodegradable materials. Yeast and fungal biomass are low-cost and abundant sources of biopolymers with highly promising properties for the development of biodegradable materials. This study aimed to select a preparation method to develop new biodegradable films using the whole biomass of Paecilomyces variotii subjected to successive physical treatments including ultrasonic homogenization (US) and heat treatment. Sterilization process had an important impact on the final filmogenic dispersion and mechanical properties of the films. Longer US treatments produced a reduction in the particle size and the application of an intermediate UT treatment contributed favorably to the breaking of agglomerates allowing the second US treatment to be more effective, achieving an ordered network with a more uniform distribution. Samples that were not filtrated after the sterilization process presented mechanical properties similar to plasticized materials. On the other hand, the filtration process after sterilization eliminated soluble and hydratable compounds, which produced a reduction in the hydration of the films.

Multi-Component Biodegradable Materials Based on Water Kefir Grains and Yeast Biomasses: Effect of the Mixing Ratio on the Properties of the Films.

The use of biopolymeric materials is restricted for some applications due to their deficient properties in comparison to synthetic polymers. Blending different biopolymers is an alternative approach to overcome these limitations. In this study, we developed new biopolymeric blend materials based on the entire biomasses of water kefir grains and yeast. Film-forming dispersions with varying ratios of water kefir to yeast (100/0, 75/25, 50/50 25/75 and 0/100) underwent ultrasonic homogenisation and thermal treatment, resulting in homogeneous dispersions with pseudoplastic behaviour and interaction between both biomasses. Films obtained by casting had a continuous microstructure without cracks or phase separation. Infrared spectroscopy revealed the interaction between the blend components, leading to a homogeneous matrix. As the water kefir content in the film increased, transparency, thermal stability, glass transition temperature and elongation at break also increased. The thermogravimetric analyses and the mechanical tests showed that the combination of water kefir and yeast biomasses resulted in stronger interpolymeric interactions compared to single biomass films. The ratio of the components did not drastically alter hydration and water transport. Our results revealed that blending water kefir grains and yeast biomasses enhanced thermal and mechanical properties. These studies provided evidence that the developed materials are suitable candidates for food packaging applications.

New Antioxidant Active Packaging Films Based on Yeast Cell Wall and Naphtho-γ-Pyrone Extract.

The main objective of this work is the development of new active films based on yeast cell wall obtained by high-pressure homogenization (YCW-H) supplemented with naphtho-γ-pyrone (CL-NGP) extract, which is a bioactive compound produced by Aspergillus tubingensis G131 with great antioxidant potential. A complete characterization of the functional properties of the bioactive films, such as their structural, colour, thermal, mechanical, hydration and water vapour transport, was carried out to evaluate the influence of the addition of the antioxidant compounds. Likewise, the antioxidant capacity of the developed materials and the specific migration of NGPs in food simulants were evaluated. The results showed that CL-NGP extract possessed an important antioxidant activity, which was maintained after incorporation in YCW-H films. The addition of 2 and 5% CL-NGPs decreased the hydration of films and consequently improved the water vapour barrier properties. It was observed that CL-NGPs migrate in fatty food simulants and retain their antioxidant capacity in the simulant. The results obtained in this work showed that bioactive films based on yeast cell walls with the addition of CL-NGPs have the potential to be used as packaging material in systems of interest in the food industry.

Bacterial cellulose films production by Kombucha symbiotic community cultured on different herbal infusions.

The symbiotic community of bacteria and yeast (SCOBY) of Kombucha beverage produces a floating film composed of bacterial cellulose, a distinctive biobased material. In this work, Kombucha fermentation was carried out in six different herbal infusions, where SCOBY was able to synthesise cellulosic films. Infusions of black and green tea, yerba mate, lavender, oregano and fennel added with sucrose (100 g/l) were used as culture media. In all cultures, film production resulted in a maximum after 21 days. Yield conversion, process productivity and antioxidant activity were quantified. Macroscopic and microscopic features of films were determined based on electronic microscopy, calorimetric and mechanical properties and hydration behaviour. Native films from yerba mate had a remarkable antioxidant activity of 93 ± 4% of radical inhibition due to plant polyphenols, which could prevent food oxidation. Results revealed that films retained natural bioactive substances preserving important physicochemical properties, essential for developing active materials.

Impact of the film-forming dispersion pH on the properties of yeast biomass films.

BACKGROUND: Yeast biomass, mainly composed of proteins and polysaccharides (mannans and ß-glucans), has been proposed to develop films. pH can affect the solubility of polysaccharides, the structure of the cell wall, and the interactions between proteins. Considering the potential impact of these effects, the pH of yeast film-forming dispersions was studied from 4 to 11. RESULTS: In tensile tests, samples increased their elongation by increasing pH, from 7 ± 2% (pH 4) to 29 ± 5% (pH 11), but Young's modulus was not significantly modified. Regarding thermal degradation, the maximum degradation rate temperature was shifted 46 °C from pH 4 to 11. Differences in water vapour permeability, colour, opacity, and roughness of films were also found. According to the results of differential protein solubility assay, hydrophobic interactions and hydrogen bonding were promoted at pH 4, but disulfide bonds were benefited at pH 11, in addition to partial ß-glucan dissolution and break-up of the alkali-sensitive linkage in molecules from the cell wall. CONCLUSION: The results lead to the conclusion that film-functional characteristics were greatly benefited at pH 11 in comparison with the regular pH of dispersion (pH 6). These results could help in understanding and selecting the pH conditions to enhance the desired properties of yeast biomass films. © 2021 Society of Chemical Industry.

Incorporation of Poly(Itaconic Acid) with Quaternized Thiazole Groups on Gelatin-Based Films for Antimicrobial-Active Food Packaging.

Poly(itaconic acid) (PIA) was synthesized via conventional radical polymerization. Then, functionalization of PIA was carried out by an esterification reaction with the heterocyclic groups of 1,3-thiazole and posterior quaternization by N-alkylation reaction with iodomethane. The modifications were confirmed by Fourier transform infrared (FTIR) and proton nuclear magnetic resonance (1H-NMR), as well as ζ-potential measurements. Their antimicrobial activity was tested against different Gram-negative and Gram-positive bacteria. After characterization, the resulting polymers were incorporated into gelatin with oxidized starch and glycerol as film adjuvants, and dopamine as crosslinking agent, to develop antimicrobial-active films. The addition of quaternized polymers not only improved the mechanical properties of gelatin formulations, but also decreased the solution absorption capacity during the swelling process. However, the incorporation of synthesized polymers increased the deformation at break values and the water vapor permeability of films. The antioxidant capacity of films was confirmed by radical scavenging ability and, additionally, those films exhibited antimicrobial activity. Therefore, these films can be considered as good candidates for active packaging, ensuring a constant concentration of the active compound on the surface of the food, increasing products' shelf-life and reducing the environmental impact generated by plastics of petrochemical origin.

Biobased Materials from Microbial Biomass and Its Derivatives.

There is a strong public concern about plastic waste, which promotes the development of new biobased materials. The benefit of using microbial biomass for new developments is that it is a completely renewable source of polymers, which is not limited to climate conditions or may cause deforestation, as biopolymers come from vegetal biomass. The present review is focused on the use of microbial biomass and its derivatives as sources of biopolymers to form new materials. Yeast and fungal biomass are low-cost and abundant sources of biopolymers with high promising properties for the development of biodegradable materials, while milk and water kefir grains, composed by kefiran and dextran, respectively, produce films with very good optical and mechanical properties. The reasons for considering microbial cellulose as an attractive biobased material are the conformational structure and enhanced properties compared to plant cellulose. Kombucha tea, a probiotic fermented sparkling beverage, produces a floating membrane that has been identified as bacterial cellulose as a side stream during this fermentation. The results shown in this review demonstrated the good performance of microbial biomass to form new materials, with enhanced functional properties for different applications.

Conformational dissection of a viral intrinsically disordered domain involved in cellular transformation.

Intrinsic disorder is abundant in viral genomes and provides conformational plasticity to its protein products. In order to gain insight into its structure-function relationships, we carried out a comprehensive analysis of structural propensities within the intrinsically disordered N-terminal domain from the human papillomavirus type-16 E7 oncoprotein (E7N). Two E7N segments located within the conserved CR1 and CR2 regions present transient α-helix structure. The helix in the CR1 region spans residues L8 to L13 and overlaps with the E2F mimic linear motif. The second helix, located within the highly acidic CR2 region, presents a pH-dependent structural transition. At neutral pH the helix spans residues P17 to N29, which include the retinoblastoma tumor suppressor LxCxE binding motif (residues 21-29), while the acidic CKII-PEST region spanning residues E33 to I38 populates polyproline type II (PII) structure. At pH 5.0, the CR2 helix propagates up to residue I38 at the expense of loss of PII due to charge neutralization of acidic residues. Using truncated forms of HPV-16 E7, we confirmed that pH-induced changes in α-helix content are governed by the intrinsically disordered E7N domain. Interestingly, while at both pH the region encompassing the LxCxE motif adopts α-helical structure, the isolated 21-29 fragment including this stretch is unable to populate an α-helix even at high TFE concentrations. Thus, the E7N domain can populate dynamic but discrete structural ensembles by sampling α-helix-coil-PII-ß-sheet structures. This high plasticity may modulate the exposure of linear binding motifs responsible for its multi-target binding properties, leading to interference with key cell signaling pathways and eventually to cellular transformation by the virus.

Protein stability and dynamics modulation: the case of human frataxin.

Frataxin (FXN) is an α/ß protein that plays an essential role in iron homeostasis. Apparently, the function of human FXN (hFXN) depends on the cooperative formation of crucial interactions between helix α1, helix α2, and the C-terminal region (CTR) of the protein. In this work we quantitatively explore these relationships using a purified recombinant fragment hFXN90-195. This variant shows the hydrodynamic behavior expected for a monomeric globular domain. Circular dichroism, fluorescence, and NMR spectroscopies show that hFXN90-195 presents native-like secondary and tertiary structure. However, chemical and temperature induced denaturation show that CTR truncation significantly destabilizes the overall hFXN fold. Accordingly, limited proteolysis experiments suggest that the native-state dynamics of hFXN90-195 and hFXN90-210 are indeed different, being the former form much more sensitive to the protease at specific sites. The overall folding dynamics of hFXN fold was further explored with structure-based protein folding simulations. These suggest that the native ensemble of hFXN can be decomposed in at least two substates, one with consolidation of the CTR and the other without consolidation of the CTR. Explicit-solvent all atom simulations identify some of the proteolytic target sites as flexible regions of the protein. We propose that the local unfolding of CTR may be a critical step for the global unfolding of hFXN, and that modulation of the CTR interactions may strongly affect hFXN physiological function.

Sedimentation velocity analytical ultracentrifugation for intrinsically disordered proteins.

The size of intrinsically disordered proteins (IDPs) is large compared to their molecular mass and the resulting mass-to-size ratio is unusual. The sedimentation coefficient, which can be obtained from sedimentation velocity (SV) analytical ultracentrifugation (AUC), is directly related to this ratio and can be easily interpreted in terms of frictional ratio. This chapter is a step-by-step protocol for setting up, executing and analyzing SV experiments in the context of the characterization of IDPs, based on a real case study of the partially folded C-terminal domain of Sendai virus nucleoprotein.

Neutron structure of type-III antifreeze protein allows the reconstruction of AFP-ice interface.

Antifreeze proteins (AFPs) inhibit ice growth at sub-zero temperatures. The prototypical type-III AFPs have been extensively studied, notably by X-ray crystallography, solid-state and solution NMR, and mutagenesis, leading to the identification of a compound ice-binding surface (IBS) composed of two adjacent ice-binding sections, each which binds to particular lattice planes of ice crystals, poisoning their growth. This surface, including many hydrophobic and some hydrophilic residues, has been extensively used to model the interaction of AFP with ice. Experimentally observed water molecules facing the IBS have been used in an attempt to validate these models. However, these trials have been hindered by the limited capability of X-ray crystallography to reliably identify all water molecules of the hydration layer. Due to the strong diffraction signal from both the oxygen and deuterium atoms, neutron diffraction provides a more effective way to determine the water molecule positions (as D(2) O). Here we report the successful structure determination at 293 K of fully perdeuterated type-III AFP by joint X-ray and neutron diffraction providing a very detailed description of the protein and its solvent structure. X-ray data were collected to a resolution of 1.05 Å, and neutron Laue data to a resolution of 1.85 Å with a "radically small" crystal volume of 0.13 mm(3). The identification of a tetrahedral water cluster in nuclear scattering density maps has allowed the reconstruction of the IBS-bound ice crystal primary prismatic face. Analysis of the interactions between the IBS and the bound ice crystal primary prismatic face indicates the role of the hydrophobic residues, which are found to bind inside the holes of the ice surface, thus explaining the specificity of AFPs for ice versus water.

Structure and interactions of fish type III antifreeze protein in solution.

It has been suggested that above a critical protein concentration, fish Type III antifreeze protein (AFP III) self-assembles to form micelle-like structures that may play a key role in antifreeze activity. To understand the complex activity of AFP III, a comprehensive description of its association state and structural organization in solution is necessary. We used analytical ultracentrifugation, analytical size-exclusion chromatography, and dynamic light scattering to characterize the interactions and homogeneity of AFP III in solution. Small-angle neutron scattering was used to determine the low-resolution structure in solution. Our results clearly show that at concentrations up to 20 mg mL(-1) and at temperatures of 20 degrees C, 6 degrees C, and 4 degrees C, AFP III is monomeric in solution and adopts a structure compatible with that determined by crystallography. Surface tension measurements show a propensity of AFP III to localize at the air/water interface, but this surface activity is not correlated with any aggregation in the bulk. These results support the hypothesis that each AFP III molecule acts independently of the others, and that specific intermolecular interactions between monomers are not required for binding to ice. The lack of attractive interactions between monomers may be functionally important, allowing for more efficient binding and covering of the ice surface.

Electro-optical properties characterization of fish type III antifreeze protein.

Antifreeze proteins (AFPs) are ice-binding proteins that depress the freezing point of water in a non-colligative manner without a significant modification of the melting point. Found in the blood and tissues of some organisms (such as fish, insects, plants, and soil bacteria), AFPs play an important role in subzero temperature survival. Fish Type III AFP is present in members of the subclass Zoarcoidei. AFPIII are small 7-kDa-or 14-kDa tandem-globular proteins. In the present work, we study the behavior of several physical properties, such as the low-frequency dielectric permittivity spectrum, circular dichroism, and electrical conductivity of Fish Type III AFP solutions measured at different concentrations. The combination of the information obtained from these measurements could be explained through the formation of AFP molecular aggregates or, alternatively, by the existence of some other type of interparticle interactions. Thermal stability and electro-optical behavior, when proteins are dissolved in deuterated water, were also investigated.

Analytical ultracentrifugation sedimentation velocity for the characterization of detergent-solubilized membrane proteins Ca++-ATPase and ExbB.

We have investigated the potential of new methods of analysis of sedimentation velocity (SV) analytical ultracentrifugation (AUC) for the characterization of detergent-solubilized membrane proteins. We analyze the membrane proteins Ca(++)-ATPase and ExbB solubilized with DDM (dodecyl-beta-D: -maltoside). SV is extremely well suited for characterizing sample heterogeneity. DDM micelles (s(20w) = 3.1 S) and complexes (Ca(++)-ATPase: s(20w) = 7.3 S; ExbB: s(20w) = 4 S) are easily distinguished. Using different detergent and protein concentrations, SV does not detect any evidence of self-association for the two proteins. An estimate of bound detergent of 0.9 g/g for Ca(++)-ATPase and 1.5 g/g for ExbB is obtained from the combined analysis of SV profiles obtained using absorbance and interference optics. Combining s(20w) with values of the hydrodynamic radius, R(s) = 5.5 nm for Ca(++)-ATPase or R(s) = 3.4 nm for ExbB, allows the determination of buoyant molar masses, M(b). In view of their M(b) and composition, Ca(++)-ATPase and ExbB are monomers in our experimental conditions. We conclude that one of the main advantages of SV versus other techniques is the possibility to ascertain the homogeneity of the samples and to focus on a given complex even in the presence of other impurities or aggregates. The relative rapidity of SV measurements also allows experiments on unstable samples.

Lactobionamide surfactants with hydrogenated, perfluorinated or hemifluorinated tails: physical-chemical and biochemical characterization.

Detergents are customarily used to solubilize cell membranes and keep membrane proteins soluble in aqueous buffers, but they often lead to irreversible protein inactivation. Hemifluorinated amphiphiles with hybrid hydrophobic chains have been specifically designed to minimize the denaturating propensity of surfactants toward membrane proteins. We have studied the physical-chemical and biochemical properties of lactobionamide surfactants bearing either a hydrogenated, a fluorinated or a hemifluorinated chain (respectively H-, F-, and HF-Lac). We show that the dual composition of the hydrophobic chain of HF-Lac endows it with unusual physical-chemical properties as regards its critical micellar concentration, interfacial area per molecule, and behavior upon reverse phase chromatography. Analytical ultracentrifugation shows that, whereas H-Lac assembles into well-defined micelles, F-Lac and HF-Lac form large and heterogeneous assemblies, whose size increases with surfactant concentration. Molecular dynamics calculations suggest that F-Lac forms cylindrical micelles. The ability of HF-Lac to keep membrane proteins soluble was examined using the cytochrome b(6) f complex from Chlamydomonas reinhardtii's chloroplast as a model protein. HF-Lac/b(6) f complexes form particles relatively homogeneous in size, in which the b(6) f complex is as stable or markedly more stable, depending on the surfactant concentration, than it is in equivalent concentrations of hydrogenated surfactants, including H-Lac.

The role of hydration on the mechanism of allosteric regulation: in situ measurements of the oxygen-linked kinetics of water binding to hemoglobin.

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98% relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.