Effect of enzymatic modification on the structure and rheological properties of diluted alkali-soluble pectin fraction rich in RG-I.

This study focuses on pectin covalently linked in cell walls from two sources, apples and carrots, that was extracted using diluted alkali, and it describes changes in the rheological properties of diluted alkali-soluble pectin (DASP) due to enzymatic treatment. Given DASP's richness of rhamnogalacturonan I (RG-I), RG-I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG-I pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM imaging revealed a significant increase in the length of branched molecules after incubation with ABF, suggesting that arabinose side chains limit RG-I aggregation. Structural modifications were confirmed by changes in the intensity of bands in the pectin fingerprint and anomeric region on Fourier transform infrared spectra. ABF treatment led to a decrease in the stability of pectic gels, while the simultaneous use of ABF, RGAE, and RGL enzymes did not increase the degree of aggregation compared to the control sample. These findings suggest that the association of pectin chains within the DASP fraction may rely significantly on intermolecular interactions. Two mechanisms are proposed, which involve side chains as short-range attachment points or an extended linear homogalacturonan conformation favoring inter-chain interactions over self-association.

Structural and rheological properties of diluted alkali soluble pectin from apple and carrot.

Pectin, a complex polysaccharide found in plant cell walls, plays a crucial role in various industries due to its functional properties. The diluted alkali-soluble pectin (DASP) fractions that result from the stepwise extraction of apples and carrots were studied to evaluate their structural and rheological properties. Homogalacturonan and rhamnogalacturonan I, in different proportions, were the main pectin domains that composed DASP from both materials. Atomic force microscopy revealed that the molecules of apple DASP were longer and more branched. A persistence length greater than 40 nm indicated that the pectin molecules deposited on mica behaved as stiff molecules. The weight-averaged molar mass was similar for both samples. Intrinsic viscosity values of 194.91 mL·g-1 and 186.79 mL·g-1 were obtained for apple and carrot DASP, respectively. Rheological measurements showed greater structural strength for apple-extracted pectin, whereas carrot pectin was characterized by a higher linear viscoelasticity limit. This comparison showed that the pectin fractions extracted by diluted alkali are structurally different and have different rheological properties depending on their botanical origin. The acquired insights can enhance the customized use of pectin residue and support further investigations in industries relying on pectin applications.

Influence of pectin and hemicelluloses on physical properties of bacterial cellulose.

The properties of bacterial cellulose (BC)-based films produced by in situ biosynthesis with various polysaccharides (water-soluble pectin, arabinan, rhamnogalacturonan I, arabinoxylan, xyloglucan, glucomannan) were investigated. The addition of the polysaccharides to the bacterial growth environment changed the composition of the films by incorporating characteristic monosaccharides. BC-based films contained up to 26.7 % of non-cellulosic polysaccharides. The applied modification had a clear impact on water sorption and caused a decrease in the thermal stability of most BC films, which was connected with the depletion of geometrical dimensions of cellulose nanofibers observed with AFM. The FT-IR and Raman spectra demonstrated a decrease in % Iα of cellulose films, most notably for xyloglucan and glucomannan, as well as a change in their degree of crystallinity and the length of cellulose chains. The addition of xyloglucan had the most pronounced effect on film hardening; the other additives had a similar but lesser effect.

A mini-review on the plant sources and methods for extraction of rhamnogalacturonan I.

Rhamnogalacturonan type I (RG-I) is one of the pectin family member abundant in plant cell walls. Process of RG-I extraction from cell walls, either as a one-step or several-stage process, conditions the structure and properties of obtained polysaccharides. In this paper, we provide comprehensive overview of the factors related to the source and extraction techniques that determine the yield and chemical composition of pectin belonging to RG-I. The role of the source material, solvent, pH, temperature, time and additional factors related to applied techniques, such as microwaves, ultrasounds, high and low pressure or enzymatic treatments are discussed.

The Influence of High-Intensity Ultrasonication on Properties of Cellulose Produced from the Hop Stems, the Byproduct of the Hop Cones Production.

The goal of this work is to evaluate the hop stems, a byproduct of hop cones production, as a potential source of cellulose. Hop stems contain up to 29% of cellulose. The cellulose isolation was conducted through the thermochemical treatment. After high-speed blending, the cellulose was characterized by 67% of crystallinity degree obtained from X-ray diffraction and median diameter of 6.7 nm obtained from atomic force microscopy imaging. The high-intensity ultrasonication (HIUS) was applied to reach further disintegration of cellulose fibers. The longer HIUS treatment resulted in decrease in crystallinity degree even up to 60% and decrease in the fiber diameter up to 4 nm. The Fourier transform infrared spectroscopy (FTIR) spectra showed that HIUS treatment led to changes in intermolecular hydrogen bonds. The stability of cellulose dispersions versus length of HIUS treatment was monitored over 14 days with back dynamic light scattering and laser Doppler electrophoresis methods. Obtained results are evidence that the hop stems are a potential source of cellulose and that it is possible to obtain stable dispersions after HIUS treatment. This was the first time that the properties of hop cellulose have been described so extensively and in detail after the use of HIUS treatment.

Structure and functionality of Rhamnogalacturonan I in the cell wall and in solution: A review.

Rhamnogalacturonan I (RG-I) belongs to the pectin family and is found in many plant cell wall types at different growth stages. It plays a significant role in cell wall and plant biomechanics and shows a gelling ability in solution. However, it has a significantly more complicated structure than smooth homogalacturonan (HG) and its variability due to plant source and physiological state contributes to the fact that RG-I's structure and function is still not so well known. Since functionality is a product of structure, we present a comprehensive review concerning the chemical structure and conformation of RG-I, its functions in plants and properties in solutions.

The primary, secondary, and structures of higher levels of pectin polysaccharides.

Pectin is a heteropolysaccharide abundant in the cell wall of plants and is obtained mainly from fruit (citrus and apple), thus its properties are particularly prone to changes occurring during ripening process. Properties of pectin depend on the string-like structure (conformation, stiffness) of the molecules that determines their mutual interaction and with the surrounding environment. Therefore, in this review the primary, secondary, and structures of higher levels of pectin chains are discussed in relation to external factors including crosslinking mechanisms. The review shows that the primary structure of pectin is relatively well known, however, we still know little about the conformation and properties of the more realistic systems of higher orders involving side chains, functional groups, and complexes of pectin domains. In particular, there is lack of knowledge on the influence of postharvest changes and extraction method on the primary and secondary structure of pectin that would affect conformation in a given environment and assembly to higher structural levels. Exploring the above-mentioned issues will allow to improve our understanding of pectin functionality and will help to tailor new functionalities for the food industry based on natural but often biologically variable source. The review also demonstrates that atomic force microscopy is a very convenient and adequate tool for the evaluation of pectin conformation since it allows for the relatively straightforward stretching of the pectin molecule in order to measure the force-extension curve which is directly related to its stiffness or flexibility.

Evaluation of Nanocomposite Made of Polylactic Acid and Nanocellulose from Carrot Pomace Modified with Silver Nanoparticles.

In this research, it was proposed to use carrot cellulose nanofibrils (CCNF) isolated from carrot pomace modified with silver nanoparticles (AgNPs) as a filler of polylactic acid (PLA) composites matrix. The new procedure was based on two steps: first, the preparation of nanocellulose modified with metal nanoparticles, and then the combination with PLA. Two concentrations-0.25 mM and 2 mM-of AgNO3 were used to modify CCNF. Then, PLA was mixed with the filler (CCNF/AgNPs) in two proportions 99:1 and 96:4. The influence of CCNF/AgNPs on mechanical, hydrophilic, thermal, and antibacterial properties of obtained nanocomposites was evaluated. The greatest improvement of mechanical properties was observed for composite containing CCNF with 2 mM of AgNPs, which obtained the lowest Young modulus and highest strain at break. The degradation temperature was lower for PLA with CCNF/AgNPs, but crystallization temperature wasn't influenced. The addition of CCNF/AgNPs also increased hydrophilicity. The transmission rates of oxygen, nitrogen, and carbon dioxide also increased after the addition of CCNF/AgNPs to PLA. The antibacterial function against Escherichia coli and Bacillus cereus was obtained after the addition of AgNPs but only at the contact surface with the material made, suggesting the lack of migration of nanoparticles from the composite.

Immunocytochemical studies on the distribution of arabinogalactan proteins (AGPs) as a response to fungal infection in Malus x domestica fruit.

Arabinogalactan proteins (AGPs) are cell components implicated in plant-microbe interactions. Despite the significance of AGPs in response to stress factors, their distribution during development of fungal disease in fruit is unknown. In our work, in situ analysis of AGP arrangement in fruit inoculated with Penicillium spinulosum during the consecutive days of infection development was carried out. For immunolocalization of AGPs, samples were incubated with JIM13, MAC207, LM2, and LM14 antibodies recognizing the AGP carbohydrate moieties. To analyse cell walls without proper action of AGP, an experiment with ß-glucosyl Yariv reagent specifically binding AGPs was performed. The results showed an increase of signal fluorescence in the fruit after 16 days of fungal disease. Higher amounts of the examined epitopes were observed in the infection-altered sites of the fruit, in close vicinity to a surface filled by fungal spores. The results indicate that the Yariv reagent treatment induced progress of the fungal disease. Changes in the AGP presence during the fungal disease confirmed their involvement in defence against pathogen attack in fruit.

Tailored nanocellulose structure depending on the origin. Example of apple parenchyma and carrot root celluloses.

Cellulose is the major polysaccharide of cell walls in every plant, making it one of the most abundant natural polymers on Earth. However, despite many decades of investigations, the supramolecular structure of cellulose and especially its variation in the cell walls of different plants have still not been fully revealed. In the present study, cellulose from the parenchymatic tissue of apple fruits and carrot roots was isolated, and nanocellulose was further prepared by high-intensity ultrasonication. AFM revealed that the obtained nanocellulose differed in dimension between the two plant species. Compared with carrot cellulose, whose nanocellulose was obtained in the form of whiskers, apple cellulose had longer and thinner nanofibrils. Both nanocellulose types also differed in terms of their crystalline structure. XRD data indicated that, compared with the apple cellulose, the carrot cellulose had a higher degree of crystallinity and larger crystallites. Moreover, FTIR and Raman spectroscopy revealed differences between the cellulose types in terms of their methine environment, hydroxymethyl conformations and skeletal vibrations. Additionally, with respect to their mechanical properties, the less crystalline apple cellulose and nanocellulose films were more elastic than the stiffer carrot cellulose and nanocellulose films. The possible reason for such differences between the two cellulose types is related to differences in plant tissue morphology and function. During development, apple fruit cell walls must withstand increasing turgor, probably higher that in the case of carrot tissue; therefore, the cellulose scaffolding must be elastic and strong. On the other hand, carrot, a root vegetable, also has to be strong enough to penetrate the soil as well as for its own growth; thus, the cell wall and cellulose scaffold have to be stiff and tough. Thus the structure of nanocellulose depends not only on the treatment but also on the cellulose source.

Compression simulations of plant tissue in 3D using a mass-spring system approach and discrete element method.

A hybrid model based on a mass-spring system methodology coupled with the discrete element method (DEM) was implemented to simulate the deformation of cellular structures in 3D. Models of individual cells were constructed using the particles which cover the surfaces of cell walls and are interconnected in a triangle mesh network by viscoelastic springs. The spatial arrangement of the cells required to construct a virtual tissue was obtained using Poisson-disc sampling and Voronoi tessellation in 3D space. Three structural features were included in the model: viscoelastic material of cell walls, linearly elastic interior of the cells (simulating compressible liquid) and a gas phase in the intercellular spaces. The response of the models to an external load was demonstrated during quasi-static compression simulations. The sensitivity of the model was investigated at fixed compression parameters with variable tissue porosity, cell size and cell wall properties, such as thickness and Young's modulus, and a stiffness of the cell interior that simulated turgor pressure. The extent of the agreement between the simulation results and other models published is discussed. The model demonstrated the significant influence of tissue structure on micromechanical properties and allowed for the interpretation of the compression test results with respect to changes occurring in the structure of the virtual tissue. During compression virtual structures composed of smaller cells produced higher reaction forces and therefore they were stiffer than structures with large cells. The increase in the number of intercellular spaces (porosity) resulted in a decrease in reaction forces. The numerical model was capable of simulating the quasi-static compression experiment and reproducing the strain stiffening observed in experiment. Stress accumulation at the edges of the cell walls where three cells meet suggests that cell-to-cell debonding and crack propagation through the contact edge of neighboring cells is one of the most prevalent ways for tissue to rupture.

Simultaneous influence of pectin and xyloglucan on structure and mechanical properties of bacterial cellulose composites.

The impact of the matrix polysaccharides on the cellulose microfibrils structure as well as on the mechanical properties of cell walls still remains an open question. Therefore, the aim of investigations was to determine the simultaneous influence of (i) different concentrations of pectins with constant concentration of xyloglucan, and (ii) different concentrations of xyloglucan with constant concentration of pectins on cellulose structure. Composites of bacterial cellulose (BC) produced by Komagataeibacter xylinus are considered to mimic natural plant cell walls. This investigation showed that the lower the ratio of xyloglucan to pectin was, the higher Young's modulus of BC composite was and also obtained cellulose microfibrils were thinner. The increasing concentration of xyloglucan to pectin also caused the drop down in microfibrils crystallinity degree with predominant structure of cellulose Iß. In that case, also the length of cellulose chains was growing and reaching the highest value among all BC composites.

Changes of pectin nanostructure and cell wall stiffness induced in vitro by pectinase.

Structural modifications of fruit cell-wall pectins are controlled by various enzymes. In this in vitro study, the cell wall material (CWM) from pear fruit (Pyrus communis L.) was treated using pectinases in two concentrations. Water soluble (WSP), chelator soluble (CSP) and sodium carbonate soluble (DASP) pectin fractions were extracted from CWM. By visualization of enzymatic-induced changes of structure and CWM stiffness using an atomic force microscopy (AFM), the role of pectins in the mechanical properties of cell walls was shown. Galacturonic acid (GalA) content in pectin fractions was assayed as well. This experiment unveiled evidence of the structural degradation of molecules in pectin fractions extracted from CWM caused by in vitro pectinase action and softening of CWM due to pectin removal that might be related to the creation of empty spaces in the cellulose-hemicellulose network.

A Comparative Study of the Application of Fluorescence Excitation-Emission Matrices Combined with Parallel Factor Analysis and Nonnegative Matrix Factorization in the Analysis of Zn Complexation by Humic Acids.

The main aim of this study was the application of excitation-emission fluorescence matrices (EEMs) combined with two decomposition methods: parallel factor analysis (PARAFAC) and nonnegative matrix factorization (NMF) to study the interaction mechanisms between humic acids (HAs) and Zn(II) over a wide concentration range (0-50 mg·dm-3). The influence of HA properties on Zn(II) complexation was also investigated. Stability constants, quenching degree and complexation capacity were estimated for binding sites found in raw EEM, EEM-PARAFAC and EEM-NMF data using mathematical models. A combination of EEM fluorescence analysis with one of the proposed decomposition methods enabled separation of overlapping binding sites and yielded more accurate calculations of the binding parameters. PARAFAC and NMF processing allowed finding binding sites invisible in a few raw EEM datasets as well as finding totally new maxima attributed to structures of the lowest humification. Decomposed data showed an increase in Zn complexation with an increase in humification, aromaticity and molecular weight of HAs. EEM-PARAFAC analysis also revealed that the most stable compounds were formed by structures containing the highest amounts of nitrogen. The content of oxygen-functional groups did not influence the binding parameters, mainly due to fact of higher competition of metal cation with protons. EEM spectra coupled with NMF and especially PARAFAC processing gave more adequate assessments of interactions as compared to raw EEM data and should be especially recommended for modeling of complexation processes where the fluorescence intensities (FI) changes are weak or where the processes are interfered with by the presence of other fluorophores.

Raman imaging of changes in the polysaccharides distribution in the cell wall during apple fruit development and senescence.

MAIN CONCLUSION: Du ring on-tree ripening, the pectin distribution changed from polydispersed in cell wall to cumulated in cell wall corners. During apple storage, the pectin distribution returned to evenly dispersed along the cell wall. The plant cell wall influences the texture properties of fruit tissue for example apples become softer during ripening and postharvest storage. This softening process is believed to be mainly connected with changes in the cell wall composition due to polysaccharides undergoing an enzymatic degradation. These changes in polysaccharides are currently mainly investigated via chemical analysis or monoclonal labeling. Here, we propose the application of Raman microscopy for evaluating the changes in the polysaccharide distribution in the cell wall of apples during both ripening and postharvest storage. The apples were harvested 1 month and 2 weeks before optimal harvest date as well as at the optimal harvest date. The apples harvested at optimal harvest date were stored for 3 months. The Raman maps, as well as the chemical analysis were obtained for each harvest date and after 1, 2 and 3 months of storage, respectively. The analysis of the Raman maps showed that the pectins in the middle lamella and primary cell wall undergo a degradation. The changes in cellulose and hemicellulose were less pronounced. These findings were confirmed by the chemical analysis results. During development changes of pectins from a polydispersed form in the cell walls to a cumulated form in cell wall corners could be observed. In contrast after 3 months of apple storage we could observe an substantial pectin decrease. The obtained results demonstrate that Raman chemical imaging might be a very useful tool for a first identification of compositional changes in plant tissue during their development. The great advantage Raman microspectroscopy offers is the simultaneous localization and identification of polysaccharides within the cell wall and plant tissue.

The stiffening of the cell walls observed during physiological softening of pears.

MAIN CONCLUSION: The Young's modulus of the primary cell walls of pears decreases linearly during the pre-harvest on-tree maturation and increases during postharvest storage, and does not correlate with firmness of fruit. The determination of mechanical properties of cell walls is indispensable for understanding the mechanism of physiological softening and deterioration of quality of fruits during postharvest storage. The Young's modulus of the primary cell walls from pear fruit (Pyrus communis L., cultivars 'Conference' and 'Xenia') during pre-harvest maturation and postharvest storage in an ambient atmosphere at 2 °C followed by shelf life was studied using atomic force microscopy (AFM). The results were related to the firmness of fruits, galacturonic acid content in water, chelator, sodium carbonate and insoluble pectin fractions, polygalacturonase and pectin methylesterase activities. The Young's modulus of the primary cell walls decreased linearly during the last month of pre-harvest maturation from 3.2 ± 1.8 to 1.1 ± 0.7 MPa for 'Conference' and from 1.9 ± 1.2 to 0.2 ± 0.1 MPa for 'Xenia' which correlated with linear firmness decrease. During postharvest storage the cell wall Young's modulus increased while firmness continued to decrease. Correlation analysis for the entire period of the experiment showed a lack of straightforward relation between the Young's modulus of primary cell walls and fruit firmness. The Young's modulus of cell walls correlated negatively either with galacturonic acid content in sodium carbonate soluble pectin ('Conference') or with insoluble pectin fractions ('Xenia') and positively with polygalacturonase activity. It was therefore evidenced that covalently linked pectins play the key role for the stiffness of fruit cell walls. Based on the obtained results, the model explaining the fruit transition from firm and crispy to soft and mealy was proposed.

Evaluation of Structure and Assembly of Xyloglucan from Tamarind Seed (Tamarindus indica L.) with Atomic Force Microscopy.

The role of xyloglucan (XG) in the cell wall of plants and its technological usability depends on several factors, pertaining to molecular structure. Therefore, the goal of this study was to evaluate the nano-structure and self-assembly of XG by atomic force microscopy (AFM). As the model, a non-modified xyloglucan from a tamarind seed (Tamarindus indica L.) was used. Samples were minimally processed, i.e., treated with low-power ultrasound and studied on the surface of mica in ambient butanol. AFM topographic images revealed rod-like nanomolecules of xyloglucan with a mean height of 2.3 ± 0.5 nm and mean length of 640 ± 360 nm. The AFM study also showed that XG chains possessed a helical structure with a period of 115.8 ± 29.2 nm. This study showed possible-bending of molecules with a mean angle of 127.8 ± 25.6°. The xyloglucan molecules were able to aggregate as cross-like and a parallel like assemblies, and possibly as rope-like structures. The self-assembled bundles of xyloglucan chains were often complexed at an angle of 114.2 ± 36.3°.

Effect of Cytochalasin B, Lantrunculin B, Colchicine, Cycloheximid, Dimethyl Sulfoxide and Ion Channel Inhibitors on Biospeckle Activity in Apple Tissue.

The biospeckle phenomenon is used for non-destructive monitoring the quality of fresh fruits and vegetables, but in the case of plant tissues there is a lack of experimentally confirmed information about the biological origin of the biospeckle activity (BA). As a main sources of BA, processes associated with the movement inside the cell, such as cytoplasmic streaming, organelle movement and intra- and extracellular transport mechanisms, are considered. The aim of this study is to investigate the effect of metabolism inhibitors, connected with intracellular movement such as cytochalasin B, lantrunculin B, colchicine, cycloheximid, dimethyl sulfoxide (DMSO) and mixture of ion channel inhibitors, injected into apples, on BA. Two methods of BA analysis based on cross-correlation coefficient and Laser Speckle Contrast Analysis (LASCA) were used. DMSO, lantrunculin B and mixture of ion channel inhibitors have a significant effect on BA, and approximately 74 % of BA of apple tissue is potentially caused by biological processes. Results indicate that the functioning of actin microfilaments and ion channels significantly affect BA.