Atomic Force Microscopy in the Characterization of the Structure of Cell Wall Components.

Atomic force microscopy (AFM) has broken boundaries in the characterization of the supramolecular architecture of cell wall assemblies and single cell wall polysaccharides at the nanoscale level. Moreover, AFM provides an opportunity to evaluate the mechanical properties of cell wall material which is not possible with any other method. However, in the case of plant tissue, the critical step is a smart sample preparation that should not affect the polysaccharide structure or assembly and on the other hand should consider device limitations, especially scanner ranges. In this chapter, the protocols from the sample preparation, including isolation of cell wall material and extraction of cell wall polysaccharide fractions, through AFM imaging of polysaccharide assemblies and single molecules until an image analysis to obtain quantitative data characterizing the biopolymers are presented.

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.

Coarse-grained molecular dynamics model to evaluate the mechanical properties of bacterial cellulose-hemicellulose composites.

The plant cell wall (PCW) inspires the preparation of fiber-based biomaterials, particularly emphasizing exploiting the intrinsic interactions within the load-bearing cellulose and hemicellulose network. Due to experimental difficulties in studying and interpreting the interaction between these polysaccharides, this research presents a numerical model based on coarse-grained molecular dynamics that evaluates the mechanical properties of fiber composites. To validate the model and explain the structural and mechanical role of hemicelluloses, bacterial cellulose (BC) was synthesized in the presence of different concentrations of xylan, arabinoxylan, xyloglucan, or glucomannan and subjected to nano- and macroscale structural and mechanical characterization. The data obtained were used to interpret the effects of each hemicellulose on the mechanics of the BC-hemicellulose composite based on the sensitivity of the model. The mechanical properties of the resulting simulated networks agreed well with the experimental observations of the BC-hemicellulose composites. Increased xylan and arabinoxylan contents increased the macroscale mechanical properties, fiber modulus (xylan), and fiber width (arabinoxylan). The addition of xyloglucan increased the mechanical properties of the composites in the elastic deformation phase, associated with an increase in the fiber modulus. Adding glucomannan to the culture medium decreased all the mechanical properties studied while the fiber width increased.

Apple metabolism under oxidative stress affects plant cell wall structure and mechanical properties.

Several studies have shown beneficial effects of short exposure to oxidative stress on stored fruit, such as better preservation, increased firmness, preservation of polyphenolic compounds, and reduced risk of postharvest disorders such as bitter pit and superficial scald in apples. In this study the effect of short-term oxidative stress conditions on the physiology of apple fruit was investigated. Apple fruit of three cultivars were exposed to hypoxic storage conditions of various lengths to induce anaerobiosis. The response of apple fruit to short-term oxidative stress was evaluated by means of cell wall immunolabeling and atomic force microscopy. In addition, the antioxidant capacity and antioxidative activity of apple peels was assessed. Through various techniques, it was shown that short-term oxidative stress conditions promote specific enzymatic activity that induces changes in the cell wall of apple fruit cells. Exposure to short-term stress resulted in the remodeling of cell wall pectic polysaccharides, observed as an increase in the size and complexity of extracted oxalate pectin. Structural changes in the cell wall were followed by an increase in Young's modulus (compressive stiffness of a solid material, expressed as the relationship between stress and axial strain) of the cell wall material. The data presented in this paper show in a novel way how storage under short-term oxidative stress modifies the cell wall of apple fruit at the molecular level.

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.

Application of polysaccharides for the encapsulation of beneficial microorganisms for agricultural purposes: A review.

Intensive farming practices have increased the consumption of chemical-based pesticides and fertilizers thereby creating health issues for humans and animals and also causing a deterioration in the natural ecosystem. The promotion of biomaterials synthesis could potentially lead to the replacement of synthetic products and improve soil fertility, protect plants from pathogen attacks, and enhance the productivity of the agricultural sector resulting in less environmental pollution. Microbial bioengineering involving the use and improvement of encapsulation using polysaccharides has the required potential to address environmental issues and promote green chemistry. This article describes various encapsulation techniques and polysaccharides which have an immense applicable capability to encapsulate microbial cells. The review elucidates the factors that may result in a reduced viable cell count during encapsulation, particularly using the spray drying method, where a high temperature is required to dry the suspension, this may damage the microbial cells. The environmental advantage of the application of polysaccharides as carriers of beneficial microorganisms, which do not pose a risk for soil due to their full biodegradability, was also shown. The encapsulated microbial cells may assist in addressing certain environmental problems such as ameliorating the unfavourable effects of plant pests and pathogens, and promoting agricultural sustainability.

Neutral and Pectic Heteropolysaccharides Isolated from Opuntia joconostle Mucilage: Composition, Molecular Dimensions and Prebiotic Potential.

Opuntia joconostle is a semi-wild cactus cultivated for its fruit. However, the cladodes are often discarded, wasting the potentially useful mucilage in them. The mucilage is composed primarily of heteropolysaccharides, characterized by their molar mass distribution, monosaccharide composition, structural features (by vibrational spectroscopy, FT IR, and atomic force microscopy, AFM), and fermentability by known saccharolytic commensal members of the gut microbiota. After fractionation with ion exchange chromatography, four polysaccharides were found: one neutral (composed mainly of galactose, arabinose, and xylose) and three acidic, with a galacturonic acid content from 10 to 35%mol. Their average molar masses ranged from 1.8 × 105 to 2.8 × 105 g·mol-1. Distinct structural features such as galactan, arabinan, xylan, and galacturonan motifs were present in the FT IR spectra. The intra- and intermolecular interactions of the polysaccharides, and their effect on the aggregation behavior, were shown by AFM. The composition and structural features of these polysaccharides were reflected in their prebiotic potential. Lactobacilli and Bifidobacteria were not able to utilize them, whereas members of Bacteroidetes showed utilization capacity. The obtained data suggest a high economic potential for this Opuntia species, with potential uses such as animal feed in arid areas, precise prebiotic, and symbiotic formulations, or as the carbon skeleton source in a green refinery. Our methodology can be used to evaluate the saccharides as the phenotype of interest, helping to guide the breeding strategy.

Novel polysaccharide and polysaccharide-peptide conjugate from Rosa rugosa Thunb. pseudofruit - Structural characterisation and nutraceutical potential.

A novel bioactive polysaccharopeptide (C1) and polysaccharide (C2) with an average molecular weight of 180 kDa and 70 kDa were isolated from R. rugosa pseudofruit. The composition of the macromolecules was established using 1H NMR, FT-IR, GC-MS, SDS-PAGE coupled with enzymatic cleavage, and proteomic analyses (LC-MS). C1 was found to contain 60.56 ± 1.82 % of sugars and 21.17 ± 0.47 % of uronic acids. Its main neutral monosaccharides were arabinose, rhamnose, galactose, glucose, fucose, and mannose. C1 was found to be a polysaccharopeptide containing pectinesterase-like protein. C2 was composed of 32.85 ± 0.97 % of sugars and 48.77 ± 1.15 % of uronic acids. Its main neutral monosaccharides were galactose, glucose, rhamnose, arabinose, and mannose. A promising nutraceutical value of the polysaccharides was revealed. Assays showed strong α-glucosidase inhibitory activity of both macromolecules and considerable antiradical potential and moderate lipoxygenase inhibitory activity of the crude polysaccharide. Moreover, antiproliferative activity of C2 was observed.

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.

Working towards arabinogalactan proteins (AGPs) from fruit: carbohydrate composition and impact on fungal growth.

BACKGROUND: Arabinogalactan proteins (AGPs) are extracellular matrix constituents involved in plant response to fungal infection. The aim of the current study was to investigate the antifungal effect of AGPs ex situ and to determine the structural features of AGPs that may have an influence on this activity. The features of AGPs isolated from fruit were investigated with molecular tools based on specific monoclonal antibodies recognizing carbohydrate AGP epitopes. The Antifungal (well-diffusion) Susceptibility Test and the Agar Invasion Test were used to assess the impact of AGPs on Penicillium notatum culture. RESULTS: The results definitely ruled out the influence of AGPs on fungal growth. The immunochemical analyses revealed that AGPs consist mainly of carbohydrate chains composed of ß-linked glucuronosyl residues recognized by LM2 and GlcA-ß(1 → 3)-GalA-α(1 → 2) Rha recognized by JIM13, which do not have the same functional properties outside the plant cell in in vitro experimental conditions. CONCLUSIONS: The action of a single cell wall component does not elicit any influence ex situ. The extensive accumulation of glycan chains of AGPs in infected tissue as a result of a complex mechanism occurring in the cell wall emphasizes the importance of dependencies between particular components of the extracellular matrix in response to fungal attack.

Analysis of the chemical composition of natural carbohydrates - An overview of methods.

Natural carbohydrates are gaining importance over a wide spectrum of human activity due to their versatile functionalities. The properties of carbohydrates are currently used in many branches of industry and new possibilities of their utilization, like in medicine or materials science, are demonstrated systematically. The attractive properties of carbohydrates result from their chemical structure and ability to form macromolecules and derivatives. Each application of carbohydrate requires a knowledge of their chemical composition, which due to the number and differentiation of monosaccharides and their spatial forms is often challenging. This review presents an overview on sample preparation and the methods used for the determination of the fine chemical structure of natural carbohydrates. Most popular and reliable colorimetric, chromatographic and spectroscopic methods are presented with an emphasis on their pros and cons.

The effect of high humidity hot air impingement blanching on the changes in cell wall polysaccharides and phytochemicals of okra pods.

BACKGROUND: Okra pods contain heat-sensitive substances, such as phenolic compounds and other phytochemicals that can be degraded when okra pods are subjected to heat treatment. The understanding of the impact of high humidity hot air impingement blanching (HHAIB) on the changes in physicochemical properties of polysaccharides and phytochemicals of okra pods is of great importance because over-blanching may result in cell membrane disruption and changes in biologically active compounds under prolonged exposure to the thermal treatment. Therefore, the present study aimed to investigate the effect of HHAIB on the changes in physicochemical properties of pectins and phytochemicals extracted from okra pods. RESULTS: Both the HHAIB time and method of extraction influenced their physicochemical characteristics and biological activity. Pectin fractions subjected to HHAIB were composed of polygalacturonic acid, rhamnogalacturonan, glucomannan, galactan, mannose, arabinose, rhamnose, calcium pectate and arabinogalactan. The contents of total phenolics, total flavonoids and antioxidant activity of extracts mostly increased during HHAIB (i.e. up to 19.0%, 13.2% and 35.3%, respectively). However, HHAIB reduced the chlorophyll-a (up to 55.7%) and lycopene (up to 52.6%) contents of okra pods. CONCLUSION: The acquired knowledge may be useful for better understanding and optimization of technologies based on HHAIB treatment. The HHAIB treated okra can be a promising natural alternative in different applications, including its use as a replacement of some ingredients in food or non-food systems as a result of richness in polysaccharides and polyphenols, as well as high antioxidant properties. © 2022 Society of Chemical Industry.

Chemical Changes in the Broccoli Volatilome Depending on the Tissue Treatment.

The storage of plant samples as well as sample preparation for extraction have a significant impact on the profile of metabolites, however, these factors are often overlooked during experiments on vegetables or fruit. It was hypothesized that parameters such as sample storage (freezing) and sample pre-treatment methods, including the comminution technique or applied enzyme inhibition methods, could significantly influence the extracted volatile metabolome. Significant changes were observed in the volatile profile of broccoli florets frozen in liquid nitrogen at -20 °C. Those differences were mostly related to the concentration of nitriles and aldehydes. Confocal microscopy indicated some tissue deterioration in the case of slow freezing (-20 °C), whereas the structure of tissue, frozen in liquid nitrogen, was practically intact. Myrosinase activity assay proved that the enzyme remains active after freezing. No pH deviation was noted after sample storage - this parameter did not influence the activity of enzymes. Tissue fragmentation and enzyme-inhibition techniques applied prior to the extraction influenced both the qualitative and quantitative composition of the volatile metabolome of broccoli.

Changes of pectin structure and microbial community composition in strawberry fruit (Fragaria × ananassa Duch.) during cold storage.

Strawberry is very perishable fruit with rapid postharvest loss of quality and high susceptibility to microbial infections. In this work we study pectin modifications and microbiota and mycobiota composition in strawberry in conventional and organic cultivation systems. The enzymatic activity during postharvest storage of both types of strawberry was divided at the fifth day of storage into two phases: postharvest changes and rotting. Pectin molecules extracted from organic strawberries were longer and more branched compared to the conventional strawberries; however a more noticeable reorganization of molecular structure occurred. The sequential action of the pectinolytic enzymes had a direct effect on the molecular structure of pectin fractions. The observed changes in pectin structure relate to the synergistic activity of pectinolytic enzymes and some microorganisms. The organic system was characterized by a greater number and variety of bacteria and fungi during storage as compared to the conventional system.

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 Use of Interactions Between Microorganisms in Strawberry Cultivation (Fragaria x ananassa Duch.).

As the market indicates a growing interest in organically grown fruit, there is a need for biostimulants to counter the adverse effects of pathogenic fungi and fungal-like-pathogens. Four microbial pathogens (Botrytis cinerea, Verticillium sp., Phytophthora sp., and Colletotrichum sp.) which are the most often causes of strawberry diseases were selected. Five kinds of biostimulants (C1, C2, C3, C4, and C5) containing bacterial consortia were developed to combat the pathogens. The antagonistic effect of selected microorganisms against strawberry pathogens was observed. The effectiveness of various beneficial bacteria in combating fungal pathogens of cv. Honeoye strawberries was compared and the impact of their activity on fruit quality was assessed. The most significant effect on the strawberry firmness was found for the C2 consortium, which provided the strawberries infected with the pathogens group (MIX: B. cinerea, Verticillium sp., Phytophthora sp., and Colletotrichum sp.) with a 140% increase in maximum load in a puncture test compared to the positive control (C0). Strawberries contaminated with Phytophthora sp. after the application of Consortium C4 (C4) showed the largest increase (127%) in soluble solid content (SSC) when compared to the C0. Fruit contaminated with Colletotrichum sp. and B. cinerea after the application of C2 and Consortium 5 (C5), respectively, had the highest levels of anthocyanins and total phenolic content, when compared to C0. The largest increase, which reached as high as 25%, in D-galacturonic acid content was observed for the group of pathogens after Consortium 1 (C1) application. The extraction of strawberry pectin allowed for the study of the rheological properties of pectin solutions; on this basis, strawberry pectin from the control (NC) was distinguished as it showed the highest viscosity (0.137-0.415 Pas). Taking into account the individual effects of bacteria on strawberry pathogenic fungi and fungal-like-pathogens, it is possible to reduce the adverse effects of fungal disease and to improve the properties of strawberries by selecting the appropriate bacterial consortium. Interactions between microorganisms are often complex and not fully understood, which suggests the need for further research in this direction.

Structural properties of diluted alkali-soluble pectin from Pyrus communis L. in water and salt solutions.

The self-assembly and gelation of low-methoxyl diluted alkali-soluble pectin (LM DASP) from pear fruit (Pyrus communis L. cv. Conference) was studied in water and salt solutions (NaCl and CaCl2, constant ionic strength) without pH adjustment at 20 °C. The samples at different LM DASP concentrations were characterized using rheological tests, Fourier-transform infrared spectroscopy, dual-angle dynamic light scattering and atomic force microscopy. LM DASP from pear fruit (Pyrus communis L.) showed gelling ability. The indices (aggregation index and shape factor) based on light scattering may be useful for the characterization of structural changes in polysaccharide suspension, particularly for the determination of a gel point. The results obtained may be important for the food, cosmetic and pharmaceutical industries where pectin is used as a texturizer, an encapsulating agent, a carrier of bioactive substances or a gelling agent.

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.

Investigations of changes in the arabinogalactan proteins (AGPs) structure, size and composition during the fruit ripening process.

Arabinogalactan proteins (AGPs) are ubiquitous cell wall and plasma membrane components and are characterised by extensive glycosylation and heterogeneity of their carbohydrate and protein units. The aim of the study was to evaluate the structural features of AGPs present in apple fruits at different stages of the ripening process. AGPs were extracted using the Yariv reagent and examined using SDS-PAGE, immunoblotting, FT-IR, and AFM. In situ analysis, immunofluorescence (CLSM) and immunogold-labelling (TEM), were performed. We demonstrated that AGPs were indeed present in apple fruits at the different stages of the ripening process. The changes in the amount (1.52-2.08 mg g-1), diameter (152.73-75.05 nm), molecular mass (50-250 kDa), and distribution in the cell of AGPs demonstrate their variable presence and changeable structure during the ripening process. We propose specific wavenumbers, i.e. 1265 cm-1, 1117 cm-1, and 960 cm-1, which could be assigned to AGPs. The immunofluorescence and immunogold-labelling results indicate that the JIM13 antibody is the most characteristic for AGPs in apple fruits. This study quantitatively demonstrated for the first time that AGP accumulation occurs in ripe fruits, which is supported by the highest AGPs content, the highest molecular mass, and the appearance of a specific distribution pattern at the cellular level.

The Effect of Cultivation Method of Strawberry (Fragaria x ananassa Duch.) cv. Honeoye on Structure and Degradation Dynamics of Pectin during Cold Storage.

The high quality and long shelf life of strawberry fruit are largely dependent on the cultivation method. The goal of this experiment was to study the effect of different cultivation methods on molecular structure and rheological properties of pectin extracted from strawberry quality parameters during cold storage. Three methods of cultivation of strawberry cv. Honeoye were tested: organic cultivation on raised beds, organic cultivation with the flat-planted method and conventional cultivation with the flat-planted method. The nanostructure of pectin (AFM), its chemical structure (FT-IR) and rheological properties were studied. The fruits were also tested by size, dry matter, firmness, acidity and the content of soluble solids, anthocyanin, phenolics, vitamin C and galacturonic acid. Pectin isolated from organic strawberries was more rapidly degraded than conventional strawberry pectin, which limits the possibilities for their processing and use as gelling or stabilizing agents at 20 °C. The differences in fruit quality were particularly noticeable with respect to the anthocyanin content, which was significantly higher for organic strawberry. The organic fruit also had better sensory properties because of its lower acidity and higher soluble solid content (SSC). These and other results from this experiment showed that strawberries produced by organic farming methods had better biochemical properties compared to conventional fruit; however, pectin transformation undergone faster limits their further technological applications.