1. The Current State of the Food and Agriculture Industry
Agriculture and the Food Industry are facing several critical challenges that have apparently no short-cut solutions at sight. It is expected that the world population will reach 9 billion by 2050. This will require raising the agricultural production by 70% to feed the population adequate.1 However, most of the lands suitable for farming are already in use. To add more, current best agricultural practices have come to their limits in enhancing the yields of vital crops such as rice and wheat, the problem which is termed as yield plateauing. Producing animal source foods presents even more complicated problems. Keeping sufficient livestock population matching even the current demand is a burden on the environment. The industry produces 14.5 % of all greenhouse-gas emissions while exploiting 30% of the world’s ice-free landmass.2 Moreover, meat production is expensive. Animals need to be supplied with enormous amounts of water and food. For example, 10 kg of feed is required for every 1 kg of live animal for beef production.2
Regarding the enormity of problems and their feeding to each other, the innovations in current technology that can disrupt current practices are essential. Higher technology will lead to control the crop production processes better to achieve higher yields with less land use. It will reduce food loss and increase efficiency from production site to dinner table due to improved storage conditions and processing methods. Understanding the structure of animal-based food products will facilitate the development of artificial, possibly plant-based products that will be healthier, cheaper, and even as tasty as meat, egg, and other animal-based products.
Much needed disrupted innovation in these areas is expected to come with the help of nanotechnology. Nanoscale analysis of structure and engineering at the nanoscale are poised to advance agricultural technology, optimize food storage/food processing, and help to invent artificial (plant-based) food products that effectively mimic animal-based products both in texture and flavor.
The use of nanostructured thin films in sensors/biosensors will make them more sensitive to soil conditions, increase the quality of data taken from farm areas, and will give rise to precision agriculture or “smart farming” together with the data-processing techniques. The use of nanoparticles for controlled release of pesticides and fertilizers, the concept that is widely used in pharmacology, would limit their hazard to health and the environment.
Nanotechnology has even more promises when it comes to inspecting food quality whether it is the storage issue of the foods or manufacturing novel food products that can emulate natural ones. There is a long way between processing & storage conditions and texture/quality. And that way passes through a macromolecular nanostructure. To make informed changes in processing & storage conditions so that to have desired texture and taste, one may look at how those conditions affect the nanoscale macromolecular structure.
In this document, we present Atomic Force Microscopy (AFM) that stands out as a unique tool to fulfill the promise of nanotechnology in the food & agriculture industry. The unmatched potential of the tool in the nanoscale analysis and characterization of the structure of food systems and the technologies used in the industry justifies the timely investment in this device.
2. Introduction to AFM
The working principle of the Atomic Force Microscope (AFM) is based on the forces that arise when a sample surface is scanned with a nanometer-sized tip (a few to 10s of nm) attached to a cantilever. The advancement of the AFM over traditional stylus surface profilers is that the former uses a feedback loop to control the forces between the surface and probe. Because the forces are controlled, very small probes may be used, and not broken while capturing an image. There are two primary modes used for measuring the topography of a sample, which are contact mode and vibrating mode.
Contact Mode
The probing tip is in contact with the surface throughout the imaging in contact mode. The short-range forces between the surface and tip cause the deflection of the cantilever, which is recorded to generate the topographical image of the surface. However, the tip-surface contact in this mode can potentially damage the surface or wear the tip. Hence, this mode may not be suitable for imaging of soft surfaces. On the other hand, continuous contact with the surface allows identifying other features such as friction (lateral force imaging) or stiffness/elasticity map of the surface (force modulation imaging). In lateral force or frictional force microscopy, lateral deflections of the cantilever, arising due to forces parallel to the plane of the sample surface such as friction force, are measured.3 This allows detecting inhomogeneities on the material which gives rise to variations in surface friction.
Vibration Mode
In this mode, a probe at the end of a cantilever is vibrated up and down. As the vibrating probe begins to interact with a surface, the vibration amplitude is dampened. The amount of damping is proportional to the amount of force placed on the surface by the probe on each oscillation of the vibrating probe. A feedback loop is used to maintain a fixed vibration amplitude as the probe is scanned across a surface. Forces between the probe and surface in vibrating mode can be as low as a few 10's of piconewtons.
Although AFM is widely known for mapping surface topography, that alone does not always provide the answers that researchers need to understand the material.4 Fortunately, as a result of its capability to measure varying forces arising between the tip and sample, AFM can characterize a wide array of mechanical properties (e.g. adhesion, stiffness, friction, dissipation, viscoelasticity), electrical properties (e.g. capacitance, electrostatic forces, work function, electrical current, conductivity, surface potential, resistance), magnetic properties, optical/spectroscopic properties, thermal properties, and solvent effects (via imaging at liquid environment) in almost real-time.5
Phase imaging
A phase difference between oscillation of the cantilever and of the signal that drives cantilever oscillation (for example, piezoelectric crystal) is measured and visualized in phase imaging.6 There is no phase contrast when the surface is homogenous, or when there is no interaction between the tip and surface (i.e., the cantilever is well above the surface). However, if specific regions of the surface have distinct mechanical properties, that could be captured with phase imaging. This is because cantilever loses a different amount of energy as probe taps to surface areas with differing mechanical properties. Hence, phase imaging could be helpful to detect variations in mechanical properties such as friction, adhesion, and viscoelasticity on surfaces. It could as well be used to detect patterns of various materials such as polymers on the surface or to identify contaminants that cannot be distinguished with topography imaging.
3. Advantages of AFM in the Agriculture and the Food Industry
Traditionally, the Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) are used for measuring nanoscale images of samples. The AFM offers an alternative to these costly techniques. Certain inherent properties of AFM make it attractive to food industry:7
Three-dimensional topography is measured with an AFM so the surface textures can be directly revealed.
AFM provides nanoscale resolution in imaging of surfaces which is not limited to topographical analysis.
Compared to other high-resolution imaging methods such as SEM or TEM, AFM requires only minimal sample preparation.
AFM obtains images in ambient air or liquids and does not require large vacuum chambers. This could be critical for testing the effects of a storage condition on food molecules or evaluating the blend of food molecules, such as for manufacturing plant-based foods, under physiological conditions.
Images of very smooth, flat materials are readily imaged with AFM.
The cost of acquisition and ownership of an AFM is a fraction of an SEM.
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AFM is the only technique to provide mechanical information on the surface.
Evaluation of effects of storage conditions on nanoscale structure of foods.
Characterization of food components down to molecules at nanoscale.
Measuring molecular interaction parameters at nanometer spatial resolution.
Investigation of morphological and mechanical characteristics of polymers, nanoparticles, and other materials obtained from and used in agriculture and the food industry.
Identifying antibacterial and antiparasitic effects of agricultural products at single cell scale.
Identification of mechanical properties of food structures.
Characterization of nanomaterials obtained from processing of agricultural products.
4. Relevant Applications of AFM in Food Industry and Agriculture
4.1 Food storage/preservation methods
The texture of food products, apart from their flavor, is central to their sensory perception. Texture changes throughout the entire supply chain of especially vegetables and fruits could have a tremendous effect on their final quality. Therefore, the development of storage and preservation methods that delay texture changes is necessary for the food industry.
It is no wonder that treatments which target the nanoscale structure underpinning the texture are more probable to deliver the desired results. Pectin nanostructure had been shown to associate with textural properties of fruits and vegetables. Chen et al. studied the effect of exogenous ATP treatment on the postharvest qualities of mung bean sprouts (Vigna radiata) which are consumed as cooked food or fresh vegetable salad.8 Due to their inherent properties, mung beans lose their quality in the form of softening, browning, and nutritional loss after three days of storage. As they form 60% of the wall mass of many fruits, authors decided to study the role of pectin molecules in the process of quality loss. By using TT-AFM (AFM Workshop), they showed that the firmness of mung bean sprouts is underpinned by the nanostructures of sodium carbonate soluble pectin (SSP) that form massive thick chains interconnected with branch structures (Fig. 1A). After storage of 3 days, this structure is compromised with the degradation of thick chains indicated by the reduction in width and height of the SSP nanostructures (Fig. 1A, B). ATP treatment delayed the degradation process, giving rise to nanostructures with higher width and height than the control group. This was in parallel with macro-scale observations where ATP treatment also reduced browning and softening in comparison to the control group.
Figure 1. AFM imaging of sodium carbonate-soluble pectin (SSP) chains in mung bean sprouts by using TT-AFM (AFMWorkshop). A) AFM images a: control group before treatment; b: 3D image of the control group before treatment; c, d: control group at day 3; e, f: ATP treatment group at day 3. Br: branched chain; Tk: thick chain; Tn: thin chain; Ls: loose structure; Ss: short straight chain. B) Measurements of width and height of thick chains (B). (taken from Ref. 8)
The association between pectin degradation and decrease in firmness has also been confirmed in fresh-cut honeydew melons. Here, Chong et al. used chitosan and calcium chloride to prevent deterioration at 7 C.9 They also employed AFM (TT-AFM) to measure the effect of quality loss and the preventive measures on the length and width of pectin (SSP) nanostructures. Control samples, that lost their firmness rapidly, had shorter and narrower chains than the samples treated with chitosan and calcium chloride which also showed better firmness (Fig. 2).
Lastly, Yang et al. investigated the effects of vacuum impregnation with calcium lactate and pectin methylesterase as a preservation method on fresh-cut papayas.10 This treatment made a tremendous difference in texture properties, such as hardness and chewiness, compared to the untreated group after storage for eight days at 4 C. AFM analysis (TT-AFM) showed that the chelate soluble pectin nanochains had higher width and branching structure in the treatment group than the control untreated group.
Figure 2. AFM (TT-AFM) imaging of SSP chains from honeydew. (a) control group at day 7; (b) 2% chitosan group at day 7; (c) 1% CaCl2 group at day 7; (d) combined treatment group at day 7; Note: lc: long chain; sc: short chain; ls: linear single fraction; br: branching structure; mb: multiple branched chain; cp: cleavage point; ag: aggregates. (taken from Ref. 9).
Another set of studies applied AFM to investigate the nanostructure of fish to evaluate the treatments for preservation of the quality. In these studies, the nanostructure of myofibril, which is the essential component of fish muscle, was analyzed using AFM. Fish fillets are prone to quality loss during storage due to enzymatic and microbiological activities.11 Several methods have been developed to extend the shelf life of fish fillets such as irradiation, edible coating, vacuum packaging, and modified atmosphere packaging.12 The edible coating is the most cost-effective way among these as it does not require advanced machines.
The most commonly used materials for edible coating are biopolymers such as proteins and carbohydrates. Feng et al. studied the effects of chitosan, a carbohydrate, and gelatin, a protein-based product, on the cold storage of golden pomfret (Trachinotus blochii) fillet.12 AFM-based (TT-AFM) analysis of fish myofibrils at nanoscale allowed researchers to identify the right combination of coating ingredients (chitosan/gelatin) for optimal storage. They compared the myofibril length, width, and height for various chitosan, gelatin, or combination treatment groups. It turned out that the combination of 0.4% chitosan with 7.2% gelatin provided the best preservation (Fig. 3). While the myofibril length reduced from more than 15 um to 5 um in the control group at day 17 (4 C), in the combination treatment group, the myofibrils preserved their length. Other combination treatments or chitosan alone or gelatin alone couldn't reach similar effectivity.
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Figure 3. AFM imaging of myofibril nanostructures extracted from fish fillet. (A) day 0; (B) day 9 control; (C) & (D) day 17 control (E) day 9 chitosan; (F) day 17 chitosan; (G) day 9 chitosan + 3.6% gelatin (H) day 17 chitosan + 3.6% gelatin; (I) & (J) day 9 chitosan + 7.2% gelatin (K) day 17 chitosan + 7.2% gelatin (taken from ref. 12).
In another study by Feng et al., the authors employed the mixture of gelatin and tea polyphenols to preserve fish fillet (golden pomphret) quality in cold storage.12 Fish gelatin is used due to its film-forming property and its resistance against drying, light, and oxygen. Tea polyphenols (TP) have antimicrobial and antioxidant activities and improve the mechanical property of films. The researchers tested different combinations of the two ingredients, where 0.4 % TP + 1.2 % gelatin mixture proved to have the most potential as a preservative. This treatment had the best effect on reducing weight loss, lowering the pH, and antimicrobial growth. By using TT-AFM, authors showed that 0.4 % TP + 1.2 % gelatin is also the most optimal treatment for preventing myofibril degradation. On day 17 of cold storage, the treatment group showed myofibril length of greater than 15 μm, while the control group (coating with deionized water) had a 5.18 μm length. This treatment group also had myofibril nanostructures of the highest diameter among all other groups.
Tilapia is a widely consumed fish species in the world. It is also susceptible to drip loss, oxidation, microorganism proliferation, and changes in texture. As a preservation technique, Zhao et al. introduced fish gelatin (FG) with grape seed extract by vacuum impregnation on tilapia fillets at cold storage.13 Grape seed extract (GSE) is a natural additive that is abundant with polyphenol compounds. It has high antioxidant and antimicrobial properties. This treatment decreased drip loss, improved texture properties, and reducing microbial survival on the fillets. Analysis of myofibril nanostructures by using AFM (TT-AFM) revealed their degradation at control treatments. On the other hand, combination treatment (3% FG + 0.9% GSE) limited reduction at all dimensions (length, height, and width).
4.2 Anti-microbial treatments for food preservation
The consumption of organic food products has been increased dramatically in recent years, shown by the sales. From 1997 to 2017, organic sales jumped from 3.6b $ to 49.4b $.14 Organic farming policies restrict the usage of chemosynthetic fertilizers and recommend organic fertilizers such as animal manure instead.15 However, this increases the risk of contamination of the food products by pathogens. Moreover, most chemical sanitizers are either prohibited or allowed for usage with limited concentrations. It leads researchers to find alternative disinfection methods. The study by Liu et al. investigated the synergistic effect of low concentration electrolyzed water (LcEW) (chlorine, 4 mg/L) and short-term heat treatment of organic carrot on inactivation of E. coli O157:H7 and S. Typhimurium pathogens.15 The authors found that the two treatments acted synergistically to reduce the bacterial load on organic carrots regarding both pathogens. Then the authors used AFM (AFM Workshop) to understand the disinfectant mechanism of the sanitizer and compare the combined treatment with each of the single treatments by imaging the bacteria at the nanoscale. As illustrated in Fig. 4, AFM images demonstrate that the combination treatment was more effective at the destruction of a bacterial cell. Although destruction mechanisms such as shrinkage of the cells, the collapse of the membrane, increased granularity, leakage of the intracellular content outside, and degradation of flagella could be detected with single treatments as well, they become more pronounced with combined treatment.
In the same line of research, Zhao et al. studied the anti-microbial effects of combined low concentration acidic electrolyzed water and levulinic acid treatment on fresh organic lettuce.14 They used AFM (TT-AFM) to study the bactericidal mechanism of treatments against the bacteria E. coli (first column) and L. innocua. The intensity of antibacterial effect was higher in combined treatment than single treatments of electrolyzed water or deionized water, while there was no significant difference from levulinic acid single treatment. This was based on both length and width measurements of cells via AFM, and anti-microbial mechanisms in action as revealed from AFM images. Similar mechanisms were detected in this study as what is observed by Liu et al., such as membrane collapse, leakage of cell content, and cellular shrinkage.
And Chen et al. tested the anti-microbial efficacy of lactic acid combined with low-concentration sodium hypochlorite in organic broccoli sprouts.16 Although bacteria (L. innocua) survival analysis indicated very small differences between combined treatment and lactic acid treatment, especially when compared to sodium hypochlorite or deionized water treatments, AFM imaging showed further contrast between antibacterial effects of these two treatments. Cell width and roughness measurements showed that bacteria exposed to combination treatment had significantly lower cell width and higher roughness values than bacteria treated with any other treatment including lactic acid treatment. These effects indicate bactericidal mechanisms such as shrinkage and membrane collapse. AFM images also demonstrated that combined treatment wreaked the greatest havoc on microbes as revealed by cracking of the cells and abundant cell leakage.
Figure 4. Topographical AFM images of E. coli O157:H7 (A–D) and S. Typhimurium (a–d), untreated (A, a) and after treatment with deionized water at 70 °C (B, b), electrolyzed water (EW) at 25 °C (C, c), and EW at 70 °C (D, d). Images were obtained at non-contact mode with AFM Workshop equipment (taken from ref. 15).
4.3 Food engineering / Food manufacturing
Having a desired texture is critical for newly manufactured food. Natural foods are biological materials composed of hierarchically organized macromolecules from which the texture emerges. It is not easy to build these complex structures from simpler macromolecules to make artificial food products.17 It requires an understanding of the nanostructure that gives rise to the desired texture and sensory quality. Therefore, AFM is a promising tool also in food engineering/manufacturing that may help to uncover the linkage between structure and texture.
Cakes have a growing market as a food that is globally consumed and favored by people. Eggs are utilized in making cakes to provide foaming capacity, emulsification, stabilization, elasticity, also as binding, coloring, and flavoring agents in yellow cakes. Using eggs in traditional cakes is not acceptable for consumers who have dietary restrictions due to health concerns, religious reasons, or personal lifestyle choices. Moreover, eggs make up 50% of the cost of cake ingredients. Therefore, developing egg substitutes for cakes using plant-based molecules, such as proteins and polysaccharides, would be welcomed by consumers. With that aim, Lin et al. studied various combinations of soybean protein isolate (SPI) and plant polysaccharides and emulsifiers.18 As a result, they concluded that SPI with 1 % Mono and diglycerides (MDG) can be used as an egg substitute in cake formulations. This combination not only recapitulated physicochemical properties, such as specific volume, texture, and specific gravity, of the traditional cake, it also proved to be less costly. Changes in these attributes are most likely derived from the changes in the molecular structure (nanostructure). Hence, authors characterized nanoscale structures formed by gluten proteins, such as gliadin and glutenin, isolated from cake samples. By using AFM (TT-AFM), they identified that gliadin proteins formed aggregates with a similar size distribution between traditional cakes and SPI + 1% MDG cakes. Five other combinations tested as egg alternatives showed significant variations. The images of glutenin networking nanostructures also indicated that SPI + 1% MDG combination has the most similarity to traditional cake (Fig. 5).
Figure 5. AFM imaging of gliadin and glutenin nanostructures in cakes by using TT-AFM. (a) AFM image of gliadin aggregates; (b) Size distribution of gliadin aggregates; Porous structures of glutenin in (c) traditional cake and eggless cakes with (d) SPI + 0.1% XN, (e) SPI + 1% MDG, (f) SPI + 1% SL, (g) SPI + 0.1% XN + 1% MDG, (h) SPI + 0.1% XN + 1% SL. SPI – soy protein isolate; XN – xanthan gum; MDG – mono, diglycerides; SL – soy lecithin. Within each size range, groups with different letters are significantly different (p < 0.05) (taken from ref. 18).
In another study by the same group, authors aimed to develop egg substitutes for cakes via testing various combinations of pea proteins and plant polysaccharide mixture.19 They found that a cake recipe containing pea protein with 0.1% xanthan gum and 1% soy lecithin instead of an egg has comparable physical properties with a traditional cake. AFM study again enabled the researchers to compare nanostructures of gliadin and glutenin proteins in control and test samples (cakes).
Most of the commercial gelatin available originates from mammalian (porcine sources), which limits its use in halal and kosher certified food. Hence, it is essential to find its substitute given the size of the market. Although gelatin from warm water fishes has similar properties to pork gelatin, there are important differences in such as gel strength, melting temperature (Tm), and gelling temperature (Tg). Thermal and rheological properties are suboptimal when compared to pork gelatin due to subtle differences in chemical composition. And these attributes have commercial value. Yang research group engineered fish (tilapia) gelatin with two different approaches to have the same physicochemical properties as porcine gelatin. In the first approach, they mixed fish gelatin with either of the polysaccharides low-acyl gellan or k-carrageenan, and salts (CaCl2 or KCl). The formulation of gelatin with gellan and CaCl2 turned out to mimic the pork gelatin most in terms of gel strength and Tm. AFM imaging (AFMWorkshop) unveiled another layer of similarity at the nanoscale structure which might be behind physicochemical properties. Both formulations had similar aggregate size distribution. In the second approach, researchers combined fish gelatin with various concentrations of alginate. However, none of the combinations, in that case, revealed similar textural or nanoaggregate properties to pork gelatin. For similar purposes, in the third study from the same group, they mixed tilapia gelatin with polysaccharide gellan and CaCl2.20 This mixture had comparable textural characteristics such as strength, hardness, cohesiveness, chewiness with commercial beef gelatin. Nanostructural analysis with AFM showed that mixing gelatin with gellan and calcium chloride decreased the diameter of spherical nanoaggregates from 472 to 249 nm. This value is closer to what is observed with beef gelatin (272 nm).
4.4 Food processing
Fish balls are widely utilized in certain regions of the world. However, a limited supply of fish such as the yellowtail fusilier that is used in making fish balls renders them expensive to utilize. The hard texture of certain fish such as golden pomfret makes them unsuitable for manufacturing fish balls. Feng et al. investigated whether the addition of gelatin into regular fish ball processing will make the golden pomfret appropriate for the job.21 They examined myofibril nanostructure, texture properties, and mass transfer. Hardness and chewiness, the texture properties, decreased with boiling time as well as gelatin addition. According to AFM imaging, the myofibrils before and after processing displayed rod-like shapes (Fig. 6). However, their size diminished as per boiling time indicating degradation with boiling; while the length of myofibrils was longer than 15 um and width 2.7 um, after 30 min boiling these values were 11 um and 0.95 um. However, gelatin addition did not change these values significantly, suggesting gelatin does not have any effect on myofibril nanostructures.
In another approach by the same group, researchers used bromelain, an enzymatic juice that degrades proteins and obtained from pineapple, to tenderize golden pomfret (GP) fish.22 This method was considered more energy-efficient and environmentally friendly than chemical and physical methods by the authors. Bromelain treatment dampened textural parameters such as hardness and chewiness of GP so that those became similar to that of yellowtail fusilier (YF). The authors also investigated the myofibril nanostructures of fish by using AFM (Fig. 7). Rod-shaped nanostructures of GP turned into ring-like structures that are characteristics of YF after 0.4% bromelain treatment. Boiling generated granular nanostructures in both types of fish. Width, height, and length of myofibrils reduced after bromelain treatment of GP to approach the values for YF. It also became clear that 0.8% bromelain treatment tenderized fish too much as both macroscale textural parameters and nanoscale myofibril dimensions indicated.
Surimi is a paste or gel of proteins obtained by processing fish (or other meat) to remove unwanted contents. Conventional processing method that relies on multiple washings has low yields. Hence, a pH shift method has been proposed that exploits the pH sensitivity of proteins. However, the high lipid content can still pose a problem due to lipid oxidation, unwanted odors, and quality deterioration in one of the widely used fish – Tilapia. Zhou et al. successfully tried to optimize the pH shift processing method by using calcium ions and citric acid to lower lipid content in the final product.23 AFM was used to study the changes in myofibril nanostructure during this process that may reveal the mechanism behind the changes at macroscale properties. Higher gel strength observed with surimi products processed with the novel method was probably caused due to the smaller height of myofibril protein nanostructures as measured with AFM imaging.
As the fish gelatin is considered as an alternative to the gelatin from mammalian sources, it is necessary to understand its interaction with basic food components such as sugar and salt. Sow & Yang studied the effects of 1.5% NaCl and 1.5% sucrose on gelatin obtained from tilapia.24 NaCl induced the formation of spherical nanoaggregates with larger diameters when compared to untreated gelatin as revealed by using AFM (TT-AFM, AFM Workshop). It is associated with reduced gel strength in NaCl-treated gelatin. On the other hand, sucrose did not cause significant changes both physiochemically and structurally.
Figure 6. AFM imaging of myofibrils extracted from fish balls by using TT-AFM (AFM Workshop). (A) before boiling; fish balls boiled for 10 min with 0 g (B), 0.75 g (C), 1.5 g (D) and 3 g (E) of added gelatin in 100 g of fish; fish balls boiled for 20 min with 0 g (F), 0.75 g (G), 1.5 g (H) and 3 g (I) of added gelatin in 100 g of fish; fish ball boiled for 30 min with 0 g (J), 0.75 g (K), 1.5 g (L) and 3 g (M) of added gelatin in 100 g of fish (taken from ref. 21).
Figure 7. AFM images of myofibrils from different groups by using TT-AFM (AFM Workshop): a. YF, b. GP, c. GP + 0.4% bromelain (BML) and d. GP + 0.8% BML before boiling (taken from ref. 22).
4.5 Processing of agricultural products
Efficient processing of agricultural products is necessary to achieve a green and sustainable economy. Degradation and fractionation of lignocellulose cell walls economically and effectively to produce useful molecules would be a good example.25 Chen et al. discovered such a method that uses the chemical p-toluenesulfonic acid (p-TsOH) and the heat treatment (≤80°C) for 20 min to process wood.25 Processing of poplar wood with this method yielded two fractions, a cellulose-rich water-insoluble solid fraction and a spent acid liquor stream containing mainly dissolved lignin. Both fractions could be used to obtain high-value materials. Researchers investigated the possibility of producing lignin nanoparticles via dilution of the second fraction. Lignin nanoparticles have many potential applications as they are biodegradable and have large surface area with anti-oxidant, UV light absorbing, and hydrophobic properties.26 For example, they could be used as a co-polymer in rubber and other composites, or as vehicles for drug delivery. Thus obtaining lignin nanoparticles from wood could be a valuable support for economic development in rural agricultural communities.26 Investigation of the second fraction mentioned above by using AFM demonstrated the formation of lignin nanoparticles via self-assembly of dissolved lignin (Fig. 8).25 Nanoparticles presented themselves as aggregates and stand-alone structures as well (Fig. 8a, 8b). Another achievement by the authors was to fractionate the nanoparticles according to their sizes by using centrifugation. Centrifugation at increasing speeds allowed to exclude the nanoparticles of smaller sizes from the supernatant, the content of which is analyzed by AFM (Fig. 8c-8f).
A similar technique was also applied to the processing of wheat straw by the same research group. Wheat straw is an abundant and renewable agricultural output that is not utilized well and even creates a disposal problem.27 On-farm valorization of wheat straw to produce high-value materials might bring sustainable economic development in rural areas. As with wood processing, wheat straw also gave rise to two fractions. Here, AFM was employed also to characterize the cellulose-rich water-insoluble solid (WIS) fraction.27 Various processing conditions were tested to understand the effects of process parameters on the properties of lignocellulose nanofibrils that could be imaged with AFM (Fig. 9). In order to study the effects of delignification, which is dependent on acid concentration, two processing batches that were treated with either 25% or 35% p-TsOH were compared (9A, 9C). Higher delignification resulted in fibrils of smaller diameter (Fig. 9A, 9C, 9D). And increasing the grinding cycles of the WIS fraction also gave rise to the finer fibrils with less entanglement (9B, 9C, 9D). As with the aforementioned example, AFM was highly indispensable to characterize the nanomaterials obtained with the processing of agricultural products and optimize the processing conditions to fine-tune the properties of these nanomaterials.
Cellulose nanocrystals and nanofibrils have promising properties, such as large surface area, high aspect ratio, high Young’s modulus, lightweight, abundance, low thermal expansion, optical transparency, renewability, biodegradability, and low toxicity. These properties give them wide-ranging applicability from polymer reinforcement to drug delivery. Jia et al. used AFM imaging to compare the cellulose nanostructures obtained from three different sources - bleached eucalyptus pulp (BEP), spruce dissolving pulp (SDP), and cotton-based qualitative filter paper (QFP) – and observe the effects of changing processing conditions.28 Nanocrystals from different sources had different sizes. For example, nanocrystals of SDP were about 100-200 nm shorter than those of BEP and QFP. Processing conditions impacted the size of crystals as revealed by AFM measurements. Increasing oxalic acid concentration from 30% to 50% made nanocrystals from all three sources have smaller dimensions. On the other hand, the authors also investigated cellulose nanofibrils from the three sources. It appeared that nanofibrils from BEP were not affected by acid treatment and cycles of microfluidization. However, fibrils from the other two sources became finer and less aggregated as a result of the mentioned processes.
Figure 8. . AFM (AFMWorkshop) images of lignin nanoparticles (LNPs) obtained by poplar wood processing. A) AFM images of LNPs in a diluted spent p-TsOH liquor of 10 wt % that is produced using milled poplar wood at p-TsOH concentration of 75 wt % at 80°C for 20 min. (B) Topographical height profiles corresponding to lines 1 and 2 in (A). (C to E) AFM images of LNPs in the supernatant from the centrifuged sample in (A) at different speeds for 10 min, at 3000g (C), at 10,000g (D), and at 15,000g (E). (F) AFM-measured topographical height distributions of samples shown in (C) to (E) (taken from ref. 25).
Figure 9.Tapping mode AFM imaging (CS-3230, AFM Workshop) was used to analyze the water-insoluble solid (WIS) fraction obtained from wheat straw processing. The lignocellulose nanofibrils (LCNF) show varying features according to the processing condition used: A) P25T90t120N8; B) P35T90t120N4; C) P35T90t120N8 (P for p-TsOH concentration (25-35%), T for processing temperature (90 °C), t for processing time (120 min), N for number of WIS grinding cycles (4-8 times); D) AFM measured LCNF height distributions (taken from ref. 27).
4.6 Anti-microbial / Anti-parasitic effects of agricultural products
The extract of the neem tree (Azadirachta indica A. Juss) is known to have anti-microbial and anti-parasitic effects.29 However, our understanding of its role against virulence factors such as biofilm formation and planktonic aggregation is limited. Quelemes et al. studied the effects of neem leaf ethanolic extract on biofilms and planktonic aggregations of pathogenic methicillin-resistant Staphylococcus aureus (MRSA).29 AFM images revealed that the aggregation capability of the MRSA reduced depending on the concentration of the extract (Fig. 10). These results indicate that the neem leaf extract can interfere with the aggregation and biofilm formation process of MRSA.
Figure 10. AFM imaging to study the effect of Neem EE sub-MIC concentrations on USA100-MRSA planktonic aggregation. A) Control (non-treated), B) 250 µg/mL, C) 500 µg/mL, and D) 1000 µg/mL. In all images, the X and Y axes are 40 µm; the Z axis is 2.2 µm. TT-AFM from AFM Workshop was used in vibrating mode to take the images (taken from ref. 29).
AFM has been also helpful to determine the anti-fungal effects of agricultural products. Terminalia fagifolia Mart is an endemic plant to Brazil and used by the local population for treating several diseases such as oral lesions caused by the strains of Candida, a fungus.30 To pinpoint these effects, researchers produced ethanolic extracts from the plant and applied the aqueous fraction obtained from the extracts on Candida albicans. AFM was used to study the morphology of the fungal cells upon the treatment (Fig. 11).30 The cells treated with the extracts displayed a radical (destructive) change in morphology apart from the reduction in cell density, while untreated cells showed standard morphological characteristics such as particular shape, smooth cell surfaces, and the presence of hyphae (Fig. 11). The data proved the anti-fungal potential of Terminalia fagifolia Mart plants.
The same group also investigated the antimicrobial effects of Terminalia fagifolia on the bacterial pathogen Staphylococcus aureus in another study.31 The methicillin-resistant strain of this species causes lethal infections through their ability to form biofilms. Researchers showed that the extract of Terminalia fagifolia inhibit biofilm formation significantly. AFM imaging revealed the morphological changes in S. aureus cells upon treatment with the extract. Among these changes, cell membranes had lost their smooth shape and become rough, on the other hand, cells which retained their shape had increased size when compared to the untreated group.
Figure 11. AFM height images of Candida albicans cells untreated (A, B) or treated with aqueous fraction of Terminalia fagifolia extracts (C, D). In B and D, the images were obtained via scanning zoomed in areas on A and C, respectively. TT-AFM (AFM Workshop, USA) in vibrating mode has been used to obtain the images (taken from ref. 30).
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