Plant-based Systems Research Articles

Biosimilars - An Overview

Biosimilars are the biologic medical products highly similar to an already approved reference biologic with no clinically meaningful difference in terms of safety,purity,and potency .they are developed after the original biologics patent expires and are subjected to rigorous regulatory evaluation to ensure their efficacy and safety .Biosimilars offer a cost - effective alternative to biologics ,fostering in the pharmaceutical market and increasing patient access to treatments fo br various chronic and life threatening diseases and auto immune disorders. Biosimilars are made from the same type of sources (eg:living organisms like bacteria(e.coli, bacilius subtilis) mammalian cells lines (HEK293,NSO) plant based system (tobacco ,corn ). Biosimilars is a biologic medication.

Cytogenetic and photosynthetic responses of plants after exposure to water from a lake environment.

Plants are sensitive to environmental pollutants and are excellent organisms for genetic and physiological testing. Plant-based test systems are often used to study aquatic, aerial, and terrestrial pollution, especially Allium cepa, but studies with Tradescatia pallida specimens have gained prominence due to their sensitivity and applicability. Among the biomarkers, cytogenetic damage and chlorophyll levels are used in stress studies due to their responses to single or combined factors. The aim of this study was to evaluate cytogenetic and photosynthetic responses in T. pallida, and cytogenetic responses in A. cepa exposed to water from three sampling stations in the Juara lagoon (Municipality of Serra, ES, Brazil), collected in two sampling campaigns. The cytotoxic, genotoxic, and mutagenic potentials were analyzed using the T. pallida root tip mitosis assay and the Allium cepa test. Chloroplast pigment levels were measured in T. pallida leaves after chronic exposure to the lagoon water. The cytogenetic tests showed cytogenetic alterations at two sampling stations in at least one sampling campaign, suggesting the presence of potential pollutants, the effects of which were maximized during the rainy season. The study of photosynthetic metabolism in T. pallida showed a relationship between the levels of chloroplast pigments and the amount of nutrients present in the water.

Ecological impacts of diversified agroforestry on soil nutrients and bacterial communities in Pinus massoniana plantations in the southern subtropics

Converting forests from single-species to mixed-species planting affects soil chemical and biological properties, yet its impacts within medicinal plant-based agroforestry systems remain largely unexamined. This research assessed the soil nutrient spectrum and bacterial community composition in a monoculture Pinus massoniana (CK) and various agroforestry models: (M1) Pinus massoniana and Alpina oxyphylla, (M2) Pinus massoniana and Ficus simplicissima, (M3) Pinus massoniana and Amomum villosum, and (M4) Pinus massoniana and Curcuma longa, within both field soil and rhizosphere environments. Results showed significant (p < 0.05) improvements in soil pH and cation exchange capacity (CEC) in agroforestry systems. Agroforestry models exhibited greater variability in soil macronutrient distribution, including nitrogen, potassium, calcium, magnesium, and sulfur (N, K, Ca, Mg, S), compared to monocultures. Specifically, Curcuma longa (M4C.RS) had 46.12 % higher total N content than monoculture Pinus massoniana. Micronutrients were higher in agroforestry rhizospheres, except for total zinc, which was higher in monoculture Pinus massoniana. Bacterial community analysis revealed dominant phyla including Acidobacteriota, Proteobacteria, Actinobacteria, and Chloroflexi. Agroforestry models had higher abundance of Proteobacteria, while monoculture had higher Acidobacteriota. Alpha diversity metrics, including Chao1 and Shannon indices, indicated higher species richness and evenness in agroforestry models, particularly in the rhizosphere of Amomum villosum (M3A.RS) and Curcuma longa (M4C.RS). Phylogenetic analysis indicated greater genetic diversity in agroforestry models, in terms of species richness and phylogenetic variation especially for Proteobacteria. Cluster analysis and NMDS revealed close grouping of agroforestry models, with dbRDA showing significant associations between environmental variables (pH, CEC, and nutrient profile), emphasizing their critical role in shaping bacterial community composition, supported by Spearman correlation. Functional prediction (PICRUSt2) indicated metabolism as the predominant functional category. Therefore, transition from monoculture to agroforestry, especially with Curcuma longa (M4), significantly enhanced soil fertility and ecosystem sustainability.

CuBe: a geminivirus-based copper-regulated expression system suitable for post-harvest activation.

The growing demand for sustainable platforms for biomolecule manufacturing has fuelled the development of plant-based production systems. Agroinfiltration, the current industry standard, offers several advantages but faces limitations for large-scale production due to high operational costs and batch-to-batch variability. Alternatively, here, we describe the CuBe system, a novel bean yellow dwarf virus (BeYDV)-derived conditional replicative expression platform stably transformed in Nicotiana benthamiana and activated by copper sulphate (CuSO4), an inexpensive and widely used agricultural input. The CuBe system utilizes a synthetic circuit of four genetic modules integrated into the plant genome: (i) a replicative vector harbouring the gene of interest (GOI) flanked by cis-acting elements for geminiviral replication and novelly arranged to enable transgene transcription exclusively upon formation of the circular replicon, (ii) copper-inducible Rep/RepA proteins essential for replicon formation, (iii) the yeast-derived CUP2-Gal4 copper-responsive transcriptional activator for Rep/RepA expression, and (iv) a copper-inducible Flp recombinase to minimize basal Rep/RepA expression. CuSO4 application triggers the activation of the system, leading to the formation of extrachromosomal replicons, expression of the GOI, and accumulation of the desired recombinant protein. We demonstrate the functionality of the CuBe system in N. benthamiana plants expressing high levels of eGFP and an anti-SARS-CoV-2 antibody upon copper treatment. Notably, the system is functional in post-harvest applications, a strategy with high potential impact for large-scale biomanufacturing. This work presents the CuBe system as a promising alternative to agroinfiltration for cost-effective and scalable production of recombinant proteins in plants.

Agriculture 4.0 adoption challenges in the emerging economies: Implications for smart farming and sustainability

To ensure food security in this age of production and supply disruption, the agricultural sectors of emerging economies are gradually adopting more smart technologies to achieve sustainability. However, literature on the challenges of adopting Agriculture 4.0-based smart farming technologies is still very limited. This research, therefore, explores the contextual interrelation among the challenges to adopting Agriculture 4.0-based smart technologies in the agricultural production system from a developing country's perspective and prioritizes the identified challenges. A case study was conducted in Bangladesh, an emerging economy, where data was collected through interviews and focus group discussion sessions. A total of 21 challenges were finalized as relevant to the country's context. The Interpretive Structural Modeling (ISM) technique was deployed to develop a hierarchical structure depicting the challenges' interrelations. The challenges were later ranked based on their relevant weight using the Best-Worst Method (BWM). This study finds technological complexity, lack of collaboration among different stakeholders, inadequate support from the government, and lack of action plans to have very high driving power. Challenges such as high initial investment and operational costs, lack of skilled workforce, and farmers' resistance were found to be dependent challenges. This study is expected to contribute by providing a deeper insight into the challenges of adopting Agriculture 4.0 in emerging economies so that practitioners can take effective mitigating measures to streamline the plant-based agricultural production systems to promote food security and sustainability.

PaMYB11 promotes suberin deposition in Norway spruce embryogenic tissue during cryopreservation: A novel resistance mechanism against osmosis.

The osmotic resistance mechanism has been extensively studied in whole plants or plant tissues. However, little is known about it in embryogenic tissue (ET) which is widely used in plant-based biotechnological systems. Suberin, a cell wall aliphatic and aromatic heteropolymer, plays a critical role in plant cells against osmosis stress. The suberin regulatory biosynthesis has rarely been studied in gymnosperms. Here, PaMYB11, a subgroup 11 R2R3-MYB transcription factor, plays a key role in the osmotic resistance of Norway spruce (Picea abies) ETs during cryoprotectant pretreatment. Thus, RNA-seq, histological, and analytical chemical analyses are performed on the stable transformations of PaMYB11-OE and PaMYB11-SRDX in Norway spruce ETs. DAP-seq, Y1H, and LUC are further combined to explore the PaMYB11 targets. Activation of PaMYB11 is necessary and sufficient for suberin lamellae deposition on Norway spruce embryogenic cell walls, which plays a decisive role in ET survival under osmotic stress. Transcriptome analysis shows that PaMYB11 enhances suberin lamellae monomer synthesis by promoting very long-chain fatty acid (VLCFA) synthesis. PaPOP, PaADH1, and PaTET8L, the first two (PaADH1 and PaPOP, included) involved in VLCFA synthesis, are proved to be the direct targets of PaMYB11. Our study identified a novel osmotic response directed by PaMYB11 in Norway spruce ET, which provides a new understanding of the resistance mechanism against osmosis in gymnosperms.

Testing the hydrological performance of live pole drains (LPD) for mitigation of slope instability

Nature-based solutions (NbS) and soil bioengineering techniques have gained considerable attention due to their relevant hydrological functions and ability to mitigate slope instability. Live pole drains (LPD), a lesser-known NbS, have traditionally been deployed on slopes to drain the excess surface water and regulate the soil's water budget, making it a suitable technique for stormwater management and landslide prevention. However, neither the LPD performance as a plant-based drainage system nor its potential to regulate the soil-water budget through hydrological processes have been thoroughly studied. This paper presents a novel pilot, lab-based approach for testing the hydrological performance of LPD under different soil hydrological conditions. We built three different treatments and investigated their hydrological performance under multiple storm events. We explored how LPD regulate the soil-water budget by partitioning the water inputs (i.e., rainfall precipitation) into water outputs (i.e., surface runoff, subsurface flow, and percolation). The study revealed that LPD can effectively manage stormwater by draining excess runoff and buffering water in the soil, outperforming fallow soil. Subsurface flow and percolation were significantly higher under LPD treatments when compared to fallow ground, suggesting that the presence of an enhanced structure in the soil results in high soil hydrological performance. The presence of a secondary species with the LPD showed a more efficient hydrological performance than an LPD alone, which aligns with the current implementation of NbS fostering biodiversity. Antecedent soil moisture impacted the hydrological performance of LPD by altering the relative infiltration capacity of the soil and by potentially modifying the availability of channels for preferential flow. Our findings provide a sound basis for future research to improve our understanding of the hydrological performance of LPD for slope instability mitigation and encourage their reproduction and upscaling.

Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension

Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.

Enhanced efficacy of glycoengineered rice cell-produced trastuzumab.

For several decades, a plant-based expression system has been proposed as an alternative platform for the production of biopharmaceuticals including therapeutic monoclonal antibodies (mAbs), but the immunogenicity concerns associated with plant-specific N-glycans attached in plant-based biopharmaceuticals has not been completely solved. To eliminate all plant-specific N-glycan structure, eight genes involved in plant-specific N-glycosylation were mutated in rice (Oryza sativa) using the CRISPR/Cas9 system. The glycoengineered cell lines, PhytoRice®, contained a predominant GnGn (G0) glycoform. The gene for codon-optimized trastuzumab (TMab) was then introduced into PhytoRice® through Agrobacterium co-cultivation. Selected cell lines were suspension cultured, and TMab secreted from cells was purified from the cultured media. The amino acid sequence of the TMab produced by PhytoRice® (P-TMab) was identical to that of TMab. The inhibitory effect of P-TMab on the proliferation of the BT-474 cancer cell line was significantly enhanced at concentrations above 1 μg/mL (****P < 0.0001). P-TMab bound to a FcγRIIIa variant, FcγRIIIa-F158, more than 2.7 times more effectively than TMab. The ADCC efficacy of P-TMab against Jurkat cells was 2.6 times higher than that of TMab in an in vitro ADCC assay. Furthermore, P-TMab demonstrated efficient tumour uptake with less liver uptake compared to TMab in a xenograft assay using the BT-474 mouse model. These results suggest that the glycoengineered PhytoRice® could be an alternative platform for mAb production compared to current CHO cells, and P-TMab has a novel and enhanced efficacy compared to TMab.

Molecular farming navigates a complex regulatory landscape.

Molecular farming, the practice of engineering plants to produce recombinant proteins, presents novel challenges and opportunities for domestic markets and international trade. This article explores the multifaceted risks associated with these biotechnological advancements, including public health concerns related to recombinant animal proteins produced in plants, cross-contamination and unintended allergens, and the necessity for stringent identity preservation systems to avoid past failures. On the global stage, the trade of such genetically engineered crops brings about unique regulatory concerns, underscoring the need for internationally harmonized policies and reevaluating existing low-level presence (LLP) thresholds to address unexpected allergens. Moreover, molecular farming ventures into complex religious and ethical territories, particularly affecting communities with strict dietary laws, such as Islamic, Jewish, and those following vegan or vegetarian lifestyles. Addressing these concerns requires a collaborative approach among scientists, regulatory bodies, industry leaders, and religious figures, aiming to foster an inclusive dialogue that navigates the ethical, religious, and environmental implications of integrating animal proteins into plant-based systems. Such efforts are essential for ensuring the responsible development of molecular farming technologies, contributing to a future of sustainable, secure, and inclusive food systems that respect diverse cultural and ethical values.

LeichtPRO-profiles: development and validation of novel linear biocomposite structural components fabricated from pultruded natural flax fibres with plant-based resin for sustainable architectural applications

Reconsidering the materials used in construction is crucial within the building industry, particularly in the context of sustainability. Recently, there has been a growing interest in exploring novel materials, with fibre-reinforced composites emerging as a prominent choice with biocomposites standing out as promising for advancing sustainability goals. This paper introduces the development of LeichtPRO-Profiles, continuous linear biocomposite profiles fabricated using the pultrusion technology. A primary focus is the application of these profiles in structural systems as load-bearing elements, emphasising the significance of understanding their mechanical properties. Specifically, an original application involves active-bending structures, necessitating a focus on the material’s bending behaviour. This study discusses the methods employed in developing the pultruded biocomposite profiles which are made from natural flax fibres and an optimised matrix formulation based on a plant-based resin system. This research also outlines the optimisation of the fabrication process of these biocomposite profiles using bio-based ingredients. The results demonstrate the material’s mechanical capabilities through extensive experiments and mechanical tests, revealing a compression strength of 31.2 kN and a flexural strength of 300 MPa, with a bending radius of up to 2.4 m, indicating its suitability for structural applications. Concepts of applications in several systems across different scales and contexts are also presented. The versatility and adaptability of this product make it suitable for a wide range of applications spanning various scales and thematic contexts.

Traditional health care systems and immunity boosting: exploring plant based indigenous knowledge systems amidst the COVID-19 pandemic

The COVID-19 outbreak has highlighted the importance of utilizing traditional Ecological knowledge (TEK) to address the health risks linked to the pandemic. Mountain communities have traditionally relied on wild plant species and indigenous crops to enhance their immune system and maintain nutritional security. Recognizing the significance of indigenous knowledge systems this study was undertaken to explore plant-based traditional healthcare systems to revitalize immunity against COVID-19. A comprehensive survey and interviews of 195 local healers (Vaidyas) and residents were carried out using structured and semi-structured questionnaires following the convenience sampling method. The survey identified 40 plant species traditionally used for treating various ailments and augmenting immune functions. This study revealed a strong interest (over 80%) among the local population in plant-based remedies to enhance immunity for COVID-19. This study underscores the necessity of revitalizing and integrating diminishing traditional knowledge into mainstream healthcare practices, emphasizing the significance of region-specific approaches aligned with local resources and requirements. Therefore, there is an urgent need for exploration and recognition of the value of traditional knowledge and indigenous foods, alongside rigorous clinical trials to ascertain their role in fortifying health resilience against COVID-19.

Carotenoids encapsulated in natural flaxseed oil body: Different colloidal interfaces induce their behavior and stability disparity

Flaxseed oil bodies (FOBs) are being explored as natural plant-based delivery systems for the encapsulation and delivery of nutrients. However, there is currently a relatively poor understanding of their interfacial interaction and how this impacts delivery systems. In this study, β-carotene and lutein with far different molecular polarity were used to provide insights into their impacts on properties of FOB. In addition to the carotenoids (>93%) embedded and dissolved in the lipid core, they also interacted with the interfacial membrane components to impact the FOB properties. Raman showed the proportion of random coil and β-sheet in FOB-L (hydrophilic domain of interfacial protein) increased by 8% and 5% respectively, but remained almost unchanged in FOB-β, indicating lutein could interact with the polar exterior of interfacial membranes. Consistently, molecular dynamic simulations and spectroscopy analysis showed that lutein containing hydroxyl in β, ε-ionone ring was more likely to approach the outer hydrophilic regions of interfacial protein, while fatty soluble β-carotene interacted with hydrophobic region of inner membrane protein and phospholipid alkyl chain (close to oil). This difference in interfacial interactions induced significant discrepancies in the lipid oxidation, rheological behavior of carotenoid-FOB assemblies. Lutein was located at the outer edge where oxidation begins, making it more effective than β-carotene in improving the lipid oxidation stability of the FOB. Moreover, lutein therefore modulated interactions between oil droplets and altered the rheological properties of the FOB, whereas β-carotene had little effect. This study may facilitate the design of oil bodies-based delivery systems that have improved functional properties.

Implementation of a botanical bioscrubber for the treatment of indoor ambient air

This study explores the effectiveness of a botanical bioscrubber system using Golden Pothos (Epipremnum aureum) in hydroponic setups to mitigate common indoor atmospheric pollutants. Over a 100-day operation, levels of SO2, NO2, O3, TVOC, CO, CO2, PM10, and PM2.5 were monitored, with a significant reduction in carbon-based compounds and particulate matter-. Notably, CO2 and PM2.5 removal efficiencies were significantly correlated with the foliar area, suggesting that the interaction between pollutants and plant leaves plays a crucial role in the phytoremediation process. In contrast, CO, PM10, and TVOC exhibited varied removal efficiencies, hinting the involvement of mechanisms beyond leaf interaction, such as adsorption in irrigation water or root system capture. The absence of significant correlations for PM10 emphasized the need for further investigation into alternative removal processes, potentially mediated by the root system. Overall, our findings suggest that botanical bioscrubbers, particularly those utilizing Golden Pothos, hold promise for indoor air purification through plant-based systems.

Utilization of lysed and dried bacterial biomass from the marine purple photosynthetic bacterium Rhodovulum sulfidophilum as a sustainable nitrogen fertilizer for plant production

Plant-based agricultural systems rely heavily on inorganic nitrogen (N) fertilizers to increase yields and ensure food security for the rapidly growing global population. However, the production and overuse of synthetic fertilizers lead to significant amounts of greenhouse gas emissions, causing a critical need for the development of alternative and sustainable organic N fertilizers. Here, we demonstrate the effective use of lysed and dried bacterial biomass from the marine purple photosynthetic bacterium Rhodovulum sulfidophilum as an alternative source of N fertilizer for the cultivation of Japanese mustard spinach (komatsuna, Brassica rapa var. perviridis). To assess the suitability of this processed bacterial biomass, containing approximately 11% N, as a N fertilizer, we examined the effects of its application on plant germination and growth (measured by leaf chlorophyll, maximum leaf length, and dry weight) under two different temperature regimes, comparing it to that of a conventional N-containing mineral fertilizer. Application at rates up to four times that of the mineral fertilizer had no negative effects on seed germination and plant growth. The bacterial biomass had to be applied at approximately twice the rate of mineral fertilizer to obtain similar plant growth parameters, roughly corresponding to the predicted 62% rate of mineralization of the bacterial N. Our findings confirm the ability of plants to take up N from the lysed and dried biomass of marine purple photosynthetic bacteria, demonstrating the potential for using R. sulfidophilum as a source of N fertilizer.

Applications of physical and chemical treatments in plant-based gels for food 3D printing.

Extrusion-based three-dimensional (3D) printing has been extensively studied in the food manufacturing industry. This technology places particular emphasis on the rheological properties of the printing ink. Gel system is the most suitable ink system and benefits from the composition of plant raw materials and gel properties of multiple components; green, healthy aspects of the advantages of the development of plant-based gel system has achieved a great deal of attention. However, the relevant treatment technologies are still only at the laboratory stage. With a view toward encouraging further optimization of ink printing performance and advances in this field, in this review, we present a comprehensive overview of the application of diverse plant-based gel systems in 3D food printing and emphasize the utilization of different treatment methods to enhance the printability of these gel systems. The treatment technologies described in this review are categorized into three distinct groups, physical, chemical, and physicochemical synergistic treatments. We comprehensively assess the specific application of these technologies in various plant-based gel 3D printing systems and present valuable insights regarding the challenges and opportunities for further advances in this field.

Suborganellar resolution imaging for the localisation of human glycosylation enzymes in tobacco Golgi bodies.

Plant cells are a capable system for producing economically and therapeutically important proteins for a variety of applications, and are considered a safer production system than some existing hosts such as bacteria or yeasts. However, plants do not perform protein modifications in the same manner as mammalian cells do. This can impact on protein functionality for plant-produced human therapeutics. This obstacle can be overcome by creating a plant-based system capable of 'humanising' proteins of interest resulting in a glycosylation profile of synthetic plant-produced proteins as it would occur in mammalian systems. For this, the human glycosylation enzymes (HuGEs) involved in N-linked glycosylation N-acetylglucosaminyltransferase IV and V (GNTIV and GNTV), β-1,4-galactosyltransferase (B4GALT1), and α-2,6-sialyltransferase (ST6GAL) were expressed in plant cells. For these enzymes to carry out the stepwise glycosylation functions, they need to localise to late Golgi body cisternae. This was achieved by a protein targeting strategy of replacing the mammalian Golgi targeting domains (Cytoplasmic-Transmembrane-Stem (CTS) regions) with plant-specific ones. Using high-resolution and dynamic confocal microscopy, we show that GNTIV and GNTV were successfully targeted to the medial-Golgi cisternae while ST6GAL and B4GALT1 were targeted to trans-Golgi cisternae. Plant cells are a promising system to produce human therapeutics for example proteins used in enzyme replacement therapies. Plants can provide safer and cheaper alternatives to existing expression systems such as mammalian cell culture, bacteria or yeast. An important factor for the functionality of therapeutic proteins though are protein modifications specific to human cells. However, plants do not perform protein modifications in the same manner as human cells do. Therefore, plant cells need to be genetically modified to mimic human protein modifications patterns. The modification of importance here, is called N-linked glycosylation and adds specific sugar molecules onto the proteins. Here we show the expression of four human glycosylation enzymes, which are required for N-linked glycosylation, in plant cells. In addition, as these protein modifications are carried out in cells resembling a factory production line, it is important that the human glycosylation enzymes be placed in the correct cellular compartments and in the correct order. This is carried out in Golgi bodies. Golgi bodies are composed of several defined stacks termed cis-, medial and trans-Golgi body stacks. For correct protein function, two of these human glycosylation enzymes need to be placed in the medial-Golgi attacks and the other two in the trans-Golgi stacks. Using high-resolution laser microscopy in live plant cells, we show here that the human glycosylation enzymes are sent within the cells to the correct Golgi body stacks. These are first steps to modify plant cells in order to produce human therapeutics.

Exploring the effect of sucrose and d-allulose addition on the gelling ability and physical properties of agar–agar vegan gels

Plant-based hydrocolloid systems which are mixed with different carbohydrate types are becoming popular due to vegan concerns for both non-food and food industry. In this study, different sugar types (d-allulose and sucrose) at different concentrations (20% and 40%) were used to explore their effects on the gelation and physical properties of agar–agar. For characterization of these agar–agar-based gel systems, in addition to common physical methods (moisture content, water activity, color, hardness, viscosity), novel methods such as TD-NMR relaxometry, and FTIR were also used to get deep insight of the microstructures and water dynamics of the gel systems. Results clearly indicated that sugar type and concentration were vital factors affecting the agar–agar gel properties and dynamics of the system. Utilization of allulose in the gel systems led to formation of less moist and softer agar–agar matrix characterized with longer T2 spin–spin relaxation times. This study will lead to designing new types of vegan agar–agar-based confectionery products such as soft candies that are considered as perfect composite gels.

Plant-based delivery systems for controlled release of hydrophilic and hydrophobic active ingredients: Pea protein-alginate bigel beads

Oral delivery systems for nutrients and nutraceuticals are required for personalized nutrition applications. In some applications, these delivery systems need to encapsulate both hydrophilic and hydrophobic bioactive agents. In this study, bigel beads (2 mm) were prepared using pea protein (PP) and sodium alginate (SA) to form the hydrogel phase, and glyceryl monostearate and soybean oil to form the oleogel phase. The hydrogel phase was crosslinked by using Ca2+ ions to form electrostatic bridges between the alginate molecules. The bigel beads had an oil-in-water type structure consisting of an oleogel dispersed phase within a hydrogel continuous phase. These beads were used to encapsulate and control the release of model hydrophobic (curcumin) and hydrophilic (epigallocatechin gallate) nutraceuticals. The textural and microstructural characteristics of the bigel beads were evaluated by dynamic shear rheology, textural profile analysis, cryo-electron microscopy, and fluorescence confocal microscopy. As the concentration of SA increasing from 0.2 to 1.0%, the hardness of bigel beads increased from 306.75 g to 792.40 g. In vitro digestion experiments showed that most of the nutraceuticals were released in the small intestine, rather than the stomach. Nutraceutical release could be controlled by manipulating bead composition, with the extent of nutraceutical release decreasing with increasing alginate concentration. The bigel beads developed in this study could be used as easy-to-swallow carriers for the controlled release of hydrophilic and hydrophobic bioactive ingredients. These kinds of products may be especially useful for people with dysphagia.

Efficient Expression of Functionally Active Aflibercept with Designed N-glycans.

Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It is a highly glycosylated protein with five N-glycosylation sites that might impact it structurally and/or functionally. Aflibercept is produced in mammalian cells and exhibits large glycan heterogeneity, which hampers glycan-associated investigations. Here, we report the expression of aflibercept in a plant-based system with targeted N-glycosylation profiles. Nicotiana benthamiana-based glycoengineering resulted in the production of aflibercept variants carrying designed carbohydrates, namely, N-glycans with terminal GlcNAc and sialic acid residues, herein referred to as AFLIGnGn and AFLISia, respectively. Both variants were transiently expressed in unusually high amounts (2 g/kg fresh leaf material) in leaves and properly assembled to dimers. Mass spectrometric site-specific glycosylation analyses of purified aflibercept showed the presence of two to four glycoforms in a consistent manner. We also demonstrate incomplete occupancy of some glycosites. Both AFLIGnGn and AFLISia displayed similar binding potency to VEGF165, with a tendency of lower binding to variants with increased sialylation. Collectively, we show the expression of functionally active aflibercept in significant amounts with controlled glycosylation. The results provide the basis for further studies in order to generate optimized products in the best-case scenario.