Phenolic Compounds Profile and Antioxidant Capacity of Plant-Based Protein Supplements.

The study aimed to determine the phenolic content and antioxidant capacity of five protein supplements of plant origin. The content and profile of phenolics were determined using the UHPLC-DAD-MS method, while antioxidant capacity (ABTS and DPPH assays) and total phenolic content (TPC) were evaluated using spectrophotometric tests. In the analyzed proteins, twenty-five polyphenols were detected, including eleven phenolic acids, thirteen flavonoids, and one ellagitannin. Hemp protein revealed the highest individual phenolics content and TPC value (1620 µg/g and 1.79 mg GAE/g, respectively). Also, hemp protein showed the highest antioxidant activity determined via ABTS (9.37 µmol TE/g) and DPPH (9.01 µmol TE/g) assays. The contents of p-coumaric acid, m-coumaric acid, kaempferol, rutin, isorhamnetin-3-O-rutinoside, kaempferol-3-O-rutinoside, and TPC value were significantly correlated with antioxidant activity assays. Our findings indicate that plant-based protein supplements are a valuable source of phenols and can also be used in research related to precision medicine, nutrigenetics, and nutrigenomics. This will benefit future health promotion and personalized nutrition in the prevention of chronic diseases.

Fabrication of an Antifouling Surface Plasmon Resonance Sensor with Stratified Zwitterionic Peptides for Highly Efficient Detection of Peanut Allergens in Biscuits.

Peanut allergen monitoring is currently an effective strategy to avoid allergic diseases, while food matrix interference is a critical challenge during detection. Here, we developed an antifouling surface plasmon resonance sensor (SPR) with stratified zwitterionic peptides, which provides both excellent antifouling and sensing properties. The antifouling performance was measured by the SPR, which showed that stratified peptide coatings showed much better protein resistance, reaching ultralow adsorption levels (<5 ng/cm2). Atomic force microscopy was used to further analyze the antifouling mechanism from a mechanical perspective, which demonstrated lower adsorption forces on hybrid peptide coatings, confirming the better antifouling performance of stratified surfaces. Moreover, the recognition of peanut allergens in biscuits was performed using an SPR with high efficiency and appropriate recovery results (98.2-112%), which verified the feasibility of this assay. Therefore, the fabrication of antifouling sensors with stratified zwitterionic peptides provides an efficient strategy for food safety inspection.

A comprehensive review on pulse protein fractionation and extraction: processes, functionality, and food applications.

The increasing world population requires the production of nutrient-rich foods. Protein is an essential macronutrient for healthy individuals. Interest in using plant proteins in foods has increased in recent years due to their sustainability and nutritional benefits. Dry and wet protein fractionation methods have been developed to increase protein yield, purity, and functional and nutritional qualities. This review explores the recent developments in pretreatments and fractionation processes used for producing pulse protein concentrates and isolates. Functionality differences between pulse proteins obtained from different fractionation methods and the use of fractionated pulse proteins in different food applications are also critically reviewed. Pretreatment methods improve the de-hulling efficiency of seeds prior to fractionation. Research on wet fractionation methods focuses on improving sustainability and functionality of proteins while studies on dry methods focus on increasing protein yield and purity. Hybrid methods produced fractionated proteins with higher yield and purity while also improving protein functionality and process sustainability. Dry and hybrid fractionated proteins have comparable or superior functionalities relative to wet fractionated proteins. Pulse protein ingredients are successfully incorporated into various food formulations with notable changes in their sensory properties. Future studies could focus on optimizing the fractionation process, improving protein concentrate palatability, and optimizing formulations using pulse proteins.

Screening of α-amylase/trypsin inhibitor activity in wheat, spelt and einkorn by high-performance thin-layer chromatography.

α-Amylase/trypsin inhibitor proteins (ATI) are discussed as possible triggers for non-celiac gluten sensitivity. The potential of high-performance thin-layer chromatography (HPTLC) was studied for the first time to analyse the inhibitory properties of ATIs from flour of wheat, spelt, and einkorn. Inhibition by each flour of the digestive enzymes trypsin or α-amylase was determined by the reduction of released metabolisation products in comparison to non-digested flour, and positive (acarbose) and negative (water) controls. Firstly, amylolysis was carried out in miniaturized form on the HPTLC surface (HPTLC-nanoGIT) after in-vial pre-incubation of the amylase with the inhibitors from flour. α-Amylase inhibition was evident via the reduction of released saccharides, as analysed by normal phase HPTLC. A strong influence of the flour matrix on the assay results (individual saccharides) was evident, caused by an increased amylolysis of further polysaccharides present, making HPTLC analysis more reliable than currently used spectrophotometric sum value assays. The detection and visualization of such matrix influence helps to understand the problems associated with spectrophotometric assays. Only maltotriose was identified as a reliable marker of the amylolysis. The highest α-amylase inhibition and thus the lowest saccharide response was detected for maltotriose in refined spelt, whereas the lowest α-amylase inhibition and thus the highest saccharide response was detected for maltotriose in refined wheat. A comparison of refined and whole grain flours showed no clear trend in the responses. Secondly, trypsin inhibition and proteolysis were performed in-vial, and any inhibition was evident via the reduction of released peptides, analysed by reversed-phase HPTLC. Based on the product pattern of the proteolysis, einkorn and whole wheat showed the highest trypsin inhibition, whereas refined wheat and refined spelt showed the lowest inhibition. Advantageously, HPTLC analysis provided important information on changes in individual saccharides or peptides, which was more reliable and sustainable than spectrophotometric in-vial assays (only sum value) or liquid column chromatography analysis (targeting only the ATI proteins).

Seaweed Proteins: A Step towards Sustainability?

This review delves into the burgeoning field of seaweed proteins as promising alternative sources of protein. With global demand escalating and concerns over traditional protein sources' sustainability and ethics, seaweed emerges as a viable solution, offering a high protein content and minimal environmental impacts. Exploring the nutritional composition, extraction methods, functional properties, and potential health benefits of seaweed proteins, this review provides a comprehensive understanding. Seaweed contains essential amino acids, vitamins, minerals, and antioxidants. Its protein content ranges from 11% to 32% of dry weight, making it valuable for diverse dietary preferences, including vegetarian and vegan diets. Furthermore, this review underscores the sustainability and environmental advantages of seaweed protein production compared to traditional sources. Seaweed cultivation requires minimal resources, mitigating environmental issues like ocean acidification. As the review delves into specific seaweed types, extraction methodologies, and functional properties, it highlights the versatility of seaweed proteins in various food products, including plant-based meats, dairy alternatives, and nutritional supplements. Additionally, it discusses the potential health benefits associated with seaweed proteins, such as their unique amino acid profile and bioactive compounds. Overall, this review aims to provide insights into seaweed proteins' potential applications and their role in addressing global protein needs sustainably.

Wheat-Based Glues in Conservation and Cultural Heritage: (Dis)solving the Proteome of Flour and Starch Pastes and Their Adhering Properties.

Plant-based adhesives, such as those made from wheat, have been prominently used for books and paper-based objects and are also used as conservation adhesives. Starch paste originates from starch granules, whereas flour paste encompasses the entire wheat endosperm proteome, offering strong adhesive properties due to gluten proteins. From a conservation perspective, understanding the precise nature of the adhesive is vital as the longevity, resilience, and reaction to environmental changes can differ substantially between starch- and flour-based pastes. We devised a proteomics method to discern the protein content of these pastes. Protocols involved extracting soluble proteins using 0.5 M NaCl and 30 mM Tris-HCl solutions and then targeting insoluble proteins, such as gliadins and glutenins, with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, and 75 mM DTT. Flour paste's proteome is diverse (1942 proteins across 759 groups), contrasting with starch paste's predominant starch-associated protein makeup (218 proteins in 58 groups). Transformation into pastes reduces proteomes' complexity. Testing on historical bookbindings confirmed the use of flour-based glue, which is rich in gluten and serpins. High levels of deamidation were detected, particularly for glutamine residues, which can impact the solubility and stability of the glue over time. The mass spectrometry proteomics data have been deposited to the ProteomeXchange, Consortium (http://proteomecentral.proteomexchange.org) via the MassIVE partner repository with the data set identifier MSV000093372 (ftp://MSV000093372@massive.ucsd.edu).

Two-Dimensional Gel Electrophoresis for Protein Separation of Plant Samples.

This chapter provides a description of the procedure for two-dimensional electrophoresis that can be performed for any given gel size and isoelectric focusing range. This will enable the operator to recognize critical steps and gain sufficient information to generate 2D images suitable for computer-assisted analysis of 2D-gel, as well as mass spectrometry analysis for protein identification and characterization.

Indophenol Blue Colorimetric Method to Determine Grain Protein Content of Cereal Plants.

Here, we propose a method to convert the organic nitrogen in maize kernels into ammonia in solution and then chlorinate it to prepare monochloride salts, which can form an oxidatively coupled blue-green mixture with sodium salicylate and sodium dichloroisocyanurate. The concentration of ammonium ions in the blue-green mixture can then be determined in the solution, and finally the protein content in maize kernels can be calculated from the nitrogen content.

One-Dimensional Acrylamide Gel Electrophoresis for Analysis of Plant Samples.

Polyacrylamide gel electrophoresis (PAGE) is a widely used technique for separating proteins from complex plant samples. Prior to the analysis, proteins must be extracted from plant tissues, which are rather complex than other types of biological material. Different protocols have been applied depending on the protein source, such as seeds, pollen, leaves, roots, and flowers. Total protein amounts must also be determined before conducting gel electrophoresis. The most common methodologies include PAGE under native or denaturing conditions. Both procedures are used consequently for protein identification and characterization via mass spectrometry. Additionally, various staining procedures are available to visualize protein bands in the gel, facilitating the software-based digital evaluation of the gel through image acquisition.

Sample Preparation and Phosphopeptide Enrichment for Plant Phosphoproteomics via Label-Free Mass Spectrometry.

Phosphopeptide enrichment is the main bottleneck of every phosphorylation study. Therefore, in this chapter, a general workflow tries to overbridge the hurdles of plant sample handling from sample collection to protein extraction, protein solubilization, enzymatic digestion, and enrichment step prior to mass spectrometry. The workflow provides information to perform global proteomics as well as phosphoproteomics enabling the researcher to use the protocol in both fields.

Biomarker profiling in plants to distinguish between exposure to chlorine gas and bleach using LC-HRMS/MS and chemometrics.

Since its first employment in World War I, chlorine gas has often been used as chemical warfare agent. Unfortunately, after suspected release, it is difficult to prove the use of chlorine as a chemical weapon and unambiguous verification is still challenging. Furthermore, similar evidence can be found for exposure to chlorine gas and other, less harmful chlorinating agents. Therefore, the current study aims to use untargeted high resolution mass spectrometric analysis of chlorinated biomarkers together with machine learning techniques to be able to differentiate between exposure of plants to various chlorinating agents. Green spire (Euonymus japonicus), stinging nettle (Urtica dioica), and feathergrass (Stipa tenuifolia) were exposed to 1000 and 7500 ppm chlorine gas and household bleach, pool bleach, and concentrated sodium hypochlorite. After sample preparation and digestion, the samples were analyzed by liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). More than 150 chlorinated compounds including plant fatty acids, proteins, and DNA adducts were tentatively identified. Principal component analysis (PCA) and linear discriminant analysis (LDA) showed clear discrimination between chlorine gas and bleach exposure and grouping of the samples according to chlorine concentration and type of bleach. The identity of a set of novel biomarkers was confirmed using commercially available or synthetic reference standards. Chlorodopamine, dichlorodopamine, and trichlorodopamine were identified as specific markers for chlorine gas exposure. Fenclonine (Cl-Phe), 3-chlorotyrosine (Cl-Tyr), 3,5-dichlorotyrosine (di-Cl-Tyr), and 5-chlorocytosine (Cl-Cyt) were more abundantly present in plants after chlorine contact. In contrast, the DNA adduct 2-amino-6-chloropurine (Cl-Ade) was identified in both types of samples at a similar level. None of these chlorinated biomarkers were observed in untreated samples. The DNA adducts Cl-Cyt and Cl-Ade could clearly be identified even three months after the actual exposure. This study demonstrates the feasibility of forensic biomarker profiling in plants to distinguish between exposure to chlorine gas and bleach.

[Analysis of the electrophoretic profiles of the protein extracts of the species Manihot esculenta (Crantz, 1975), Persea Americana (Mill., 1768) and Actinidia delicious (A. Chevalier., 1984), and their association with the latex-fruit syndrome for searching for potential allergens]. / Análisis de los perfiles electroforéticos de los extractos proteicos de las especies Manihot esculenta (Crantz,1975), Persea americana (Mill.,1768) y Actinidia deliciosa (A. Chevalier., 1984), y su asociación con el síndrome látex-fruta para la búsqueda de alérgenos potenciales.

OBJECTIVE: Determine the electrophoretic profiles of the extracts of Manihot esculenta, Actinidia Deliciosa and Persea Americana and their possible relationship with Latex-Fruit Syndrome. METHODS: Protein extracts of M. esculenta, P. Americana and A. Deliciosa were prepared through the processes of maceration and solvent extraction from plant samples. In the case of the avocado, a prior extraction by soxhlet was carried out to eliminate the fat. The extracts were vacuum filtered, dialyzed and finally lyophilized. Separation of proteins based on molecular weight was performed by SDS PAGE electrophoresis. The electrophoretic profiles obtained were compared with the allergenic proteins previously identified in the latex extract, in order to determine a possible relationship with Latex-Fruit Syndrome, depending on the molecular weight. RESULTS: The extracts of M. esculenta and P. Americana showed a wide range of protein fractions with molecular weights varying from 10 to 250 KD, finding that the region with the highest concentration of bands was between 20 and 89 KD, (60 and 65%), respectively. A 20-band profile was obtained for the M. esculenta extract (Figure 1), with seven bands sharing similar weights with the latex allergens (Hev b 1, Hev b 2, Hev b3, Hev b 4, Hev b 5, Hev b 6.03, Hev b 8 and Hev b 10) (3-5). For the P. Americana extract, 20 bands were also observed (Figure 2), seven of which presented approximate weights to the Latex allergens (Hev b 1, Hev b 2 Hev b 4 Hev b 6.01 Hev b 6.03 Hev b 8 , Hev b 10 Hev b 11 Hev b 14). The Kiwi extract showed two bands of 19.1 and 22.9 KD, with weights close to latex proteins (figure 3), (Hev b 3 and Hev b 6.01), and allergens (Act d 2 and Act d 6), reported in the literature for this fruit. CONCLUSIONS: When analyzing the relationship between the separated protein fractions and the latex allergens described in the literature, a possible association of 35% was found for the extracts of M. esculenta and P. Americana, and 10% for A. Delicious, with great relevance being the association found with the allergens Hev b 4, Hev b 2, Hev 8 and Hev b 11, which are involved in Latex-Fruit Syndrome. The electrophoretic profiles of the prepared extracts were determined and compared with the Latex allergens. This information generates a contribution for the development of new research and advances in the standardization of these extracts on a large scale and for their future use in diagnostic tests.

OBJETIVO: Determinar los perfiles electroforéticos de los extractos de Manihot esculenta, Actinidia deliciosa y Persea americana y su posible relación con el Síndrome de Látex ­ Fruta. MÉTODOS: Se prepararon extractos proteicos de M. esculenta, P. Americana y A. Deliciosa, a través de los procesos de macerado y extracción con solventes a partir muestras vegetales. En el caso del aguacate, se realizó una extracción previa por soxhlet, para eliminar la grasa. Los extractos se filtraron al vacío, se sometieron a diálisis y por último se liofilizaron. La separación de las proteínas en función del peso molecular se realizó mediante electroforesis SDS PAGE. Se compararon los perfiles electroforéticos obtenidos con las proteínas alergénicas previamente identificadas en el extracto de látex, con el fin de determinar una posible relación con el Síndrome de Látex-Fruta, en función del peso molecular. RESULTADOS: Los extractos de M. esculenta y P. americana mostraron una amplia gama de fracciones proteicas con pesos moleculares que varían desde 10 a 250 KD, encontrando que la región con mayor concentración de bandas se situó entre 20 y 89 KD, (60 y 65 %), respectivamente. Se obtuvo un perfil de 20 bandas para el extracto de M. esculenta (figura 1), con siete bandas que comparten pesos similares con los alérgenos del látex (Hev b 1, Hev b 2, Hev b3, Hev b 4, Hev b 5, Hev b 6.03, Hev b 8 y Hev b 10) (3-5). Para el extracto de P. americana, también se observaron 20 bandas (figura 2), siete de las cuales presentaron pesos aproximados a los alérgenos de Látex (Hev b 1, Hev b 2 Hev b 4 Hev b 6.01 Hev b 6.03 Hev b 8, Hev b 10 Hev b 11 Hev b 14). El extracto de Kiwi mostró dos bandas de 19,1 y 22,9 KD, con pesos cercanos a proteínas de látex (figura 3), (Hev b 3 y Hev b 6.01), y los alérgenos (Act d 2 y Act d 6), reportados en la literatura para esta fruta. CONCLUSIONES: Al analizar la relación existente entre las fracciones proteicas separadas y los alérgenos de los látex descritos en la literatura, se encontró una posible asociación del 35% para los extractos de M. esculenta y P. Americana, y del 10% para A. Deliciosa, siendo de gran relevancia la asociación encontrada con los alérgenos Hev b 4, Hev b 2, Hev 8 y Hev b 11, los cuales se encuentran implicados en el Síndrome de Látex-Fruto. Se lograron determinar los perfiles electroforéticos de los extractos elaborados y se compararon con los alérgenos del Látex. Está información genera un aporte para el desarrollo de nuevas investigaciones y avances en la estandarización de estos extractos a gran escala y para su uso futuro en pruebas diagnósticas.

Effect of dehulling and germination on the functional properties of grass pea (Lathyrus sativus) flour.

The compositional, bioactive, functional, pasting, and thermal characteristics of native, dehulled, and germinated grass pea flour were examined. Germination significantly improved the protein content and bioactive properties while simultaneously reducing total carbohydrate and fat levels. However, dehulling increased the fat content, foaming, and emulsion properties. Dehulling and germination significantly increased (p < 0.05) the functional properties by improving flowability and cohesiveness. Although processing methods enhance functional properties, the pasting properties of dehulled and germinated flours differ significantly (p < 0.05) from the native flour. The X-ray diffraction patterns indicate a reduction in percentage crystallinity in germinated flours. Overall, the study suggests that the dehulling and germination processes enhanced the quality of grass peas by improving nutritive value and functional attributes.

Ratiometric Fluorescence Aptasensor of Allergen Protein Based on Multivalent Aptamer-Encoded DNA Flowers as Fluorescence Resonance Energy Transfer Platform.

Life-threatening allergic reactions to food allergens, particularly peanut protein Ara h1, are a growing public health concern affecting millions of people worldwide. Thus, accurate and rapid detection is necessary for allergen labeling and dietary guidance and ultimately preventing allergic incidents. Herein, we present a novel ratiometric fluorescence aptasensor based on multivalent aptamer-encoded DNA flowers (Mul-DNFs) for the high-stability and sensitive detection of allergen Ara h1. The flower-shaped Mul-DNFs were spontaneously packaged using ultralong polymeric DNA amplicons driven by a rolling circle amplification reaction, which contains a large number of Ara h1 specific recognition units and has excellent binding properties. Furthermore, dual-color fluorescence-labeled Mul-DNFs probes were developed by hybridizing them with Cy3- and Cy5-labeled complementary DNA (cDNA) to serve as a ratiometric fluorescence aptasensor platform based on fluorescence resonance energy transfer. Benefiting from the combined merits of the extraordinary synergistic multivalent binding ability of Mul-DNFs, the excellent specificity of the aptamer, and the sensitivity of the ratiometric sensor to avoid exogenous interference. The developed ratiometric aptasensor showed excellent linearity (0.05-2000 ng mL-1) with a limit of detection of 0.02 ng mL-1. Additionally, the developed ratiometric fluorescence aptasensor was utilized for quantifying the presence of Ara h1 in milk, infant milk powder, cookies, bread, and chocolate with recoveries of 95.7-106.3%. The proposed ratiometric aptasensor is expected to be a prospective universal aptasensor platform for the rapid, sensitive, and accurate determination of food and environmental hazards.

An Evaluation of the Physicochemical Properties of Sesame Paste Produced by Ball Milling Compared against Conventional Colloid Milling.

The physicochemical characteristics and general food quality were greatly impacted by milling. In order to investigate the effect of milling technique for physicochemical properties of sesame paste of sesame paste, samples were prepared using ball mill and colloid mill by varying grinding times. The samples prepared by ball milling had the higher moisture contents (0.07% - 0.14%) than colloid milling (p < 0.05), except for colloid milling for one cycle (0.11%). The particle size curves showed the multimodal distributions. Compared to colloid milled samples, ball milled samples have smaller particle sizes and more uniform particle distribution. The L* values of samples prepared by ball milling were higher than colloid milling. The ball mill produced sesame paste with a wider range of hardness and silkier texture, and the samples made by ball milling for 30 min had the highest hardness. And the hardness of both CMS and BMS showed a decreasing trend with increasing grinding time. During ball milling, high-speed cutting and collision caused breakage of disulfide bonds, and the sesame proteins were decomposed to their subunits. In conclusions, ball milling may be an alternative and promising process for the preparation of sesame paste.

Estimation of wheat protein content and wet gluten content based on fusion of hyperspectral and RGB sensors using machine learning algorithms.

The protein content (PC) and wet gluten content (WGC) are crucial indicators determining the quality of wheat, playing a pivotal role in evaluating processing and baking performance. Original reflectance (OR), wavelet feature (WF), and color index (CI) were extracted from hyperspectral and RGB sensors. Combining Pearson-competitive adaptive reweighted sampling (CARs)-variance inflation factor (VIF) with four machine learning (ML) algorithms were used to model accuracy of PC and WGC. As a result, three CIs, six ORs, and twelve WFs were selected for PC and WGC datasets. For single-modal data, the back-propagation neural network exhibited superior accuracy, with estimation accuracies (WF > OR > CI). For multi-modal data, the random forest regression paired with OR + WF + CI showed the highest validation accuracy. Utilizing the Gini impurity, WF outweighed OR and CI in the PC and WGC models. The amalgamation of MLs with multimodal data harnessed the synergies among various remote sensing sources, substantially augmenting model precision and stability.

Protein extracts from amaranth and quinoa as novel fining agents for red wines.

Due to allergenic concerns, only pea, potato, and wheat proteins have been approved as alternatives for replacing animal-based fining agents in wines. In pursuit of other substitutes, this work aimed to determine the fining ability of amaranth (Amaranthus caudatus L.) proteins (AP) in red wine, compared to quinoa (Chenopodium quinoa Willd.) (QP) and a commercial pea protein. Phenolic and volatile composition, as well as color characteristics, were analyzed. AP was as effective as QP at decreasing condensed tannins, with AP at 50 g/hL being the most effective treatment (25.6% reduction). QP and AP produced a minor or no statistical change in the total anthocyanins and wine color intensity. They reduced the total ester concentration, but the total alcohols remained unchanged. The outcomes of AP and QP were similar, and sometimes better than the pea proteins, thus suggesting that they could be promising options for the development of novel fining agents.

Development of a Mass Spectrometry-Based Method for Quantification of Total Cashew Protein in Roasting Oil.

BACKGROUND: Food allergen cross-contact during food preparation and production is one of the causes of unintentional allergen presence in packaged foods. However, little is known about allergen cross-contact in shared frying or roasting oil, which prevents the establishment of effective allergen controls and may put allergic individuals at risk. To better understand the quantity of allergen transferred to frying oil and subsequent products, an analytical method is needed for quantifying protein in oil that has been exposed to frying/roasting conditions. OBJECTIVE: The goal of this study was to develop a parallel reaction monitoring LC-MS/MS method to quantify the amount of cashew protein in shared roasting oil. METHODS: The sample preparation method was evaluated to improve protein extractability and peptide performance. Four quantitative peptides representing cashew 2S and 11S proteins were selected as targets based on their sensitivity, heat stability, and specificity. A calibration strategy was developed to quantify the amount of total cashew protein in oil. Method performance was evaluated using a heated cashew-in-oil model system. RESULTS: The method showed high recovery in oil samples spiked with 100 or 10 parts per million (ppm) total cashew protein heated at 138 or 166°C for 2-30 min. Samples (100 ppm total cashew protein) heated for 30 min had more than 90% recovery when treated at 138°C and more than 50% when heated at 166°C. CONCLUSION: The method is fit-for-purpose for the analysis of cashew allergen cross-contact in oil. HIGHLIGHTS: A novel MS-based method was developed that can accurately quantify the amount of cashew protein present in heated oil.

Streamlining assays of glycosyltransferases activity using in vitro GT-array (i-GT-ray) platform: Application to family GT37 fucosyltransferases.

Numerous putative glycosyltransferases (GTs) have been identified using bioinformatic approaches. However, demonstrating the activity of these GTs remains a challenge. Here, we describe the development of a rapid in vitro GT-array screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates precoated with a N-terminal Halo- or a C-terminal GST-tagged GT-encoding plasmid DNA and a capture antibody. These arrays are then used for screening of transferase activities and the reactions are monitored by a luminescence GLO assay. The products formed by these reactions can be analyzed directly from the microplates by mass spectrometry. Using this platform, a total of 280 assays were performed to screen 22 putative fucosyltransferases (FUTs) from family GT37 (seven from Arabidopsis and 15 from rice) for activity toward five acceptors: non-fucosylated tamarind xyloglucan (TXyG), arabinotriose (Ara3), non-fucosylated rhamnogalacturonan I (RG-I), and RG-II from the mur1-1 Arabidopsis mutant, and the celery RG-II monomer lacking Arap and MeFuc of chain B and l-Gal of chain A. Our screen showed that AtFUT2, AtFUT5, and AtFUT10 have activity toward RG-I, while AtFUT8 was active on RG-II. Five rice OsFUTs have XyG-FUT activity and four rice OsFUTs have activity toward Ara3. None of the putative OsFUTs were active on the RG-I and RG-II. However, promiscuity toward acceptors was observed for several FUTs. These findings extend our knowledge of cell wall polysaccharide fucosylation in plants. We believe that in vitro GT-array platform provides a valuable tool for cell wall biochemistry and other research fields.

Development of validated sandwich ELISA for detecting peanut allergen Ara h 3 in food.

Peanut is an important food that can cause food allergies, often leading to moderate and severe allergic symptoms such as skin rashes, asthma, and even anaphylactic shock.Research indicates that Ara h 3 is one of the major peanut allergen. In order to establish a simple analytical method for detecting Ara h 3, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) with antibodies that were induced from purified Ara h 3. The experimental results showed that the purified Ara h 3 had good purity, and we successfully prepared capture and detection antibodies. The method established in this study exhibited high specificity and did not cross-react with soybeans, cashew nuts, and sesame. For validation, including precision, recovery and sensitivity were in good condition. We also detected the Ara h 3 in peanut related foods. Overall, the ELISA developed in this study is a reliable method for Ara h 3 detection.