Protein Extraction Methods Research Articles

Valorization of soursop (Annona muricata) seeds as alternative oil and protein source using novel de-oiling and protein extraction techniques

In this research, for the first time, the valorization of soursop seeds, a poorly studied biomass, was investigated to simultaneously produce oil and proteins within a biorefinery approach. A new mild protein extraction method (Pressurized Water Extraction, PWE) was developed and optimized via response surface methodology and compared with conventional alkaline extraction. Furthermore, the impact of the de-oiling technique, i.e., n-hexane or cold pressing, was investigated. The extracted proteins were evaluated in terms of physicochemical composition, amino acid content, and techno-functional properties. The type of de-oiling technique was found to have a minor impact. Instead, considerable differences were observed between the two protein extraction methodologies. At optimized conditions (pH 8.1 and 40 °C), PWE resulted in a more protein-rich extract (48 vs. 30 wt% for alkaline water), better preservation of the amino acid content, and higher solubility and in vitro digestibility. A mixed behavior was found for the techno-functional properties. Emulsification and oil holding capacity were similar for both techniques, while improved foaming was observed after alkaline water extraction. This research showed that the non-exploited soursop seeds could be a valuable source of oil and protein. Moreover, the novel PWE enables protein extraction with reduced impact on the protein structure.

Salt Solubilization Coupled with Membrane Filtration-Impact on the Structure/Function of Chickpea Compared to Pea Protein.

The demand for pulse proteins as alternatives to soy protein has been steeply increasing over the past decade. However, the relatively inferior functionality compared to soy protein is hindering the expanded use of pulse proteins, namely pea and chickpea protein, in various applications. Harsh extraction and processing conditions adversely impact the functional performance of pea and chickpea protein. Therefore, a mild protein extraction method involving salt extraction coupled with ultrafiltration (SE-UF) was evaluated for the production of chickpea protein isolate (ChPI). The produced ChPI was compared to pea protein isolate (PPI) produced following the same extraction method in terms of functionality and feasibility of scaling. Scaled-up (SU) ChPI and PPI were produced under industrially relevant settings and evaluated in comparison to commercial pea, soy, and chickpea protein ingredients. Controlled scaled-up production of the isolates resulted in mild changes in protein structural characteristics and comparable or improved functional properties. Partial denaturation, modest polymerization, and increased surface hydrophobicity were observed in SU ChPI and PPI compared to the benchtop counterparts. The unique structural characteristics of SU ChPI, including its ratio of surface hydrophobicity and charge, contributed to superior solubility at both a neutral and acidic pH compared to both commercial soy protein and pea protein isolates (cSPI and cPPI) and significantly outperformed cPPI in terms of gel strength. These findings demonstrated both the promising scalability of SE-UF and the potential of ChPI as a functional plant protein ingredient.

Optimization of Targeted Plant Proteomics Using Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS).

This study was conducted to optimize a targeted plant proteomics approach from signature peptide selection and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analytical method development and optimization to sample preparation method optimization. Three typical protein extraction and precipitation methods, including trichloroacetic acid (TCA)/acetone method, phenol method, and TCA/acetone/phenol method, and two digestion methods, including trypsin digestion and LysC/trypsin digestion, were evaluated for selected proteins related to the impact of engineered nanomaterials (ENMs) on wheat (Triticum aestivum) plant growth. In addition, we compared two plant tissue homogenization methods: grinding freeze-dried tissue and fresh tissue into a fine powder using a mortar and pestle aided with liquid nitrogen. Wheat plants were grown under a 16 h photoperiod (light intensity 150 μmol·m-2·s-1) for 4 weeks at 22 °C with a relative humidity of 60% and were watered daily to maintain a 70-90% water content in the soil. Processed samples were analyzed with an optimized LC-MS/MS method. The concentration of selected signature peptides for the wheat proteins of interest indicated that the phenol extraction method using fresh plant tissue, coupled with trypsin digestion, was the best sample preparation method for the targeted proteomics study. Overall, the optimized approach yielded the highest total peptide concentration (68,831 ng/g, 2.4 times the lowest concentration) as well as higher signature peptide concentrations for most peptides (19 out of 28). In addition, three of the signature peptides could only be detected using the optimized approach. This study provides a workflow for optimizing targeted proteomics studies.

Effect of canola meal fermentation and protein extraction method on the functional properties of resulting protein products

AbstractThe present study investigated the effect of solid‐state fermentation (SSF) of cold‐pressed (CP) and hexane‐extracted (HE) canola meals with Aspergillus niger NRRL 334 and Aspergillus oryzae NRRL 5590 on the functionalities of protein products extracted from them. After SSF, proteins were recovered using alkaline extraction‐isoelectric precipitation (AE‐IP) or salt extraction‐dialysis (SE). SSF of the two meal types reduced the protein content of the extracts produced by AE‐IP. There were varied effects to solubility, foaming, and emulsifying properties as a result of SSF under the combined influence of functionality pH, strain, meal type, and protein extraction method. The protein isolate produced from CP meal using SE had increased solubility at pH 7 (from 51.8% to 90.7%) when the meal was fermented with A. oryzae. Both strains resulted in an over twofold increase in the emulsifying activity index (at pH 7) of AE‐IP products from CP meal. For both protein extraction methods, the protein products from A. niger fermented HE meal had better foaming capacity (FC) at pH 7 than the controls (non‐fermented), but reduced FC at pH 3. Overall, regardless of meal fermentation, the SE products were richer in protein and had higher oil holding capacity (OHC), whereas the water holding capacity (WHC) was higher for AE‐IP isolates. SSF of the meals generally improved the O/WHC of the extracted proteins. The findings suggest that canola protein functionality could be effectively modulated by SSF with different microbial strains under various processing conditions to enhance their applicability in the food industry.

A Path to Protein Quantitation: PBMCs to Western Blot

Proteins are biopolymers made up of one or more long chains of amino acid residues. These proteins mediate thousands of metabolic pathways that determine how an organism functions. When proteins are thoroughly investigated, they can provide information about health and disease. A variety of methods have been developed to quantify both complex protein mixtures for total protein content as well as a single type of protein. This manuscript covers the methodologies standardized in our lab based on previously published protocols, beginning with the isolation of peripheral blood mononuclear cells (PBMCs) from human blood samples, cell viability testing, cell counting, total protein extraction methods, and protein quantification and estimation techniques, which enable a researcher to find the protocol from PBMCs to Western blot in a single manuscript.

Comparison of different protocols for protein extraction from murine ovarian tissue: A preliminary study

Objective: Extraction of proteins from tissues is a key step to study the protein structure and function. There is no set of universal protocols because of the enormous chemical and physical variety of both proteins and the sample sources. Hence, in this study a comparison of different methods for protein extraction from mouse ovarian tissue was carried out. Methods: Intravaginal dose of 20µl of Phosphate buffer saline was given to 7 mice for ten consecutive days followed by extraction of ovaries. For comparison, seven different protein extraction protocols from Protocol 1 to Protocol 7 were used. The supernatant was first quantified with Bradford assay to determine the total protein yield followed by SDS-PAGE analysis. Results: Highest protein yield of 1.13 mg/ml was attained after supplementing 1% v/v protease inhibitor cocktail in PBS. In total, 13 protein bands were detected by the extraction protocol 2 and 11 protein bands by the extraction protocol 1. 11 protein bands were detected in the range 180 kDa to 22 kDa following Protocol 3 and 4. Extraction with lysis buffer 1 resulted in 7 visible protein bands of molecular weight 182 kDa to 35 kDa range. Only 6 protein bands were observed in the range 182 kDa - 35 kDa following lysis buffer 2 method. For lysis buffer 3, ten visible protein bands were detected in range of 195 kDa - 13 kDa. Conclusion: Based on the protein extraction efficiency of Protocol 1, optimal combination of Phosphate buffer saline (pH 7.2) and protease inhibitor cocktail followed by hand-homogenization is suggested for protein extraction from mouse tissues.

Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma

Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry, requiring additional purification and buffer-exchange steps. This study compares two well-established detergent-based methods for protein extraction (in-solution sodium deoxycholate (SDC); suspension trapping (S-Trap)) with the recently developed sample preparation by easy extraction and digestion (SPEED) method, which uses strong acid for denaturation. We compared the quantitative performance of each method using label-free mass spectrometry in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED quantified the most unique proteins (SPEED 1250; S-Trap 1202; SDC 1197). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 150; SDC 148; SPEED 137). Protein quantifications were reproducible across biological replicates in both tissue (R2 = 0.85-0.90) and plasma (SPEED R2 = 0.84; SDC R2 = 0.76, S-Trap R2 = 0.65). Our data suggest SPEED as the optimal method for proteomic preparation in kidney tissue and S-Trap or SPEED as the optimal method for plasma, depending on whether a higher number of protein quantifications or greater reproducibility is desired.

Inter-Regional Proteomic Profiling of the Human Brain Using an Optimized Protein Extraction Method from Formalin-Fixed Tissue to Identify Signaling Pathways.

Proteomics offers vast potential for studying the molecular regulation of the human brain. Formalin fixation is a common method for preserving human tissue; however, it presents challenges for proteomic analysis. In this study, we compared the efficiency of two different protein-extraction buffers on three post-mortem, formalin-fixed human brains. Equal amounts of extracted proteins were subjected to in-gel tryptic digestion and LC-MS/MS. Protein, peptide sequence, and peptide group identifications; protein abundance; and gene ontology pathways were analyzed. Protein extraction was superior using lysis buffer containing tris(hydroxymethyl)aminomethane hydrochloride, sodium dodecyl sulfate, sodium deoxycholate, and Triton X-100 (TrisHCl, SDS, SDC, Triton X-100), which was then used for inter-regional analysis. Pre-frontal, motor, temporal, and occipital cortex tissues were analyzed by label free quantification (LFQ) proteomics, Ingenuity Pathway Analysis and PANTHERdb. Inter-regional analysis revealed differential enrichment of proteins. We found similarly activated cellular signaling pathways in different brain regions, suggesting commonalities in the molecular regulation of neuroanatomically-linked brain functions. Overall, we developed an optimized, robust, and efficient method for protein extraction from formalin-fixed human brain tissue for in-depth LFQ proteomics. We also demonstrate herein that this method is suitable for rapid and routine analysis to uncover molecular signaling pathways in the human brain.

Peanut oil and protein extraction using an aqueous enzymatic method and analysis of the characteristics of the emulsions produced

AbstractBackground and ObjectiveThe high cost of enzyme preparation and the stable emulsion produced in the process limit the large‐scale application of aqueous enzymatic method. In this study, a new aqueous enzymatic method for efficient peanut oil and protein extraction was optimized. And the composition, structure, and stability of the produced emulsions were characterized.FindingsPeanut emulsion proteins were mainly composed of oleosin, steroleosin, caleosin, lipoxygenase, and arachin. The composition of phospholipids in the emulsions was essentially the same as that in peanut raw materials. A surface protein wrapped the oil droplets. The ζ‐potential, particle size, and surface protein concentration properties of the second step extracted the emulsion (E2) were consistent with those produced using the conventional aqueous enzymatic method, whereas the first step extracted the emulsion (E1) was more prone to unstable aggregation between oil droplets. The mechanical properties of shear deformation resistance (viscosity, storage modulus, and loss modulus) were better in E1 than in E2.ConclusionsFollowing new method, highest yields obtained for oil and protein were 95.48% and 80.74%, respectively. The new method reduced the conditions of enzymatic hydrolysis and accelerated the demulsification of emulsions.Significance and NoveltyThe study provides a two‐step aqueous enzymatic method for extracting emulsions and proteins from peanut. Process cost and efficiencies ensure industrial scale‐up of the method.

Green Biomass-Based Protein for Sustainable Feed and Food Supply: An Overview of Current and Future Prospective.

It is necessary to develop and deploy novel protein production to allow the establishment of a sustainable supply for both humans and animals, given the ongoing expansion of protein demand to meet the future needs of the increased world population and high living standards. In addition to plant seeds, green biomass from dedicated crops or green agricultural waste is also available as an alternative source to fulfill the protein and nutrient needs of humans and animals. The development of extraction and precipitation methods (such as microwave coagulation) for chloroplast and cytoplasmic proteins, which constitute the bulk of leaf protein, will allow the production of leaf protein concentrates (LPC) and protein isolates (LPI). Obtained LPC serves as a sustainable alternative source of animal-based protein besides being an important source of many vital phytochemicals, including vitamins and substances with nutritional and pharmacological effects. Along with it, the production of LPC, directly or indirectly, supports sustainability and circular economy concepts. However, the quantity and quality of LPC largely depend on several factors, including plant species, extraction and precipitation techniques, harvest time, and growing season. This paper provides an overview of the history of green biomass-derived protein from the early green fodder mill concept by Károly Ereky to the state-of-art of green-based protein utilization. It highlights potential approaches for enhancing LPC production, including dedicated plant species, associated extraction methods, selection of optimal technologies, and best combination approaches for improving leaf protein isolation.

Identification of Relevant Protein Interactions with Partial Knowledge: A Complex Network and Deep Learning Approach.

Protein-protein interactions (PPIs) are the basis for understanding most cellular events in biological systems. Several experimental methods, e.g., biochemical, molecular, and genetic methods, have been used to identify protein-protein associations. However, some of them, such as mass spectrometry, are time-consuming and expensive. Machine learning (ML) techniques have been widely used to characterize PPIs, increasing the number of proteins analyzed simultaneously and optimizing time and resources for identifying and predicting protein-protein functional linkages. Previous ML approaches have focused on well-known networks or specific targets but not on identifying relevant proteins with partial or null knowledge of the interaction networks. The proposed approach aims to generate a relevant protein sequence based on bidirectional Long-Short Term Memory (LSTM) with partial knowledge of interactions. The general framework comprises conducting a scale-free and fractal complex network analysis. The outcome of these analyses is then used to fine-tune the fractal method for the vital protein extraction of PPI networks. The results show that several PPI networks are self-similar or fractal, but that both features cannot coexist. The generated protein sequences (by the bidirectional LSTM) also contain an average of 39.5% of proteins in the original sequence. The average length of the generated sequences was 17% of the original one. Finally, 95% of the generated sequences were true.

Isolation and Identification of Plasma Extracellular Vesicles Protein Biomarkers.

Extracellular vesicles (EVs) have emerged as a valuable source for disease biomarkers and an alternative drug delivery system due to their ability to carry cargo and target specific cells. Proper isolation, identification, and analytical strategy are required for evaluating their potential in diagnostics and therapeutics. Here, a method is detailed to isolate plasma EVs and analyze their proteomic profiling, combining EVtrap-based high-recovery EV isolation, phase-transfer surfactant method for protein extraction, and mass spectrometry qualitative and quantitative strategies for EV proteome characterization. The pipeline provides a highly effective EV-based proteome analysis technique that can be applied for EV characterization and evaluation of EV-based diagnosis and therapy.

Bidimensional Analyses of the Intra- and Extracellular Proteomes of Steroid Producer Mycobacteria.

The importance of the pathogenic mycobacteria has mainly focused the omic analyses on different aspects of their clinical significance. However, those industrially relevant mycobacteria have received less attention, even though the steroid market sales in 2021 were estimated in $56.45billion.The extracellular proteome, due to its relevance in the sterol processing and uptake, and the intracellular proteome, because of its role in steroids bioconversion, are the core of the present chapter. Both, monodimensional gels, as preparatory analysis, and bidimensional gels as proteome analysis are described. As a proof of concept, the protein extraction methods for both sub-proteomes of Mycobacterium are described. Thus, procedures and relevant key points of these proteome analyses are fully detailed.

Determination and evaluation of whey protein content in matured cheese via liquid chromatography

To date, there is no valid analytical method available to determine the total whey protein (TWP) content in cheese. Therefore, the aim of this study was to develop HPLC methods for whey protein determination in mature cheese. For that purpose, foil-ripened whey protein-enriched model cheese and traditional Edam-type cheese were produced and analyzed. Suitable protein extraction methods and subsequent analytical methods to determine the acid-soluble whey protein (ASWP) and TWP content in cheese were developed. To characterize the influence of proteolysis on native and denatured whey proteins, the ASWP and TWP contents were determined throughout the ripening process. Both chromatographic methods showed that the individual whey proteins (α-lactalbumin and β-lactoglobulin) were not degraded during ripening. However, the analyzed ASWP content increased by up to 25% throughout ripening. Compared to traditional Edam-type cheese, the β-lactoglobulin content in whey protein-enriched cheese (containing 30% high-heat milk) increased by a factor of 3.5. To evaluate the chromatographic results, two different calculation models were used to estimate a reference value for the TWP content in the manufactured cheese. Further studies are required to optimize the quantification of TWP content in hard, semihard, soft, and cream cheeses.

Proteomic Landscape of Human Spermatozoa: Optimized Extraction Method and Application

Human spermatozoa proteomics exposed to some physical, biological or chemical stressors is being explored. However, there is a lack of optimized sample preparation methods to achieve in-depth protein coverage for sperm cells. Meanwhile, it is not clear whether antibiotics can regulate proteins to affect sperm quality. Here, we systematically compared a total of six different protein extraction methods based the combination of three commonly used lysis buffers and physical lysis strategies. The urea buffer combined with ultrasonication (UA-ultrasonication) produced the highest protein extraction rate, leading to the deepest coverage of human sperm proteome (5685 protein groups) from healthy human sperm samples. Since the antibiotics, amoxicillin and clarithromycin, have been widely used against H. pylori infection, we conduct a longitudinal study of sperm proteome via data-independent acquisition tandem mass spectrometry (DIA-MS/MS) on an infected patient during on and off therapy with these two drugs. The semen examination and morphological analysis were performed combined with proteomics analysis. Our results indicated that antibiotics may cause an increase in the sperm concentration and the rate of malformed sperm and disrupt proteome expression in sperm. This work provides an optimized extraction method to characterize the in-depth human sperm proteome and to extend its clinical applications.

DEVELOPMENT OF ALLERGEN EXTRACTION METHOD IN PEEL AND PULP OF FRESH TOMATOESTHAT CAUSE ALLERGY IN HUMAN

Background: Tomatoallergens have been extensively studied but the simple and advanced extraction method from peel and pulp of fresh tomatoes has yet to be performed. Objects: To develop the proteinextraction method fromtomato peel and pulp and to investigate the allergenicity of the tomato peel and pulp extracts.Methods: Applying two different protocols and SDS-PAGE for extracting allergens from the peel and pulp of fresh tomato and performing immunoblotting them with sera of 9 allergic patients with tomato. Results: The samples extracted by thesecond protocol showed clearer and more bands than thoseby the first protocol. The immune reaction measured by Immunoblotting analysis with tomatopeel extracts exhibited more specific and visualized bands on membranes compared with those from pulp extracts. In detail, most of allergic patients to tomato reacted with peel’s allergens such as Profilin and Sola I5 Cyclophilin. Conclusion: Confirm thatthe second protocol in this research can be considered as the effective method for proteinextractionfrom freshtomato peel and pulp; and the main cause of tomato allergy comes from tomato’s peel.

Enzymatic cell wall degradation combined with pulsed electric fields increases yields of water-soluble-protein extraction from the green marine macroalga Ulva sp.

Marine macroalgae are an attractive source of alternative protein. However, protein extraction from macroalgae is challenging. In this work, we investigated a combination of enzymatic cell wall degradation and high voltage Pulsed Electric Fields (PEF), to enhance yields of water-soluble-protein extraction from the green marine macroalga Ulva sp. The combined process showed a considerably higher protein extraction yield (19.6 ± 0.33%) compared to that of PEF alone (10.8 ± 0.37%) and enzyme pretreatment alone (9.7 ± 0.42%). Moreover, the water-soluble-protein extract obtained by PEF, which followed enzymatic cell wall degradation, had significant antioxidant activity. These results indicate that PEF combined with enzymatic pretreatment could contribute to protein extraction yields from Ulva sp., as a part of sustainable seaweed biorefinery. Industrial relevanceAlthough several previous works reported on methods for protein extraction from seaweeds for food application, the commercialization of the seaweed proteins is challenging due to multiple challenges in the extraction process development. In this work we show that a combination of enzymes for cell wall degradation with high voltage pulsed electric fields for membrane permeabilization lead to higher yields of water-soluble proteins. Both enzyme treatment and PEF are scalable processes, which do not modify proteins chemically, potentially leading to higher quality of the extract in comparison to standard alkaline extraction with a need to treat chemical waste.

PlasmaCap EBA: An innovative method of isolating plasma proteins from human plasma.

The growing demand for immunoglobulin (IG) requires development of improved plasma fractionation methods to provide higher yields in a cost effective, scalable manner without compromising product purity and efficacy. A novel protein extraction method, utilizing expanded bed adsorption (EBA) chromatography, has been developed. PlasmaCap IG (10% liquid formulation intravenous IG [IVIG]) is the first plasma-derived product manufactured using PlasmaCap EBA technology. The PlasmaCap EBA platform consists of a series of consecutive columns which bind a target protein, or group of proteins, in their native state directly from cryo-poor plasma. EBA chromatography includes five key steps: (1) expand, (2) sanitize and equilibrate, (3) load, (4) wash and (5) elute. These steps are made possible using high-density tungsten-carbide agarose beads, suspended by upward flow. The PlasmaCap EBA process was evaluated during Evolve's clinical campaign for scalability, product quality and yield. PlasmaCap EBA technology can be predictably scaled by maintaining the minimum residence time and residence time distribution for EBA columns of different diameters. Scalability of the manufacturing process was demonstrated by the 50-fold volumetric increase from laboratory-scale lots to clinical-scale lots. The process is also associated with enhanced product purity, such as lower aggregates. The PlasmaCap EBA process is expected to have the same or better yield and purity at commercial scale production compared to the clinical campaign. The PlasmaCap EBA platform was used to successfully develop PlasmaCap IG (10% liquid formulation IVIG) with proven scalability, product quality and yield.

The impact of heating and freeze or spray drying on the interface and foam stabilising properties of pea protein extracts: Explained by aggregation and protein composition

The processing of plant protein extracts can affect the protein structure, leading to altered functional properties. In this work, we evaluated the impact of two common processes in pea protein extraction: heating and drying. Non-heated and heated (5 min at 95 °C) samples were compared, which were either freeze- or spray-dried. These processes led to alterations of the proteins, and resulted in changes of their interface and foam-stabilising properties. A mild protein extraction method was used to preserve the native protein structure during aqueous extraction, allowing the extraction of both albumin and globulin proteins. Spray-drying of these fractions led to higher surface hydrophobicity, which resulted in increased surface activity and stiffer interfacial layers at pH 3.8 and 7.0. The heating step induced aggregation of the globulins, while albumins remained soluble. Here, we demonstrated that the albumins had a dominant effect on the interfacial (rheology and ellipsometry) and foaming properties after heating, as the globulin aggregates were too large for effective interface stabilisation. A similar mechanism was also shown at pH 3.8, where the globulins precipitated, as the pH was close to their pI, while albumins remained soluble. Again, the albumins dictated the interfacial properties, leading to highly stable foams after removing the insoluble globulins. We have shown marginal differences in protein functionality after freeze- or spray-drying. More importantly, the changes in soluble protein composition dictate the protein functionality after heating or pH shifts.

New insight for spent hops utilization: simultaneous extraction of protein and xanthohumol using deep eutectic solvents

Agro-food by-products are a cheap source of bioactive and functional compounds that should be used via a biorefinery approach to produce a range of bio-based products. Spent hops (SH) are an important by-product of the brewing industry and are rich in valuable ingredients such as proteins and xanthohumol (XN). Considering the increasing demand for plant-based proteins and the broad spectrum of XN bioactivity, it is important to develop eco-friendly and cost-effective techniques to extract these components. In this work, a simple, one-pot, green method for the simultaneous extraction of proteins and XN from SH using deep eutectic solvents (DESs) was developed. Protein and XN-rich precipitates were obtained by adding water (antisolvent) to the DES extracts. To determine the XN content, the precipitates were extracted with methanol and the extracts were analyzed by HPLC. The presence of protein in the methanol-insoluble fractions was confirmed by techniques such NMR, IR, elemental analysis, and SDS-PAGE. The protein content varied between 40 and 64%, which was up to 87% higher than the protein content in the SH. This study demonstrates the potential use of SH and DESs to develop an environmentally friendly method to obtain protein and XN-rich products.Graphical abstract