Protein Extraction Methods Research Articles

A comprehensive review on composition to application of pea protein and its components

Pea protein, a valuable plant-based protein source, is notable for its nutritional value, essential amino acids, and low allergenicity, making it widely applicable in food, medicine, and materials. It consists mainly of globulin and albumin, which influence its functional properties and applications. However, there is a lack of comprehensive reviews on its extraction methods, functional properties, modification techniques, and applications in food. This paper aims to fill these gaps by detailing pea protein composition, extraction methods, functional properties, and modification impacts while summarizing its food applications and proposing future research directions. The goal is to enhance pea protein's functionality and expand its applications through optimized extraction and advanced technology. By improving extraction techniques and adapting pea protein for better functionality, we aim to develop high-quality market applications, ensuring the growth and sustainability of the pea protein industry globally. This approach promises a flourishing future for pea protein, meeting global competition demands and driving industry advancement.

Insights into type 2 diabetes mellitus in atrial fibrillation patients: a proteomics approach

Abstract Introduction Atrial fibrillation (AF) and Type 2 Diabetes Mellitus (T2DM) are two closely interconnected conditions, sharing a complex relationship within cardiac pathophysiology. Common risk factors such as obesity, hypertension, inflammation, and endothelial dysfunction contribute to their coexistence. T2DM is known to drive cardiac fibrosis and promote electrical and structural remodeling of the heart, creating a substrate conducive to AF onset, while AF in T2DM patients increases the risk of adverse cardiovascular outcomes, such as stroke. Understanding this complex relationship is pivotal for improving the therapeutic and preventive strategies for these patients. This study aims to examine the proteomic differences between AF patients with and without T2DM, providing novel insights into pathophysiological mechanisms and identifying novel therapeutic targets and biomarkers. Methods In total, 14 AF patients undergoing open-heart surgery, comprising of 6 T2DM individuals and 8 non-T2DM individuals have been included. The mean age of the T2DM patients was 69.5 ± 10 years, while the non-T2DM was 69.3 ± 8.2 and BMI was 29.2 ± 2.9 and 29.5 ± 3 respectively. Right atrial appendages (RAA) from these patients were obtained in an RNA stabilization solution during open-heart surgery and processed using the phenol-based method for protein extraction. The proteins were digested with trypsin to generate peptides, followed by an additional clean-up step using C18 pipette tips. The prepared peptide samples were analyzed by employing a label-free-based quantitative (LFQ) approach, using a C18 (75cmx75cm) column. Proteomic analysis was performed using a Nano liquid chromatography system coupled to tandem mass spectrometer (nano LC-MS/MS), followed by a comprehensive bioinformatics analysis, using appropriate software. Results A total of 1548 proteins were identified, of which 899 could be quantified using LFQ analysis. Applying strict significance criteria, including FDR <0.1 and Abundance ratio (AR)>2 or <0.5, we identified 100 differential expressed proteins, with 21 upregulated and 79 downregulated in T2DM (Fig.1). Most notably, DPP9 and ATP1A2 were overexpressed in hearts of AF patients with T2DM (AR=100), while INPPL1 and ARID5A were underexpressed (AR=0.01), thereby representing attractive therapeutic targets. In pathway enrichment analysis (Fig.2), the dysregulated proteins participate in noteworthy pathways, including Non-alcoholic fatty liver disease (NAFLD), Type II diabetes mellitus, and Insulin signaling pathway. Conclusion Given the increased morbidity associated with the coexistence of AF and T2DM, there is a pressing need for novel therapeutic strategies tailored to this patient population. While AF and T2DM share certain molecular characteristics in the heart, this study emphasizes the necessity for further evaluation and validation of the distinct proteomic signatures identified.Fig.1:Volcano PlotFig.2:Protein Enrichment Network

The protamines of the spider Steatoda sp. provide an example of liquid-liquid phase separation chromatin transitions during spermiogenesis.

While there is extensive information about sperm nuclear basic proteins (SNBP) in vertebrates, there is by comparison, very little information in Arthropoda. This paper aims to contribute to filling this gap by analyzing these proteins in the sperm of the noble false widow spider Steatoda nobilis (Order Araneae, Family Theridiidae). To this end, we have developed a protein extraction method that allows the extraction of both cysteine-containing and non-Cysteine-containing protamines that is suitable for the preparation and analysis of SNBPs from samples where the amount of starting tissue material is limited. We carried out top-down mass spectrometry sequencing and molecular phylogenetic analyses to characterize the protamines of S. nobilis and other spiders. We also used electron microscopy to analyse the chromatin organization of the Steatoda sperm and we found it to exhibit liquid-liquid phase spinodal decomposition during the late stages of spermiogenesis. These studies further our knowledge on the distribution of SNBPs within the animal kingdom and provide additional support for a proposed evolutionary origin of many protamines from a histone H1 (H5) replication independent precursor.

Impact of different food-grade protein extraction methods on the proteomic profile and potential allergenicity of Spirulina (Arthrospira platensis)

Various optimized methods for cell disintegration and protein extraction of algae have been reported, but there is a lack of information regarding the proteomic characterization of the extracted proteins; thus, systematic verification of the protein profiles to find potentially exploitable proteins, while minimizing allergenicity risks, is profitable. The method described in this study was developed to thoroughly investigate the proteome characterization of A. platensis following aqueous protein extraction by high-shear homogenization and pH shift. Liquid chromatography-tandem mass spectrometry was applied to assess the proteome obtained from four protein extraction methods using data-dependent proteome analysis. Overall, 699 proteins were identified; however, each extraction method identified unique proteins. The maximum number of exclusive proteins was observed using a combination of high-shear homogenization and acidic pH. The evaluation of protein abundance revealed a clear distinction between proteins isolated from pH 2 treatment compared to those obtained via neutral or basic pH treatment. The applied extraction methods affected the in silico computed physicochemical properties of the proteins. Using the AllerCatPro database, 12 putative allergenic proteins were detected, the most predominant of which were related to the C-phycocyanin beta subunit (P72508). This study demonstrated that extraction methods affect the detection, physicochemical properties, and potential allergenicity of the obtained proteins. Hence, the extraction methods used to obtain novel proteins must consider these aspects before using them as functional foods.

Determination of nut varieties and their detection in festive cookies by MALDI-TOF mass spectrometry.

Nuts contain a large amount of essential fatty acids, amino acids, and whole range of minerals and vitamins valuable for human health, yet certain risks are associated with their consumption, of which allergic reaction is the most important. Considering the growing number of people suffering from allergies caused by allergens of protein origin, the aim of this work is to find out whether nuts can be distinguished from each other on the basis of contained proteins. A total of eleven raw and subsequently heat-treated nuts (almonds, Brazil nuts, cashews, coconuts, hazelnuts, macadamia nuts, peanuts, pecans, pine nuts, pistachios, and walnuts) were analyzed using MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry with the subsequent finding of characteristic m/z values for each analyzed nut. No previous method for protein extraction was used. The characteristic values were used to verify the composition of seven types of festive cookies - six commercial products and one "unknown" cookie, where it was not known in advance, which nut it was made from. The procedure, together with the found characteristic m/z values, could serve to rapidly identify the plant origin of nut products.

Proteomic Analysis of Human Serum Proteins Adsorbed onto Collagen Barrier Membranes.

Collagen barrier membranes are frequently used in guided tissue and bone regeneration. The aim of this study was to analyze the signature of human serum proteins adsorbed onto collagen membranes using a novel protein extraction method combined with mass spectrometry. Native porcine-derived collagen membranes (Geistlich Bio-Gide®, Wolhusen, Switzerland) were exposed to pooled human serum in vitro and, after thorough washing, subjected to protein extraction either in conjunction with protein enrichment or via a conventional surfactant-based method. The extracted proteins were analyzed via liquid chromatography with tandem mass spectrometry. Bioinformatic analysis of global profiling, gene ontology, and functional enrichment of the identified proteins was performed. Overall, a total of 326 adsorbed serum proteins were identified. The enrichment and conventional methods yielded similar numbers of total (315 vs. 309), exclusive (17 vs. 11), and major bone-related proteins (18 vs. 14). Most of the adsorbed proteins (n = 298) were common to both extraction groups and included several growth factors, extracellular matrix (ECM) proteins, cell adhesion molecules, and angiogenesis mediators involved in bone regeneration. Functional analyses revealed significant enrichment of ECM, exosomes, immune response, and cell growth components. Key proteins [transforming growth factor-beta 1 (TGFβ1), insulin-like growth factor binding proteins (IGFBP-5, -6, -7)] were exclusively detected with the enrichment-based method. In summary, native collagen membranes exhibited a high protein adsorption capacity in vitro. While both extraction methods were effective, the enrichment-based method showed distinct advantages in detecting specific bone-related proteins. Therefore, the use of multiple extraction methods is advisable in studies investigating protein adsorption on biomaterials.

Evaluation of protein extraction protocols for MALDI-TOF Biotyper analysis of mycobacteria

Infections caused by Mycobacterium tuberculosis and nontuberculous mycobacteria represent a significant global threat and medical concern. Therefore, accurate and reliable methods must be employed to identify mycobacteria rapidly. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technique that compares the cellular protein profiles of unknown isolates with reference mass spectra in a database to identify microorganisms. However, the thick and waxy lipid layer, which is rich in mycolic acids and is present in mycobacterial cells, makes protein extraction challenging. To identify the optimal protocol for correctly identifying bacilli using MALDI-TOF mass spectrometry, this study compared four different cellular protein extraction methods. Four strains of M. bovis BCG were selected as representatives of slow-growing mycobacteria, while three strains of fast-growing mycobacteria were also included: M. peregrinum, M. smegmatis, and M. farcinogenes. The extraction method that proved most effective was the extraction of inactivated cells with chloroform and methanol, which partially delipidates the cells. These cells were then extracted with formic acid, as is standard practice for protein extraction. The advantage of this method is that it allows the parallel analysis of cellular lipids and proteins from a single sample. It is therefore important to optimize mycobacterial protein extraction for MALDI-TOF MS analysis in clinical microbiology laboratories.

Proteolytic degradation of seed proteins of vetch species (Vicia L. subgenus Vicia) of section Peregrinae Kupicha during SDS-electrophoresis and its prevention

Background. Due to its simplicity and good reproducibility, SDS-electrophoresis of seed proteins is widely used for investigating the gene pool of legumes and other plants, for species and varietal identification, analyzing the intraspecific variability, and registering collection material. The data obtained by this method agree well with the DNA analysis results complement them. Typically, legume seed proteins, including representatives of the genus Vicia L., show clear informative SDS electrophoretic profiles. When analyzing seed accessions of vetch species of the section Peregrinae Kupicha using standard approaches previously developed at VIR and approved by ISTA (the International Seed Testing Association), clear electrophoretic protein profiles could not be obtained for many accessions. This called into question the applicability of standard approaches to identifying vetch species in the section Peregrinae. The objective of the work was to clarify the nature of seed proteins degradation in representatives of the Peregrinae section and to find ways to prevent it to ensure the possibility of carrying out species identification and registration of all accessions in the vetch collection using a unified approach. Material and methods. Seed proteins of a number of vetch species Vicia L. from sections Bithynicae (B. Fedtsch.) Maxted, Hypechusa (Alef.) Aschers. et Graebner, Microcarinae Maxted and Peregrinae, members of the subgenus Vicia, were analyzed by SDS-electrophoresis using the standard method of protein extraction from flour with 0.025 M Tris-glycine buffer pH 8.3 at room temperature and its modifications, including heating the extract at 80°C or 100°C with or without the addition of 2-mercaptoethanol, as well as the addition of cysteine and serine protease inhibitors. Results and discussion. An analysis of seed proteins of representatives of most sections of the subgenus Vicia yielded informative species-specific protein profiles, whereas species of the section Peregrinae were characterized by the protein profiles, which indicated protein degradation, and species of this section differed in the frequency of such profile occurrence. While such profiles were obtained for all seeds of seven accessions of V. aintabensis Boiss & Hausskn. ex Boiss differing in geographical origin, year and place of regeneration, and 12 out of 13 of V. peregrine L. accessions demonstrated profiles of partially or completely degraded proteins, complete seed protein profiles were obtained for six out of nine V. michauxii Sprengel accessions. A change in conditions for protein isolation, namely replacement of their extraction from flour with Tris-glycine buffer pH 8.3 at room temperature with extraction in the same buffer by a short-term heating at 100°C in the presence of 2-mercaptoethanol, made it possible to obtain complete protein profiles for all accessions representing the section Peregrinae. The protein profiles of representatives of other vetch sections, as well as the profile of soybean proteins used as a standard for legume species identification, did not differ from the original ones under the modified conditions. Conclusions. The obtained results suggest that protein degradation in species of the Peregrinae section is associated with the abnormal activity of endogenous seed proteases under standard protein extraction conditions, and this trait is determined genotypically. A new modification of the method for isolating proteins from seeds makes it possible to apply the generally accepted approaches based on SDS-electrophoresis in the analysis of the gene pool of the Peregrinae section of the subgenus Vicia, as well as other vetch species.

A new method for protein extraction from sea lettuce (Ulva fenestrata) via surfactants and alkaline aqueous solutions

Alternative protein sources such as seaweed can help relieve the pressure on land-based protein supply. This proof-of-concept study developed an extraction method to recover soluble and lipophilic proteins from the seaweed Ulva fenestrata. The method consisted of processing U. fenestrata with 0.1–0.5 % aqueous Triton X-114 solution and reprocessing the pellet with an alkaline aqueous solution. Then, the solubilized proteins were precipitated via acidification. The new method extracted 3.4-times more protein, measured as total amino acids, compared to the control with two alkaline aqueous extraction cycles. Triton disrupted the chloroplasts and likely solubilized lipophilic membrane proteins as supported by microstructure and polypeptide pattern analysis. Triton-derived protein extracts contained lipids inside the precipitates/aggregates and were richer in fatty acids typical of photosynthetic membranes. The higher extraction yields are proposed to result from membrane charge neutralization upon acidification, triggering interactions between the membrane lipids and their subsequent precipitation with the lipophilic membrane protein.

Plant Protein Resources, Novel Extraction and Precipitation Methods: A Review

ABSTRACTThis comprehensive review explores the diverse landscape of plant protein resources, focusing on novel extraction and precipitation methods that contribute to the advancement of sustainable and efficient protein production. The increasing global demand for plant‐based proteins as a key component of balanced diets has spurred research into optimizing extraction processes from various plant sources. The review encompasses a wide range of plant protein resources, including legumes, cereals, oilseeds, and other plant‐based alternatives. Special emphasis is given to innovative techniques in protein extraction, such as enzyme‐assisted extraction, aqueous extraction, and emerging technologies like ultrasound and microwave‐assisted methods. Additionally, the review investigates novel precipitation methods employed to isolate and purify plant proteins, shedding light on techniques like isoelectric precipitation, salting‐out, and membrane filtration. The integration of sustainable practices in protein extraction, coupled with a focus on minimizing environmental impact, is a central theme throughout the review. By synthesizing current research findings, this review aims to provide a comprehensive overview of the state‐of‐the‐art in plant protein extraction and precipitation methods, offering valuable insights for researchers, industry professionals, and policymakers involved in the rapidly evolving field of plant‐based protein production.

Leg muscles of migratory locust (Locusta migratoria) as a protein source: Extraction, protein composition and foaming properties

Migratory locust (Locusta migratoria) is an edible insect species that is usually consumed at the adult developmental stage and represents a valuable source of proteins. Locusts can be processed into food ingredients or directly consumed after removal of legs and wings. Legs represent a unique matrix, which comprises a chitin-based exoskeleton typical for insects and muscle tissues commonly occurring also in other animals. Muscle proteins can be easily extracted from traditional animal sources, but the efficiency of these methods for locust legs as well as the functional characteristics of the resulting protein extracts have not been previously explored. This study first compared protein extraction methods at acid, alkaline, and salt-assisted conditions. All three methods resulted in a significantly higher extraction yield from locust leg muscles (0.465–0.595 g/g) than from whole locusts (0.061–0.125 g/g). Shotgun proteomics of the protein fraction extracted at alkaline conditions assigned 310 muscle proteins, comprising components assigned to energy and carbon metabolism, as well as to skeletal, protein folding, membrane trafficking, and cell adhesion functions. The techno-functional potential of locust leg muscles was assessed by a foamability assay. Foam stability of locust leg extracts varied as a function of pH extraction and re-solubilization conditions and was significantly higher (58.3–70.8 %) than for whey proteins taken as a benchmark (38.3–51.7 %); foam capacity at t = 0 h was in the range of 48.6–68.8 %. This study demonstrates that locust legs, which can be a by-product of locust consumption or processing, should be considered a rich source of muscle proteins with promising technological functionality.

PH shift extraction technique for plant proteins: A promising technique for sustainable development

With the recognition of animal-derived foods as unhealthy and their association with climate change, researchers are increasingly focusing on plant-based protein as a sustainable alternative. Plant proteins offer versatile functional and dietary benefits, making them suitable for various food applications. This study investigates the influence of alkaline and acidic pH conditions on the extraction yield and the functional and nutritional properties of plant-based proteins. The primary sources of plant protein include cereals, legumes, and oilseeds, which can be used to address essential amino acid deficiencies through blending. Several methods have been employed for protein extraction from plant sources, such as salt extraction, microwave-assisted extraction, ultrasound-assisted extraction, and micellar precipitation. Among these techniques, the pH shift method stands out due to its non-thermal nature, sustainability, and cost-effectiveness. In this method, proteins are solubilized at alkaline pH and then precipitated at their isoelectric point, resulting in a collection of protein precipitate. It is crucial to optimize extraction techniques based on qualitative and quantitative analysis to enhance protein yield, characteristics, and nutritional value. Most conventional protein extraction methods require a large quantity of chemicals, which imposes the issue of safe disposal, compromising environmental sustainability. Traditional methods also produce protein with non-proteinaceous constituents, adding another purification step and resulting in increased overall cost. However, the pH shift method utilizes comparatively less harsh chemicals and has a high protein extraction yield, which makes it comparatively more environmentally and economically sustainable. The sustainable extraction of plant-based proteins addresses the health and environmental concerns associated with animal-derived foods and offers a promising solution to promote sustainability in the food industry.

Protein Extraction Methods Suitable for Muscle Tissue Proteomic Analysis.

Muscle tissue is one of the most dynamic and plastic tissues of the mammalian body and covers different roles, such as force generation and metabolic control. Muscular proteomics provides an important opportunity to reveal the molecular mechanisms behind muscle pathophysiology. To ensure successful proteomic analysis, it is necessary to have an efficient and reproducible protein extraction method. This study aimed to evaluate the efficacy of two different extraction protocols of muscle samples for two-dimensional gel electrophoresis. In particular, mouse muscle proteins were extracted by an SDS-based buffer (Method A) and by a UREA/CHAPS/DTE/TRIS solution (Method B). The efficacies of the methods were assessed by performing an image analysis of the 2DE gels and by statistical and multivariate analyses. The 2DE gels in both preparations showed good resolution and good spot overlapping. Methods A and B produced 2DE gels with different means of total spots, higher for B. Image analysis showed different patterns of protein abundance between the protocols. The results showed that the two methods extract and solubilize proteins with different chemical-physical characteristics and different cellular localizations. These results attest the efficacy and reproducibility of both protein extraction methods, which can be parallelly applied for comprehensive proteomic profiling of muscle tissue.

Legume protein gelation: The mechanism behind the formation of homogeneous and fractal gels

Protein gelation can provide texture in plant-based foods and can be influenced by many factors, including protein extraction method and salt addition. However, the protein gelation mechanism is still not well understood for plant proteins, especially for isolates obtained using commercial protein extraction processes. Therefore, the structural changes that legume sources such as yellow pea, faba bean, and soybeans undergo during the gelation process to understand the differences in their gelation mechanisms was investigated herein by using small-angle neutron scattering. Among these protein sources, the commercial extraction method was found to play a major part in the gelation pathway. Intensive protein extraction methods involving isoelectric precipitation led to lower protein solubility (∼1–38% w/w), larger insoluble protein particle sizes (60–100 μm), and a gelation pathway that is dependent on the changes of the insoluble protein particles. In contrast, extraction using ultrafiltration instead of precipitation resulted in higher protein solubility (∼18–88% w/w) and smaller insoluble protein particle sizes (40–70 μm), and the structural changes observed during gelation involved the structural changes of both soluble proteins and insoluble proteins. SEM imaging also showed different gel networks, with fractal networks formed by insoluble proteins (for sources with low solubility) or more homogenous networks formed by the interactions between the soluble proteins (for sources with high solubility). Despite the differences observed in the gelation network and the protein solubility, the gelation strength exhibited by protein sources at low protein solubility were similar to the ones by protein sources at high protein solubility, demonstrating the potential of fractal gel networks in providing texture.

Efficient protein extraction for assessing food allergy risk in complex alternative protein matrices

Allergy to novel food proteins, due to diverse ingredients and innovative food processing technologies employed to achieve desired functional properties, is a major safety concern. Current allergy testing methods (ELISA and mass spectrometry) depend on high-quality protein extracts, meaning existing methods are often tailored to specific matrices. Therefore, a more efficient and general protein extraction method is desirable for comprehensive allergy risk assessment. Here, we developed a highly efficient and reproducible protein extraction method which achieved at least 80 % efficiency across several food matrices. Proteomics analysis of a plant-based meat using our optimized extraction method showed that higher extraction efficiency improved reproducibility of identified proteins. Moreover, higher protein extraction efficiency resulted in increased abundances of individual allergenic proteins. This underscores the relevance of our method for more accurate measurements of allergenic protein concentrations in allergy risk assessments.

Simple and eco-friendly fabrication of hempseed protein by one-step low-temperature-induced phase separation and centrifugal removal of oil

Hempseed protein is an emerging plant-derived alternative protein with unmatched nutritional and health benefits. However, the currently available industrial hempseed protein production relies on a defatting step using an organic solvent, hexane with health and environmental risks, which also damages the proteins’ structural and functional properties. In this study, we describe a successful method for the simple and eco-friendly simultaneous extraction of proteins and removal of oil based on the “hydrophilic” properties of hempseed oil. Both hempseed protein isolates (HPIs) extracted from hexane-defatted hempseed flour (HHPI) and non-defatted hempseed flour (NHHPI) exhibited great similarities in protein contents (both >90%), amino acid composition, protein subunits and functional properties (solubility, water retention and foaming). However, NHHPI significantly outperformed HHPI in keeping native structural and some processing properties. NHHPI exhibited a higher percentage of α-helix (44.29%) with lower amount of random coil (15.33%) than HHPI (α-helix 41.71%, random coil 19.07%), higher fluorescence intensity with lower incident wavelength (λmax = 326) than HHPI (incident wavelength λmax = 329). Meanwhile, NHHPI displayed higher surface hydrophobicity (807.01), fat absorption (1.35 g/g) and emulsion activity (2.36 m2/g) as compared to HHPI (743.48, 1.14 g/g, 1.78 m2/g, respectively). These findings highlight the importance of a new defatting method for HPI extraction, which may also be applicable to other oilseed proteins.

Characterisation, antibacterial activity, and prebiotic potential of dried Hermetia illucens L. larvae and of their fractions

Abstract In this study, black soldier fly (BSF) dried larvae were fractionated into their defatted, lipid, protein, and chitin-rich fractions. The samples were then characterised in terms of proximate composition, analysis of amino acids, analysis of the molecular weight distribution of proteins via gel electrophoresis, and fatty acids analysis of the lipid fraction. The antibacterial activity of all fractions was determined against Escherichia coli F4 and Streptococcus suis, two common swine pathogens. Finally, the prebiotic activity of the whole larvae and of their protein isolate and chitin-rich pellet was determined for Limosilactobacillus reuteri, a naturally occurring species in the gastro-intestinal tract of the pig. The results on amino acid profile showed that the protein extraction method used was not fully effective in cleaving chitin-bound proteins, as expected, as the true protein content of the chitin-rich sample was 48.5%. This was in accordance with the SDS-PAGE analysis, where the molecular weight distribution of proteins in the protein isolate sample did not show band at higher molecular weight, which were instead present in the chitin-rich sample. The analysis of fatty acid (FA) profile showed that lauric acid was the most abundant FA, in accordance with the literature. The results on microbiological analyses highlighted how all nitrogen-containing samples showed antibacterial activity against E. coli, the highest being the defatted fraction at 1% with a decrease of −0.77 log CFU/mL, while only the lipid fraction against S. suis (−0.48 log CFU/mL at 1%). All tested samples showed prebiotic activity for L. reuteri, but it was noticed that the increase in chitin concentration resulted in the decrease of the prebiotic effect. By combining the results of both antibacterial and prebiotic tests, it could be inferred that chitin might be considered as an antibacterial compound.

Anti-inflammatory and anti-hyperglycemia effects of mealworm (Tenebrio molitor larvae) protein extracted by four methods: alkali, salt, enzyme, and screw press.

The use of edible insect protein in food products is contingent on their biological effects. Conventional protein extraction methods are not only time-consuming and costly but also energy-intensive. There is a need for alternative techniques that maintain the bioactivities of insect proteins and are environmentally sustainable. This study compares the health functionality of mealworm (Tenebrio molitor larvae) concentrates obtained by conventional methods-alkali and salt (MS) extraction-and nonconventional methods-enzyme (ME) and screw press (MP)-to enhance their applicability despite lower protein concentration. Overall, MP exhibited the highest essential amino acids content, whereas ME showed the highest in vitro digestibility, total phenolic contents, and antioxidant capacities among all the concentrates. ME also had a significant cell proliferative capacity at concentrations ≥500µg/mL. MS significantly inhibited tumor necrosis factor-alpha and interleukin-1beta secretion in lipopolysaccharide-treated Hep3B cells compared to other samples. As for anti-hyperglycemia effects, treatment with MS and ME for 2 and 5min significantly increased the p-Akt/Akt ratio (MS, 1.34- and 1.61-fold; ME, 2.26- and 2.70-fold, respectively). In conclusion, enzyme treatment enhanced nutritional value and antioxidant capacity, whereas salt treatment potentially contributed to anti-inflammatory and anti-hyperglycemia activities. Hybrid extraction techniques combining conventional and nonconventional methods are suggested based on target applications, considering health benefits, environmental impact, costs, and efficiencies. PRACTICAL APPLICATION: Four mealworm protein extraction methods (alkali/salt/enzyme/screw press) were compared for their nutritional and biological properties. Alkali extraction enhanced protein content, enzyme treatment improved nutritional value and antioxidant capacity, and salt-assisted extraction exhibited immunomodulatory effects in vitro. Notably, enzyme and salt treatments produced protein concentrates with significant antidiabetic and anti-hyperglycemic properties.

Integration of MALDI-TOF MS and machine learning to classify enterococci: A comparative analysis of supervised learning algorithms for species prediction

This research focused on distinguishing distinct matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectral signatures of three Enterococcus species. We evaluated and compared the predictive performance of four supervised machine learning algorithms, K-nearest neighbor (KNN), support vector machine (SVM), and random forest (RF), to accurately classify Enterococcus species. This study involved a comprehensive dataset of 410 strains, generating 1640 individual spectra through on-plate and off-plate protein extraction methods. Although the commercial database correctly identified 76.9% of the strains, machine learning classifiers demonstrated superior performance (accuracy 0.991). In the RF model, top informative peaks played a significant role in the classification. Whole-genome sequencing showed that the most informative peaks are biomarkers connected to proteins, which are essential for understanding bacterial classification and evolution. The integration of MALDI-TOF MS and machine learning provides a rapid and accurate method for identifying Enterococcus species, improving healthcare and food safety.

Enhancing protein extraction from heterotrophic Auxenochlorella protothecoides microalgae through emerging cell disruption technologies combined with incubation

This study evaluated pulsed electric fields (PEF) and ultrasonication (US) combined with incubation to enhance cell disruption and protein extraction from Auxenochlorella protothecoides, comparing them to conventional high-pressure homogenization (HPH). A 5 h incubation enhanced protein yield by 79.4 % for PEF- and 27.2 % for US-treated samples. Extending the incubation to 24 h resulted in a total yield increase of 122 % for PEF (0.25 ± 0.03 kgEP kgTP−1) and 51.9 % for US (0.20 ± 0.02 kgEP−1 kgTP−1). Autofermentation in untreated cells after 24 h resulted in protein release with lower yields than all other treated and incubated samples. While HPH had the highest protein yield (0.58 ± 0.04 kgEP kgTP−1), PEF-incubation after 5 h (56.6 ± 5.3 MJ kgEP−1) and 24 h (49.5 ± 3.7 MJ kgEP−1) were 1.5 and 1.7-times more energy-efficient than HPH (82.9 ± 7.8 MJ kgEP−1). PEF combined incubation is an energy-efficient and targeted protein extraction method in heterotrophic A. protothecoides biorefinery.