Protein Extraction Methods Research Articles

Research progress and latest application of ultrasonic treatment on protein extraction and modification

SummaryThe advancement of the modern food industry has prompted a shift away from traditional protein extraction methods due to their inherent limitations. As a result, there is a growing interest in exploring alternative methods for protein extraction and modification such as ultrasonic (US) treatment. This shift towards more sustainable and efficient protein extraction methods is a key research focus in obtaining safe proteins in an environmentally friendly manner. Furthermore, enhancing the functional properties of proteins through modification can expand their application. This paper reviews recent research progress and applications of US treatment on protein extraction and modification as well as the impact on protein structure and functional properties. In addition, the latest applications of ultrasound‐modified‐proteins, including nano‐proteins, 3D printing and interactions between proteins and other food components, are reviewed. This review may provide some inspiration for the potential application of US technologies in food fields.

Exploring Edible Insects: A Review on Protein Diversity, Extraction Techniques, and Health Benefits.

This study investigates whether edible insects may be used as a sustainable protein source. This study covers insect proteins, their defatting process and extraction, health advantages, economic and environmental impacts, safety considerations, and regulatory aspects. This review also investigates the ecological benefits of insect farming and consumer acceptance of this dietary trend. A systematic search of the pertinent literature was conductedto collect data for this review. Current research examines the effects of using edible insects as functional, sustainable reservoirs of proteins, including protein screening, extraction methods, health benefits, and social recognition. The varied isolation techniques considerably affect protein amount and quality. . This review clarifies the broad spectrum of proteins in edible insects, particularly their nutritional importance and effective extraction methods. The viability of insects as a sustainable source of protein is highlighted by the safety considerations and the interaction of economic and ecological considerations with complex consumer characteristics.

Decoding the Duality of Antinutrients: Assessing the Impact of Protein Extraction Methods on Plant-Based Protein Sources.

This review aims to provide an updated overview of the effects of protein extraction/recovery on antinutritional factors (ANFs) in plant protein ingredients, such as protein-rich fractions, protein concentrates, and isolates. ANFs mainly include lectins, trypsin inhibitors, phytic acid, phenolic compounds, oxalates, saponins, tannins, and cyanogenic glycosides. The current technologies used to recover proteins (e.g., wet extraction, dry fractionation) and novel technologies (e.g., membrane processing) are included in this review. The mechanisms involved during protein extraction/recovery that may enhance or decrease the ANF content in plant protein ingredients are discussed. However, studies on the effects of protein extraction/recovery on specific ANFs are still scarce, especially for novel technologies such as ultrasound- and microwave-assisted extraction and membrane processing. Although the negative effects of ANFs on protein digestibility and the overall absorption of plant proteins and other nutrients are a health concern, it is also important to highlight the potential positive effects of ANFs. This is particularly relevant given the rise of novel protein ingredients in the market and the potential presence or absence of these factors and their effects on consumers' health.

The feasibility and applicability of sequential extraction of high value-added biogenic materials from sewage sludge.

The sequential extraction routes of biogenic materials from sewage sludge (SS) were investigated. Physical methods (ultrasound, heating) and chemical methods (sodium hydroxide, sodium carbonate) were used to extract extracellular polymeric substances (EPS) and alginate-like extracellular polymers (ALEs) from SS. The residues after extraction were further subjected to physical methods (heating) and chemical methods (sulfuric acid, sodium hydroxide) for protein extraction. A comparison was made between sequential extraction routes and direct extraction of biomaterials from sludge in terms of extraction quantity, material properties, and applicability. The results showed that sequential extraction of biomaterials is feasible. The highest extraction quantities were obtained when using sodium carbonate for EPS and ALE extraction and sodium hydroxide for protein, reaching 449.80 mg/gVSS, 109.78 mg/gVSS, and 5447.08 mg/L, respectively. Sequential extraction procedures facilitate the extraction of biomaterials. Finally, suitable extraction methods for different application scenarios were analyzed.

A methodological exploration of distinguishing hair quality based on hair proteomics

Hair is an advantageous biological sample due to its recordable, collectable, and storable nature. Hair's primary components are keratin and keratin-associated proteins. Owing to its abundance of cystine, keratin possesses impressive mechanical strength and chemical stability, formed by creating disulfide bonds as crosslinks within the protein peptide chain. Furthermore, keratin is cross-linked with keratin-associated proteins to create a complex network structure that provides the hair with strength and rigidity. Protein extraction serves as the foundation for hair analysis research. Bleaching hair causes damage to the structure between keratin and keratin-associated proteins, resulting in texture issues and hair breakage. This article outlines various physical treatment methods and lysate analysis that enhance the efficiency of hair protein extraction. The PLEE method achieves a three-fold increase in hair protein extraction efficiency when using a lysis solution containing SDS and combining high temperatures with intense shaking, compared to previous methods found in literature. We utilized the PLEE method to extract hair from both normal and damaged groups. Normal samples identified 156–157 proteins, including 51 keratin and keratin-associated proteins. The damaged group consisted of 155–158 identified proteins, of which 48–50 were keratin and keratin-associated proteins. Bleaching did not cause any notable difference in the protein identification of hair. However, it did reduce coverage of keratin and keratin-associated proteins significantly. Our hair protein extraction method provides extensive coverage of the hair proteome. Our findings indicate that bleaching damage results in subpar hair quality due to reduced coverage of protein primary sequences in keratin and keratin-associated proteins.

Brewers' spent grain proteins: The extraction method determines the functional properties

Brewers' spent grains (BSG) are a major byproduct from the brewing industry that are currently discarded or used as animal feed. This study systematically explored the effects of BSG protein extraction methods using alkali, ethanol or enzymes on the protein's structure, composition and function. Proteins extracted at pH 12 were partially unfolded and glutelin-rich while those extracted by 55% ethanol containing 2-mercaptoethanol were highly aggregated and hordein-rich. Enzymatic-assisted extraction resulted in peptides below 10 kDa that had no distinct structural elements. Being completely water-soluble, these peptides gave good emulsifying properties (activity: 83 m2/g protein; stability: 35 min) and a high antioxidant activity. On the other hand, alkali-extracted and ethanol-extracted proteins were mostly insoluble but exhibited high water holding capacities (2.5–4.0 g/g) that enabled gelation. The results highlighted that selection of the extraction method is critical, as the composition, structure and function of proteins are modified, which affects its potential applications. Industrial relevanceAs a major side stream from the production of beer, brewers' spent grains are present in huge amounts. These residues are a promising source of alternative proteins, but before separating them, it is important to first consider an extraction method that does not compromise on its functionality. This study offered insights into the influence of extraction methods on the structure and function of the resulting protein concentrates as well as its future applications.

Sustainable Valorization of Coffee Silverskin: Extraction of Phenolic Compounds and Proteins for Enzymatic Production of Bioactive Peptides.

Coffee silverskin (CS), a by-product of the coffee roasting process, has high protein content (16.2-19.0%, w/w), making it a potential source for plant protein and bioactive peptide production. This study aims to develop innovative extraction methods for phenolic compounds and proteins from CS. The conditions for hydrothermal (HT) extraction of phenolic compounds from CS were optimized by varying CS loading (2.5-10%, w/v), temperature (110-130 °C), and time (5-30 min) using a one-factor-at-a-time (OFAT) approach. The highest TPC of 55.59 ± 0.12 µmole GAE/g CS was achieved at 5.0% (w/v) CS loading and autoclaving at 125 °C for 25 min. Following hydrothermal extraction, CS protein was extracted from HT-extracted solid fraction by microwave-assisted alkaline extraction (MAE) using 0.2 M NaOH at 90 W for 2 min, resulting in a protein recovery of 12.19 ± 0.39 mg/g CS. The CS protein was then subjected to enzymatic hydrolysis using protease from Bacillus halodurans SE5 (protease_SE5). Protease_SE5-derived CS protein hydrolysate had a peptide concentration of 0.73 ± 0.09 mg/mL, with ABTS, DPPH, and FRAP values of 15.71 ± 0.10, 16.63 ± 0.061, and 6.48 ± 0.01 µmole TE/mL, respectively. Peptide identification by LC-MS/MS revealed several promising biological activities without toxicity or allergenicity concerns. This study's integrated approach offers a sustainable and efficient method for extracting valuable compounds from CS, with potential applications in the food and pharmaceutical industries.

Fibrillization of lentil proteins is impacted by the protein extraction conditions and co-extracted phenolics

Legume proteins can be induced to form amyloid-like fibrils upon heating at low pH, with the exact conditions greatly impacting the fibril characteristics. The protein extraction method may also impact the resulting fibrils, although this effect has not been carefully examined. Here, the fibrillization of lentil protein prepared using various extraction methods and the corresponding fibril morphology were characterized. It was found that an acidic, rather than alkaline, protein extraction method was better suited for producing homogeneous, long, and straight fibrils from lentil proteins. During alkaline extraction, co-extracted phenolic compounds bound proteins through covalent and non-covalent interactions, contributing to the formation of heterogeneous, curly, and tangled fibrils. Recombination of isolated phenolics and proteins (from acidic extracts) at alkaline pH resulted in a distinct morphology, implicating a role for polyphenol oxidase also in modifying proteins during alkaline extraction. These results help disentangle the complex factors affecting legume protein fibrillization.

The Effect of Ultrasound on the Extraction and Functionality of Proteins from Duckweed (Lemna minor).

The inclusion of protein in the regular human diet is important for the prevention of several chronic diseases. In the search for novel alternative protein sources, plant-based proteins are widely explored from a sustainable and ecological point of view. Duckweed (Lemna minor), also known as water lentil, is an aquatic plant with potential applications for human consumption due to its protein content and carbohydrate contents. Among all the conventional and novel protein extraction methods, the utilization of ultrasound has attracted the attention of scientists because of its effects on improving protein extraction and its functionalities. In this work, a Box-Behnken experimental design was proposed to optimize the alkaline extraction of protein from duckweed. In addition, an exploration of the effects of ultrasound on the morphological, structural, and functional properties of the extracted protein was also addressed. The optimal extraction parameters were a pH of 11.5 and an ultrasound amplitude and processing time of 60% and 20 min, respectively. These process conditions doubled the protein content extracted in comparison to the value from the initial duckweed sample. Furthermore, the application of ultrasound during the extraction of protein generated changes in the FTIR spectra, color, and structure of the duckweed protein, which resulted in improvements in its solubility, emulsifying properties, and foaming capacity.

Evaluation of different fractionation methods for the simultaneous protein and carbohydrate extraction from microalgae

AbstractThe production of high-value products from microalgae, one of the preferred emerging biorefineries’ feedstocks, relies on the crucial step of biomass fractionation. In this work, the fractionation of Chlorella vulgaris and Scenedesmus obliquus biomass was tested for protein extraction using a wide range of physical, chemical, and enzymatic treatment combinations, including ultrasound, cell homogenizer, cellulase, and alcalase combinations in aqueous and alkali extraction conditions. The impact of these processes on biomass carbohydrates was also evaluated. Alkaline-assisted ultrasound treatments using alcalase presented the highest protein extraction yield, reaching 90 g/100 g protein on C. vulgaris, closely followed by the same treatment in aqueous conditions (85 g/100 g protein). The same aqueous treatment achieved the best performance on S. obliquus, reaching 82 g/100 g protein. All treatments on both microalgae partially solubilized the polysaccharide fraction with all alkaline treatments solubilizing over 50 g/100 g sugars for all conditions. Overall, all the treatments applied were effective methods for biomass fractionation, although they showed low selectivity regarding the individual extraction of protein or carbohydrates.

The extraction of buckwheat protein and its interaction with kappa-carrageenan: Textural, rheological, microstructural, and chemical properties

Current plant-based foods use plant proteins as a key structuring and texturing ingredient. The use of water for extraction can replace conventional protein extraction methods. Water extraction of protein is environmentally friendly and could prevent the loss of protein functionality due to extreme pH changes. This study demonstrates an aqueous extraction method, coupled with ultrasound as pre-treatment, to obtain buckwheat protein (BWPE) and assess its gelling property and composited gel with kappa-carrageenan (k-carr). Textural and rheological analyses showed that the hardness and storage modulus of the composited gel containing 1 % w/w BWPE and 1 % w/w k-carr was 4.2-fold and 100-fold, respectively, higher than k-carr gel at 1 % w/w. Light microscopy showed a mixed bi-continuous gel system, with k-carr reinforcing the protein gel network. Besides volume exclusion effects, chemical bond and FTIR analyses revealed that adding k-carr to BWPE altered the protein's secondary structure and mediated protein denaturation during heating. This results in greater β-sheet content, which creates a more organised gel structure. These results demonstrated that ultrasound-assisted water-extracted BWPE, together with varying concentrations of k-carr, can be used to develop composited gels of tailorable textural and rheological properties to suit different food applications.

Hemp (Cannabis sativa L.) protein: Impact of extraction method and cultivar on structure, function, and nutritional quality

Hemp (Cannabis sativa L.) is increasingly gaining traction as a novel and sustainable source of plant protein. Accordingly, the aim of this study was to investigate the effectiveness of two protein extraction methods, alkaline extraction coupled with isoelectric precipitation (AE-IEP) and salt extraction coupled with ultrafiltration (SE-UF) in producing hemp protein isolates (pH-HPI and salt-HPI) with high purity and yield. Structural characterization as impacted by extraction method and cultivar was performed and related to functional performance and nutritional quality. Both extraction methods, with carefully selected parameters, resulted in HPI with high purity (86.6–88.1% protein) and protein extraction yields (81.6–87.3%). All HPI samples had poor solubility (∼9–20%) at neutral pH compared to commercial soy protein and pea protein isolates (cSPI, cPPI). A relatively high surface hydrophobicity and low surface charge contributed to such poor solubility of HPI. However, HPI demonstrated similar solubility at acidic pH (50–67%) and comparable gel strength (up to 24 N) to cSPI. Comparing experimental amino acid composition to the theoretical amino acid distribution in hemp protein provided insights to the functional performance of the protein isolates. While pH-HPI demonstrated better functionality than salt-HPI, minimal structural, functional, and nutritional differences were noted among the pH-HPI samples extracted from four different cultivars. Overall, results from this work could be used to guide future attempts to further develop successful protein extraction processes, and to provide valuable insights to propel breeding efforts that target enhanced hemp protein characteristics for food applications.

Phenol-Based Protein Extraction Method for Plant Proteomic Studies.

The protein extraction method based on the phenol solution and combined with protein precipitation with ammonium acetate in methanol and purification in the same solution, and additionally in acetone and ethanol, is recommended for proteomic studies of plant tissues. The obtained protein samples do not require additional nucleic acid digestion and removal of interfering contaminations. The presented protocol was used to analyze the proteome of common buckwheat flowers and leaves.

An Efficient Protein Extraction Method from Astragalus armatus Willd. Roots for Proteomic Analysis

Abstract The first step of the proteomic study is the extraction and the success of this technique was based mainly on the choice of the best extraction. The purpose of this study was to determine the simplest and lowest-cost method of total protein extraction. Initially, various extraction methods were performed for protein extraction from roots of Astragalus armatus Willd. The methods employed were extraction by RIPA buffer, hypotonic buffer and distilled water. After determination of protein concentration by Bradford method and SDS PAGE electrophoretic analysis, the quantity and quality of extracted proteins using different protocols from A. armatus were determined and compared. The protein yield of RIPA buffer method was higher than the yields of hypotonic buffer method and distilled water method. The best protein patterns were produced by RIPA buffer method. The extract obtained by RIPA buffer was the optimal protocol for protein extraction.

Improved methods for total and chloroplast protein extraction from Cajanus species for two-dimensional gel electrophoresis and mass spectrometry.

The recent advances in pigeon pea genomics, including high-quality whole genome and chloroplast genome sequence information helped develop improved varieties. However, a comprehensive Cajanus proteome, including the organelle proteome, is yet to be fully mapped. The spatial delineation of pigeon pea proteins at sub-cellular levels and inter-organelle communication could offer valuable insights into its defense mechanism against various stresses. However, the major bottleneck in the proteomic study is the lack of a suitable method of protein extraction and sample preparation compatible with two-dimensional gel electrophoresis (2D-PAGE), liquid chromatography-mass spectrometry (LCMS), or matrix-assisted laser desorption ionization-time of flight (MALDi-ToF). Our study introduces two efficient methods, one for isolating total proteins and another for organelle (chloroplast) proteins from various Cajanus spp. For total protein extraction, we have optimized a protocol using phenol in combination with a reducing agent (DTT) and protease inhibitor cocktail, also washing (6-7 times) with ice-cold acetone after overnight protein precipitation of total proteins. Our modified extraction method using phenol for total leaf protein yielded approximately 2-fold more proteins than the previously reported protocols from C. cajan (3.18 ± 0.11 mg/gm) and C. scarabaeoides (2.06 ± 0.08 mg/gm). We have also optimized a protocol for plastid protein extraction, which yielded 1.33 ± 0.25 mg/10 gm plastid proteins from C. cajan and 0.88 ± 0.19 mg/10 gm plastid proteins from C. scarabaeoides. The 2D-PAGE analysis revealed 678 ± 08 reproducible total protein spots from C. cajan and 597 ± 22 protein spots from C. scarabaeoides. Similarly, we found 566 ± 10 and 486 ± 14 reproducible chloroplast protein spots in C. cajan and C. scarabaeoides, respectively. We confirmed the plastid protein fractions through immunoblot analysis using antibodies against LHCb1/LHCⅡ type Ⅰ protein. We found both methods suitable for 2D-PAGE and mass spectrometry (MS). This is the first report on developing protocols for total and chloroplastic protein extraction of Cajanus spp. suitable for advanced proteomics research.

Pea protein extraction method impacts the protein (micro)structural organisation and in vitro digestion kinetics.

There is increasing interest in including pulse proteins into food products due to their nutrient-rich and sustainable character. However, little is known regarding the consequences of different extraction approaches on the pulse protein structure and the subsequent protein (micro)structural organization and protein digestion kinetics. Therefore, three green pea protein extracts were created: (i) cooking followed by cotyledon cell isolation, (ii) alkaline extraction followed by isoelectric precipitation, or (iii) salt extraction, and compared to the original pea flour as well as to sodium caseinate. The results showed that encapsulated, denatured protein inside pea cotyledon cells presented the (s)lowest digestion, while accessible and more native protein (e.g., pea flour, pea protein salt extract) presented much faster and higher digestion. Moreover, the alkali extracted pea protein was denatured to some extent, significantly lowering in vitro digestion kinetics. In the second part, three different in vitro approaches were applied to digest the salt extracted pea protein. Semi-dynamic gastric digestion approaches simulate in vivo conditions more closely which especially impacted the rate of digestion.

Protein-Based Films and Coatings: An Innovative Approach

Protein-based films and coatings are highly biodegradable and represent sustainable alternatives to petroleum-based materials. These materials possess commendable barrier properties, effectively safeguarding against oxygen, moisture, and aroma compounds, rendering them well-suited for various food packaging applications. Beyond their role in food packaging, coatings and films have significant applications in the biomedical and pharmaceutical domains. Their inherent biocompatibility and controlled release properties make them valuable for applications such as drug-delivery systems, wound dressings, and tissue-engineering scaffolds. Moreover, the adaptability of these films to exhibit stimuli-responsive behavior opens avenues for on-demand drug release and sensing capabilities. Despite these promising attributes, challenges persist in terms of the mechanical strength, water resistance, and scalability of the processing of protein-based films and coatings. Ongoing research endeavors are dedicated to refining protein extraction methods, incorporating reinforcing agents, and implementing strategies to optimize the overall performance of these materials. Such efforts aim to overcome existing limitations and unlock the full potential of protein-based films and coatings in diverse applications, contributing to the advancement of sustainable and versatile biomaterials.

A Review of the Utilization of Canola Protein as an Emulsifier in the Development of Food Emulsions.

Canola is the second-largest cultivated oilseed crop in the world and produces meal consisting of about 35-40% proteins. Despite this, less than 1% of the global plant-based protein market is taken up by canola protein. The reason behind such underutilization of canola protein and its rapeseed counterpart could be the harsh conditions of the industrial oil extraction process, the dark colour of the meal, the presence of various antinutrients, the variability in the protein composition based on the source, and the different properties of the two major protein components. Although academic research has shown immense potential for the use of canola protein and its rapeseed counterpart in emulsion development and stabilization, there is still a vast knowledge gap in efficiently utilizing canola proteins as an effective emulsifier in the development of various emulsion-based foods and beverages. In this context, this review paper summarizes the last 15 years of research on canola and rapeseed proteins as food emulsifiers. It discusses the protein extraction methods, modifications made to improve emulsification, emulsion composition, preparation protocols, and emulsion stability results. The need for further improvement in the scope of the research and reducing the knowledge gap is also highlighted, which could be useful for the food industry to rationally select canola proteins and optimize the processing parameters to obtain products with desirable attributes.

Tea (Camellia sinensis (L.) Kuntze) as an emerging source of protein and bioactive peptides: A narrative review.

Tea residues represent one of the major agricultural wastes that are generated after the processing of tea. They account for 21-28% of crude protein and are often discarded without the extraction of valuable proteins. Due to various bioactivity and functional properties, tea proteins are an excellent alternative to other plant-based proteins for usage as food supplements at a higher dosage. Moreover, their good gelation capacity is ideal for the manufacturing of dairy products, jellies, condensation protein, gelatin gel, bread, etc. The current study is the first to comprehend various tea protein extraction methods and their amino acid profile. The preparation of tea protein bioactive peptides and hydrolysates are summarized. Several functional properties (solubility, foaming capacity, emulsification, water/oil absorption capacity) and bioactivities (antioxidant, antihypertensive, antidiabetic) of tea proteins are emphasized.

MALDI-TOF MS analysis of Burkholderia pseudomallei and closely related species isolated from soils and water in Khon Kaen, Thailand

Melioidosis is caused by Burkholderia pseudomallei (Bp) acquired from the environment. Conventional identification methods for environmental Bp are challenging due to the presence of closely related species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is accurate for bacterial identification, but has been little used to identify Bp from environmental samples. This study aims to evaluate MALDI-TOF MS for the identification of Bp and closely related species isolated from environmental samples in Thailand using whole-genome sequencing (WGS) as the gold standard, including determining the best sample preparation method for this purpose. We identified Bp (n = 22), Burkholderia spp. (n = 28), and other bacterial species (n = 32) using WGS. MALDI-TOF analysis of all Bp isolates yielded results consistent with WGS. A decision-tree algorithm identified 16 important variable peaks, using the protein extraction method (PEM), demonstrating distinct MALDI-TOF profiles for the three categories (Bp, Burkholderia spp. and “other bacterial species”). Three biomarker peaks (4060, 5196, and 6553 Da) could discriminate Bp from other Burkholderia and closely related species with 100% sensitivity and specificity. Hence, the MALDI-TOF technique has shown its potential as a species discriminatory tool, providing results comparable to WGS for classification and surveillance of environmental Bp.