Protein Extracts Research Articles

Grazing-Induced Habitat Degradation: Challenges to Giant Panda Survival Resulting from Declining Bamboo and Soil Quality

Grazing is the primary human-induced disturbance affecting giant panda (Ailuropoda melanoleuca) habitats and has a severe impact on the long-term sustainability of the giant panda population. To address the lack of quantitative studies on grazing’s impact on habitat quality, we selected China’s most heavily grazed giant panda nature reserve. Utilizing the Maxent model and stoichiometric analysis, we investigated habitat quality degradation caused by grazing and quantified changes in bamboo nutritional quality and soil physicochemical properties. The results indicate that grazing has significantly reduced the suitable habitat area for giant pandas from 101.87 km2 to 80.64 km2. Specifically, high-suitability habitats declined by 14.14%, moderate-suitability habitats declined by 22.70%, and low-suitability habitats declined by 22.88%. Grazing has forced pandas to move to higher altitudes (2650–3057 m) with taller (12–20 m) trees, denser (28–55 plants) shrubs, and sparser (30–69%) bamboo. Additionally, the soil water content has decreased, while soil bulk density, total N, available N, and pH have significantly increased. Reductions in crude protein and ether extract, along with increased crude fiber and ash, have lowered bamboo’s nutritional value and palatability. This study elucidates how grazing degrades giant panda habitat quality and provides a scientific basis for its conservation management.

Effective Mixed-Type Tissue Crusher and Simultaneous Isolation of RNA, DNA, and Protein from Solid Tissues Using a TRIzol-Based Method

One of the major challenges of studying biomarkers in tumor samples is the low quantity and quality of isolated RNA, DNA, and proteins. Additionally, the extraction methods ideally should obtain macromolecules from the same tumor biopsy, allowing better-integrated data interpretation. In this work, an in-house, low-cost, mixed-type tissue crusher combining blade and beating principles was made and the simultaneous isolation of macromolecules from human cells and tissues was achieved using TRIzol. RT-qPCR, genotyping, SDS-PAGE, and Western blot analysis were used to validate the approach. For tissue samples, RNA, DNA, and proteins resulted in an average yield of 677 ng/mg, 225 ng/mg, and 1.4 µg/mg, respectively. The same approach was validated using cell lines. The isolated macromolecule validation included the detection of mRNA levels of ATP-binding cassette (ABC) transporters through RT-qPCR, genotyping of TNFR1 (rs767455), and protein visualization through SDS-PAGE following Coomassie blue staining and Western blot. This work contributed to filling a gap in knowledge about TRIzol efficiency for the simultaneous extraction of RNA, DNA, and proteins from a single human tissue sample. A low-cost, high yield, and quality method was validated using target biomarkers of multidrug resistance mechanisms. This approach might be advantageous for future biomarker studies using different tissue specimens.

Dietary Fiber-Rich Spartina anglica Improves Intestinal Health and Antioxidant Capacity of Zhedong White Geese

Spartina anglica (SA), a plant rich in dietary fiber, has demonstrated considerable potential for enhancing gut health and antioxidant capacity in animals. This study investigates the integration of SA as a novel dietary ingredient for Zhedong white geese, with a specific focus on evaluating its effects on growth performance, nutrient digestibility, antioxidant capacity, intestinal health, and cecal microbiota composition. A total of 360 1-day-old Zhedong white geese with an average weight of 114.94 ± 0.81 g were randomly allocated to 4 dietary treatments, with 6 replicates per treatment and 15 geese per pen. The 4 dietary treatments included different SA supplement levels: a control group receiving a basal diet (CON), and three experimental groups supplemented with 3% SA (SA3), 6% SA (SA6), and 12% SA (SA12). Supplementation with 6% SA significantly enhanced the final body weight, average daily gain, and feed conversion ratio (FCR) compared to the CON group (p < 0.05). In contrast, the SA12 group exhibited reduced digestibility of crude protein and ether extract, relative to the SA3 and SA6 groups (p < 0.05). The highest antioxidant capacity was observed in the SA6 and SA12 groups, while the lowest was recorded in the CON group. SA supplementation did not significantly influence serum biochemical parameters or organ indices but increased cecum length (p < 0.05). Notably, SA supplementation markedly improved intestinal morphology, although excessive levels appeared to compromise these benefits. Additionally, SA supplementation significantly enhanced the richness and diversity of cecal microbiota and increased short-chain fatty acid concentrations. In conclusion, SA at an optimal supplementation level of 6% may be effectively utilized in Zhedong white geese diets to improve growth performance, gut health, and antioxidant capacity.

Effect of Defatting Method on Japanese Quince (Chaenomeles japonica) Fruit Seed Protein Isolate Technological Properties

Fruit seeds are often an underutilized side-stream of fruit processing. The most common approach to seed valorization is oil extraction due to the relative simplicity of the process. The partially or fully defatted seed meal is rarely further processed, even though seeds generally contain more protein and fiber than oil. The present study used single-screw extrusion (oil press), supercritical CO2 extraction, and a combination of the two, to defat Japanese quince (Chaenomeles japonica) seeds, and evaluated the defatted meals as sources of functional protein. Defatting with oil press and CO2 extraction proved similarly effective (reduced seed flour fat content from 11.75% to 6.40% and 5.32%, respectively); combining the two methods reduced fat content to 0.90%. The yield was minimally affected, but protein extract purity was defined by defatting efficiency (65.05% protein from non-defatted versus 82.29% protein from a combination-defatted meal). Defatting did not significantly affect amino acid composition but had a significant effect on every tested functional property (solubility, water, and oil binding capacity, apparent viscosity, foaming capacity, and emulsifying activity index). Of the tested defatting methods, supercritical CO2 extraction and the combination provided the best results from most aspects.

Effects of periodontal disease on the proteomic profile of the periodontal ligament.

Periodontal disease affects over 1 billion people globally. This study investigated how periodontitis affects the protein profile of the periodontal ligament (PDL) in rats. Eight Holtzman rats were divided into the control and experimental periodontitis groups. The PDL was isolated using laser capture microdissection, and protein extracts were analyzed by mass spectrometry. Data analysis utilized specialized software, and Gene Ontology enrichment analysis identified significant protein functions. The data are available via ProteomeXchange with identifier PXD055817. Proteins such as SerpinB1, C5, and Lgals3 were validated through immunohistochemistry, and their gene expression was examined in an in vitro human PDL cell line. This study identified 1326 proteins, with 156 unique to the control group, 294 unique to the periodontitis group, and 876 common to both groups. Enrichment analysis revealed that proteins associated with the regulation of enzyme activity and RNA binding were significantly represented in the periodontitis group. There were increased levels of SerpinB1, C5, and Lgals3 in the periodontitis group based on proteomic and immunohistochemical analyses. Furthermore, these targets showed increased gene expression in stimulated human PDL cells. This study provides insights into the periodontitis-related alterations in the protein composition of the PDL and PDL cells, identifying both novel and previously known disease-associated proteins. SIGNIFICANCE: The periodontal ligament plays a crucial role in oral functions by providing structural support to the tooth. Due to the presence of undifferentiated mesenchymal cells, research into its regenerative capacity is ongoing. Pathological conditions can affect these functions and protein composition. Currently, there is a lack of comprehensive research specifically focusing on evaluating the periodontal ligament in both healthy and diseased states. This pioneering study screened for protein alterations and the mechanisms related to periodontitis. The possibility of using proteomic analysis to evaluate the protein alterations that occur in periodontitis-a disease with a high global incidence-could provide therapeutic targets and new biomarkers for future clinical studies.

Investigation of the anti-aging effects of active components of Artemia franciscana loaded in hyalurosome

With the advancement of biotechnology in the marine industry, an increasing utilization of marine ingredients in skincare products has been observed in recent years. Encapsulating Artemia franciscana extract and its derivatives in a novel phospholipid vesicle called hyalurosome presents innovative strategies for drug delivery systems and anti-aging products. In this study, we developed nano hyalurosomes containing Artemia franciscana active components. Partially purification of proteins in the Artemia franciscana extract was performed using ion exchange chromatography, specifically targeting Hsp40 and Artemin. The physicochemical properties of the hyalurosomes were characterized, revealing nanoparticle sizes ranging from 100 to 130 nm, zeta potential between − 57 and − 41.2 mV. The biological compatibility of the fabricated hyalurosomes was tested in vitro on mice fibroblast cells. Results indicated that formulations containing hyalurosomes exhibited no cytotoxicity. In-vivo studies employing H&E and Mason’s trichrome staining demonstrated an increase in the dermal layer of the skin on male mice and collagen production following treatment with different formulations containing hyalurosomes. Therefore, these formulations are considered promising candidates for anti-aging effects. Stability study at 4 °C for 60 days validated by FE-SEM imaging. In conclusion, hyalurosomes fabricated with Artemia franciscana extract and its diverse active molecules successfully achieved enhanced loading and penetration into the deeper layers of the skin, and it can be a suitable candidate for the treatment of skin aging and rejuvenation.

Digitalisation, bioinformatics, and delivery systems in sustainable nonthermal extraction of proteins

Digitalisation, bioinformatics, and delivery systems in sustainable nonthermal extraction of proteins

Integral-Omics: Serial Extraction and Profiling of Metabolome, Lipidome, Genome, Transcriptome, Whole Proteome and Phosphoproteome Using Biopsy Tissue.

The integrative multiomics characterization of minute amounts of clinical tissue specimens has become increasingly important. Here, we present an approach called Integral-Omics, which enables sequential extraction of metabolites, lipids, genomic DNA, total RNA, proteins, and phosphopeptides from a single biopsy-level tissue specimen. We benchmarked this method with various samples, applied the workflow to perform multiomics profiling of tissues from six patients with colorectal cancer, and found that tumor tissues exhibited suppressed ferroptosis pathways at multiomics levels. Together, this study presents a methodology that enables sequential extraction and profiling of metabolomics, lipidomics, genomics, transcriptomics, proteomics, and phosphoproteomics using biopsy tissue specimens.

Protein extraction technologies for foxtail, pearl, and proso millets along with their structural and techno-functional properties: a review

Abstract As the world’s population grows, so does the demand for protein, and the growing population is actively seeking alternative protein sources that are affordable and available locally. Millet, a resilient crop with a remarkable ability to adapt to unfavourable conditions, has emerged as a potential alternative in recent years. Millet contains protein levels ranging from 7% to 21%, depending on the variety, growing condition, maturity of seeds, and species. This review looks closely into the current protein extraction technologies used on three popular millets: pearl, foxtail, and proso millet. Aside from the extraction technologies, this review provides a thorough critical analysis of structural and techno-functional properties of millet proteins, namely, solubility, emulsifying activity, water and oil holding capacity, and foaming ability, which are important attributes impacting the potential use of millet proteins in food and beverages. Thus, the insights gained from this review provide a strong foundation for future research and development on pearl, foxtail, and proso millet as an ingredient in a wide range of gluten-free food products, meat analogue formulations, and nutraceuticals.

Maceration and fractionation technologies in a demonstration-scale green biorefinery: Proteins, sugars, and lipids extraction and energy efficiency

Different configurations of maceration and wet fractionation of green biomasses were assessed at a demonstration scale intending to extend the extraction of leaf protein concentrate. Two maceration methods, one-stage and a multistage maceration, were investigated on six distinct green forage types. The study aimed to enhance the extraction of leaf protein concentrate for use as a soybean replacement in monogastric feed, while also extracting simple sugars and lipids for potential further value creation. The use of multistage maceration scenarios promoted a more robust mechanical disintegration of the plant biomass, leading to higher extraction of the soluble compounds. The highest crude protein extraction efficiencies were seen in red clover (29 %) and festulolium (28 %). While grass fiber had higher lignocellulosic content than legume fiber, additional maceration and fractionation did not further break down cellulose, which remained predominantly in the fiber fraction across all biomasses. The higher energy requirements associated with more advanced processes are offset by the significant increase in protein yields.

Protein extraction from lupin (Lupinus angustifolius L.) using combined ultrasound and microwave techniques: Impact on protein recovery, structure, and functional properties

Protein extraction from lupin (Lupinus angustifolius L.) using combined ultrasound and microwave techniques: Impact on protein recovery, structure, and functional properties

Comparative extraction of antioxidant proteins from whole frogs (Rana ridibunda Pollas).

The forest frog (Rana ridibunda Pollas) is a traditional medicinal source rich in active protein compounds. In order to extract these compounds, six extraction methods were employed, including freeze-thaw and stirring techniques. Three different solvents were utilized in this process: 0.15 M sodium chloride (NaCl), 0.05 M phosphate buffer (PB), and 0.05 M phosphate-buffered saline (PBS). The objective was to identify the most effective extraction method. The extraction efficiencies, protein content, structure, and physicochemical properties of the extracts were compared. Additionally, antioxidant activity and free amino acid composition were analyzed. The highest-scoring extract, denoted as M1, obtained through freeze-thaw extraction using 0.15 M NaCl, exhibited an extraction rate of 7.79 ± 0.71% and a protein content of 60.36 ± 2.12%. M1 also showed antioxidant activity against DPPH˙, ABTS+˙, and ˙OH free radicals, with IC50 values of 0.41, 0.41, and 0.39 mg mL-1, respectively. The freeze-thaw extraction method utilizing 0.15 M NaCl has been identified as effective for extracting proteins from dried forest frogs, confirming their potential as a source of antioxidant proteins for scientific research and application.

Assembly of oleosin during efficient extraction: Altering the sequence of defatting solvents

During the extraction of membrane proteins from oil bodies (OBs), organic solvents dissolve the lipid core and precipitate proteins through solvent stress. Here the effects of solvent type and defatting sequence on the composition and structure of membrane proteins were investigated via SDS-PAGE, FTIR, and SEM-EDS. High purity oleosin (86 %) was obtained by treatment first with a Floch solution and then with cold acetone and petroleum ether after twice washing OBs with urea. The 3D spatial structure of oleosin was predicted using AlphaFold 2, revealing that the secondary structure of oleosin was dominated by α-helices (>60 %). Oleosin consisted of two district types, with oleosin-H (16–17 kDa) being the part of the molecule with limited water solubility, while oleosin-L (13–14 kDa) constituted the non-soluble part. The results provided a technical means of efficient extraction of Camellia oleosins and selective separation of oleosin-L and oleosin-H.

Extraction of Total Protein from Cardiomyocytes and Western Blotting Analysis.

Using total protein lysates for Western blotting confers greater benefits than traditional approaches that use fractionated samples, where a portion of the sample is discarded during processing. By incorporating low-volume inputs, it is possible to identify any protein present in the sample where a suitable antibody is available. The inclusion of a calibration curve allows quantitative analyses to be undertaken. There are additional benefits including that only requiring a small input saves on precious samples. Using whole muscle lysates is necessary in order to ascertain specificity of antibodies for subsequent immunofluorescent methods.

Immunoproteomic analysis and identification of possible allergenic proteins in Artemisia annua pollen.

Artemisia annua (A. annua) is a wind-pollinated weed and a major allergen responsible for allergic respiratory diseases in Northern China. This study involved the separation of pollen proteins from A. annua utilizing SDS-PAGE and Coomassie Blue staining techniques. The effectiveness of extracting allergens from Artemisia annua pollen (AAP) were confirmed both in vivo and in vitro. A mouse model was established using A. annua pollen (AAP) extracts. In vitro, the interaction between allergenic proteins in the AAP extract and specific IgE antibodies present in patients' sera was analyzed, using IgE-immunoblotting and ELISA methods. Protein bands were subsequently analyzed by mass spectrometry. The recombinant Art an 3 (rArt an 3) protein was obtained via recombination, expression, and purification. Finally, the binding activity of rArt an 3 specific IgE was subsequently examined using Western blot and inhibition ELISA (iELISA). The electrophoretic profiles of AAP showed band patterns ranging from 10 to 70kDa, with the most prominet IgE-binding pollen proteins detected at approximately 12kDa. After stimulation of AAP, the sensitized group of mice exhibited significant allergic symptoms compared to the control group. Mass spectrometry analysis identified 7 proteins, including putative aldehyde oxidase Art an 7, Art v 1-like protein, Art an 3.0101 allergen precursor, Art an 3.0102 allergen precursor, Art an 3.0201 allergen precursor, lipid transfer protein 3, and non-specific lipid-transfer protein. The results of immunoblotting and iELISA showed that rArt an 3 could bind to IgE antibodies in the patient sera, and co-incubation of rArt an 3 with serum, the binding serum significantly inhibited IgE binding to the crude AAP. The Art an 3 is a key allergenic protein in AAP with a high IgE sensitization rate in the studied population sample. These findings enhance our understanding of the sensitization components of AAP and its sensitization characteristics within the Chinese population, thereby promoting the development of precise molecular diagnostics and therapeutic interventions.

Effects of Bacillus subtilis addition to milk replacer on growth performance, nutrient digestibility, intestinal microbiota, and short-chain fatty acid concentration of Hu lambs

We aimed to investigate the effects of adding Bacillus subtilis to milk replacers on the incidence of diarrhea, growth performance, intestinal microbiota composition, and short-chain fatty acid (SCFA) production in newborn Hu lambs. Thirty newborn lambs with similar genetic backgrounds and initial body weights were selected and nursed by their dams for 4 days before transitioning to a milk replacer. At age 7 days, the lambs were randomly assigned to two experimental groups of 15 lambs each: CON (basic milk replacer feeding) and BS (basic milk replacer feeding supplemented with 0.2% B. subtilis). At age 28 days, eight lambs in each group were randomly euthanized, and their colonic and cecal contents were collected for microbiological and SCFA analyses. The lambs were monitored daily to determine the incidence of diarrhea. The BS group showed significantly increased average daily feed intake and gain and significantly reduced incidence of diarrhea. Furthermore, dry matter digestibility and crude protein and ether extract content increased in the BS group compared with those in the CON group. Increased acetate and total SCFA concentrations were noted in the colon and cecum, with elevated propionate levels specifically in the cecum. Microbiota analysis revealed that B. subtilis supplementation enhanced microbial diversity and beneficially modulated bacterial communities. Potentially harmful bacteria showed significantly decreased abundance in the colon (Campylobacterota by 11.09% and Fusobacteriota by 2.98%), whereas beneficial bacteria exhibited increased abundance (Firmicutes by 8.9%, Actinobacteriota by 3.59%, Lactobacillus by 5.92%, and Bifidobacterium by 3.18%). Similar trends were observed in the cecum, with decreases in Campylobacterota (4.82%) and Fusobacteriota (3.12%) and increases in Bacteroidota (4.11%), Lactobacillus (3.75%), and Prevotella (3.59%). These findings indicate that adding 0.2% B. subtilis to the milk replacer is an effective approach for enhancing lamb growth performance, maintaining intestinal health, and improving the intestinal microbiota.

Application of Proteomic Methods in Oomycete Biology.

The biochemical makeup of any organism provides insight into key factors regarding its biological functions. These factors can be explored using proteomics, which allows us to obtain a snapshot of the protein content and abundance in an organism, cell type or sub-cellular compartment. Here, we describe proteomic methodologies that can be used to dissect the biochemical mechanism of phytopathogenicity in oomycetes. These methodologies include protein extraction, purification, subsequent processing, mass spectrometry analysis, and qualitative and quantitative data processing of oomycete proteomes for comparative studies. Additionally, the use of mass spectra to assist in gene validation and modelling in unfinished oomycete genomes is also described.

An Assay System for Plate-based Detection of Endogenous Peptide:N-glycanase/NGLY1 Activity Using A Fluorescence-based Probe.

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals), an amidase classified under EC:3.5.1.52, is a highly conserved enzyme across eukaryotes that catalyzes the removal of N-glycans from glycoproteins, converting N-glycosylated asparagine residues into aspartic acid. This enzyme also plays a role in the quality control system for nascent glycoproteins. Despite the development of non-radioisotope-based assay systems such as those using S-alkylated RNase or fluorescent-labeled glycopeptides as substrates, these methods are incompatible with crude enzyme sources, primarily due to the degradation of reaction products by contaminating endogenous proteases. We previously developed an assay system using a 5-carboxyfluorescein-labeled glycosylated cyclo-heptapeptide (5FAM-GCP), a substrate remarkably resistant to endogenous peptidase activity. This system enables the accurate measurement of endogenous NGLY1 activity in various samples, including cell lines, tissues, peripheral blood mononuclear cells, and NGLY1-deficient patient-derived cells, without the interference of proteolytic degradation. We recently advanced this approach by producing a novel fluorescence resonance energy transfer (FRET)-based GCP probe (fGCP) and demonstrated its ability to detect endogenous NGLY1 activity across diverse enzyme sources via fluorescence on multiarray plates. This innovative and straightforward assay now offers reliable disease diagnostics and also allows the measurement of endogenous PNGase/NGLY1 activities across various organisms. Key features • fGCP assay enables measurement of endogenous PNGase/NGLY1 activity in cells and tissues. • An aliquot of 1-5 × 106 cells or 50-100 μg of protein extract from tissues is used for this assay. • This assay enables microplate-based real-time measurement of endogenous PNGase/NGLY1 activities. • This protocol requires a fluorescence plate reader equipped with an incubation function.

Recovery of Proteases and Protease Inhibitors from Ganoderma spp. Cultivated in Amazonian Lignocellulose Wastes.

Ganoderma spp. are a great source of bioactive molecules. The production and recovery of bioactive molecules vary according to strain, growth substrate, and extraction solution. Variations in protease and their inhibitors in basidiomata from a commercial strain (G. lingzhi) and an Amazonian isolate (Ganoderma sp.) cultivated in Amazonian lignocellulosic wastes and extracted with different solutions are plausible and were investigated in our study. Basidiomata from cultivation in substrates based on açaí seed, guaruba-cedro sawdust and three lots of marupá sawdust were submitted to extraction in water, Tris-HCl, and sodium phosphate. Protein content, proteases, and protease inhibitors were estimated through different assays. The samples were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR). Tris-HCl provided higher protein extraction from Ganoderma sp. and higher caseinolytic, gelatinolytic, and fibrinolytic activity for G. lingzhi cultivated in açaí. Water extracts of Ganoderma sp., in general, exhibited higher trypsin and papain inhibitor activities compared to G. lingzhi. Extracts in Tris-HCl and sodium phosphate showed more intense protein bands in SDS-- PAGE, highlighting bands of molecular weights around 100, 50, and 30 kDa. FTIR spectra showed patterns for proteins in all extracts, with variation in transmittance according to substrate and extractor. Water extract from Amazonian Ganoderma sp. cultivated in marupá wastes are promising as a source of protease inhibitors, while the Tris-HCL extract of G. lingzhi from açaí cultivation stands out as a source of proteases with fibrinolytic, caseinolytic, and gelatinolytic activities.

Efeito do ultrassom aplicado a baixa temperatura na extração e modificação de proteinas de sub-produtos da indústria pesqueira

ABSTRACT: This study explored the use only of ultrasound (US) for extracting proteins from grass carp (Ctenopharyngodon idella) backbones at low temperature and examined its impact on hydrophobicity, solubility, electrophoretic profile, and sulfhydryl levels. Backbones were treated with 35 and 130 kHz for 20, 30, and 40 minutes at 14 ºC, resulting in two protein fractions: solid (TPS) and liquid (TPL). US increased yield compared to the non-sonicated fraction. TPL (35 kHz) exhibited a 16% reduction in total and free sulfhydryl levels and a 25% increase in hydrophobicity. US induced protein conformation and band intensity alterations, particularly between 25 to 100 kDa for TPL at 130 kHz and below 30 kDa for TPS at 35 kHz. This study demonstrated the efficacy of US for protein extraction from fish by-products and its capacity to modify protein characteristics, facilitating the development of innovative food products.