Soluble Protein Fraction Research Articles

Peptidyl arginine deiminase 4 deficiency protects against subretinal fibrosis by inhibiting Müller glial hypercitrullination.

Retinal scarring with vision loss continues to be an enigma in individuals with advanced age-related macular degeneration (AMD). Müller glial cells are believed to initiate and perpetuate scarring in retinal degeneration as these glial cells participate in reactive gliosis and undergo hypertrophy. We previously showed in the murine laser-induced model of choroidal neovascularization that models wet-AMD that glial fibrillary acidic protein (GFAP) expression, an early marker of reactive gliosis, increases along with its posttranslational modification citrullination. This was related to increased co-expression of the citrullination enzyme peptidyl arginine deiminase-4 (PAD4), which also colocalizes to GFAP filaments. However, whether such hypercitrullination in Müller glial drives fibrotic pathology has remained understudied. Here, using male and female C57Bl6 mice subjected to laser injury, we investigated in a temporal study how citrullination impacts GFAP and PAD4 dynamics. We found that high molecular weight citrullinated species that accumulate in Müller glia corresponded with dynamic changes in GFAP and PAD4 showing their temporal redistribution from polymeric cytoskeletal to soluble protein fractions using immunostaining and western blot analysis. In conditional glial-specific PAD4 knockout (PAD4cKO) mice subjected to laser injury, there was a stark reduction of citrullination and of polymerized GFAP filaments. These injured PAD4cKO retinas showed improved lesion healing, as well as reduced fibronectin deposition in the subretinal space at 30 days. Taken together, these findings reveal that pathologically overexpressed PAD4 in reactive Müller glia governs GFAP filament dynamics and alters their stability, suggesting chronic PAD4-driven hypercitrullination may be a target for retinal fibrosis.

Development of a protein concentrate for human consumption by direct enzymatic hydrolysis of antarctic krill (Euphausia superba)

Krill protein concentrates could be part of human diets as high-quality food ingredients. We have focused on food-grade enzyme hydrolysis of Antarctic krill Euphausia superba, aiming at the production of well-defined protein, phospholipid, and shell fractions. Different enzyme combinations, concentrations, and reaction times of three commercial proteases have been tested for effects on protein yield and lipid content in the soluble protein fraction. The highest protein yield (10.1 g/100 g of the initial wet weight) was obtained in the highest enzyme concentration and longest reaction time treatment, however, associated with lower cost benefits. The lipid content in the protein fractions was not statistically different among treatments, ranging within 9.2–15.8 g/100 g wet weight. The protein fractions were screened for antioxidative capacity by ORAC and minimum stable astaxanthin content. Two of the protein concentrates were fractionated by size exclusion chromatography, to define their peptide profiles. 91% of the total peptides for the single enzyme treatment and 95% after combined enzyme treatment had predominant molecular weights below 1 kDa. The detection of crustacean tropomyosin was strongly reduced after enzymatic hydrolysis in all samples. As part of food safety assessment, all hydrolysates were well tolerated by human monocytes, having no impairing effect on their viability.

Complex coacervation of pea protein and pectin: Effect of degree and pattern of free carboxyl groups on biopolymer interaction

In the field of microencapsulation, recently there has been an increasing interest in using plant-based biopolymers for complex coacervation. Since complex formation is based on electrostatic interactions, pectin and pea protein are generally considered as promising materials. However, electrostatic interactions are influenced by several biopolymer characteristics such as net charge and local charge density. Therefore, the aim of the study was to investigate the impact of degree of methoxylation (DM) and pattern of free carboxyl groups of pectin on complex coacervation with soluble pea protein fractions. Six pectin samples varying in DM 62%, 50%, 34% and local charge density (random or blockwise pattern) as well as soluble pea protein fractions were prepared under defined conditions. Interactions were qualitatively tested by turbidity measurement, by visual observation and microscopy. Isothermal titration calorimetry (ITC) allowed a quantitative evaluation of the interactions. A high protein share enhanced protein-pectin interactions, the optimum depended on pectin DM and was found for a mass ratio of 8:1 to 12:1. With respect to pectin, highest level of interaction was observed for pectin with a DM of 50%. The interaction was more pronounced for pectin with random than with blockwise pattern of free carboxyl groups. ITC measurements revealed a higher number of binding sites and a higher binding affinity with protein for pectin with random pattern of free carboxyl groups.

Physico-chemodiversity variation between the most common calcareous red seaweed, Eastern Harbor, Alexandria, Egypt

The present study sought to detect the difference in physicochemical properties and the proximate compositions of common calcareous red seaweeds "Corallina officinalis, Jania rubens, and Amphiroa rigida". J. rubens was recorded highly water (WHC), and oil holding capacity (OHC) (1.619 ± 0.08 g g−1 DW, 3.1 ± 0.50 g g−1 DW, respectively) than those other species relating to the hydrophilic nature of polysaccharides, whereas swelling water capacity (SWC) was higher in A. rigida (5.25 ± 0.38 mL g−1 DW). A higher value of carbohydrates (49.88 ± 2.56% DW) was observed in J. rubens, Contrariwise, protein (36.99 ± 1.36% DW) and lipid contents (5.85 ± 0.49% DW) were higher in C. officinalis. Albumin and protamine protein fractions were higher in J. rubens specimens (45.11 ± 2.29 mg g−1 & 0.0014 ± 0.0007 mg g−1 DW, respectively) than other species. While globulin and glutilin (31.70 ± 1.90 mg g−1 DW & 41.93 ± 2.20 mg g−1 DW, respectively) were high in A. rigida. Contrariwise, insoluble protein fraction was high in C. officinalis (9.50 ± 0.50 mg g−1 DW). J. rubens specimens were recorded maximum values of the photosynthetic pigments. The different surface types and elemental analysis of three species were examined by Scanning electron microscopy (SEM) and Energy dispersive X-ray spectroscopy (EDX). From fourier transform infrared (FTIR) spectroscopy the S–S stretching peak of disulfides group at 462 cm−1 was the fingerprint of J. rubens. From GC-MS data A.rigida possesses 16 bioactive components with biological properties. As a result, J. rubens and A. rigida could be employed as an ingredient in functional foods and drug manufacture.

APOE4 exacerbates α‐synuclein seeding activity and contributes to neurotoxicity in Alzheimer’s disease with Lewy Body pathology

AbstractBackgroundAccumulating evidence demonstrates that 40‐50% of Alzheimer’s disease (AD) brains present with concomitant Lewy pathologies at autopsy, suggesting that α‐synuclein (α‐SYN) aggregation is likely involved in the pathogenesis of AD. Interestingly, the genome‐wide association study (GWAS) revealed that the ε4 allele of APOE (APOE4) gene, the strongest genetic risk factor for AD, is also the most replicated genetic risk factor for Lewy body dementia (LBD), signifying the important role of APOE4 in both amyloid β (Aβ) and α‐SYN pathogenesis. However, how APOE4 modulates the α‐SYN aggregation in AD is still unclear.MethodWe performed biochemical analyses measuring α‐SYN levels on a pathologically confirmed human AD cohort (N = 469) obtained from the Mayo Clinic Brain Bank, wherein 54.4% of our cohort presented with co‐morbid α‐SYN pathology upon diagnosis of AD with Lewy Body pathology (hereinafter referred to as AD+LB), which was subdivided into APOE4 non‐carriers (APOE4−) and carriers (APOE4+). To determine the seeding activity of α‐SYN, we performed Real‐time quaking‐induced conversion (RT‐QuIC) assays with the soluble TBS protein fractions of the brain lysates. The cytotoxicities of the amplified α‐SYN seeds were tested with MTT assay for cell viability and neuronal outgrowth measurement by immunofluorescence staining with human induced pluripotent stem cells (iPSC)‐derived neurons.ResultWe found that AD+LB cases showed the significantly higher seeding activity of α‐SYN compared to AD cases without LB pathologies. Furthermore, APOE4+ AD+LB brains had the highest α‐SYN seeding activity compared to AD+LB APOE4− cases. The α‐SYN seeding activity was associated with the level of α‐SYN, tau, or APOE aggregates, but not Aβ. Finally, we showed that the amplified α‐SYN aggregates derived from AD+LB APOE4+ had highest protease resistance and cytotoxicities in iPSC‐derived neurons.ConclusionThese findings suggest that APOE4 modulates the conformation of α‐SYN seeds which leads to high seeding activity and toxicity of α‐SYN in AD.

The impact of the soluble protein fraction and kernel hardness on wheat flour starch digestibility

Wheat is the staple crop for 35% of the world’s population, providing a major source of calories, mainly in the form of starch. The digestibility of wheat starch varies between different flours and products. Wheat products that are rapidly digested elicit large post-prandial glucose peaks associated with metabolic disorders. We investigated the impact of protein on starch digestion in three commercial flours with different grain hardness. A soluble extract of wheat proteins reduced starch digestion, even following gastric proteolysis. This extract was enriched in proteinaceous α-amylase inhibitors which were partially degraded during gastric proteolysis. Starch digestion kinetic analysis was carried out for flour samples pre-treated with different pepsin activities. The rate of starch digestion was altered following pepsin pre-digestion, and the extent of starch digestion increased in response to pepsin pre-digestion. We conclude that soluble proteinaceous alpha-amylase inhibitors present in wheat can escape gastric digestion and significantly contribute to reducing starch digestion in the small intestine.

Stress-inducible phosphoprotein 1 (HOP/STI1/STIP1) regulates the accumulation and toxicity of α-synuclein in vivo

The predominantly pre-synaptic intrinsically disordered protein α-synuclein is prone to misfolding and aggregation in synucleinopathies, such as Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB). Molecular chaperones play important roles in protein misfolding diseases and members of the chaperone machinery are often deposited in Lewy bodies. Here, we show that the Hsp90 co-chaperone STI1 co-immunoprecipitated α-synuclein, and co-deposited with Hsp90 and Hsp70 in insoluble protein fractions in two mouse models of α-synuclein misfolding. STI1 and Hsp90 also co-localized extensively with filamentous S129 phosphorylated α-synuclein in ubiquitin-positive inclusions. In PD human brains, STI1 transcripts were increased, and in neurologically healthy brains, STI1 and α-synuclein transcripts correlated. Nuclear Magnetic Resonance (NMR) analyses revealed direct interaction of α-synuclein with STI1 and indicated that the STI1 TPR2A, but not TPR1 or TPR2B domains, interacted with the C-terminal domain of α-synuclein. In vitro, the STI1 TPR2A domain facilitated S129 phosphorylation by Polo-like kinase 3. Moreover, mice over-expressing STI1 and Hsp90ß presented elevated α-synuclein S129 phosphorylation accompanied by inclusions when injected with α-synuclein pre-formed fibrils. In contrast, reduced STI1 function decreased protein inclusion formation, S129 α-synuclein phosphorylation, while mitigating motor and cognitive deficits as well as mesoscopic brain atrophy in α-synuclein-over-expressing mice. Our findings reveal a vicious cycle in which STI1 facilitates the generation and accumulation of toxic α-synuclein conformers, while α-synuclein-induced proteostatic stress increased insoluble STI1 and Hsp90.

Dynamic Change in Starch Biosynthetic Enzymes Complexes during Grain-Filling Stages in BEIIb Active and Deficient Rice.

Starch is the predominant reserve in rice (Oryza sativa L.) endosperm, which is synthesized by the coordinated efforts of a series of starch biosynthetic-related enzymes in the form of a multiple enzyme complex. Whether the enzyme complex changes during seed development is not fully understood. Here, we investigated the dynamic change in multi-protein complexes in an indica rice variety IR36 (wild type, WT) and its BEIIb-deficient mutant (be2b) at different developmental stages. Gel permeation chromatography (GPC) and Western blotting analysis of soluble protein fractions revealed most of the enzymes except for SSIVb were eluted in smaller molecular weight fractions at the early developing stage and were transferred to higher molecular weight fractions at the later stage in both WT and be2b. Accordingly, protein interactions were enhanced during seed development as demonstrated by co-immunoprecipitation analysis, suggesting that the enzymes were recruited to form larger protein complexes during starch biosynthesis. The converse elution pattern from GPC of SSIVb may be attributed to its vital role in the initiation step of starch synthesis. The number of protein complexes was markedly decreased in be2b at all development stages. Although SSIVb could partially compensate for the role of BEIIb in protein complex formation, it was hard to form a larger protein complex containing over five proteins in be2b. In addition, other proteins such as PPDKA and PPDKB were possibly present in the multi-enzyme complexes by proteomic analyses of high molecular weight fractions separated from GPC. Two putative protein kinases were found to be potentially associated with starch biosynthetic enzymes. Collectively, our findings unraveled a dynamic change in the protein complex during seed development, and potential roles of BEIIb in starch biosynthesis via various protein complex formations, which enables a deeper understanding of the complex mechanism of starch biosynthesis in rice.

Protein citrullination marks myelin protein aggregation and disease progression in mouse ALS models

Increased protein citrullination (PC) and dysregulated protein arginine deiminase (PAD) activity have been observed in several neurodegenerative diseases. PC is a posttranslational modification catalyzed by the PADs. PC converts peptidyl-arginine to peptidyl-citrulline, thereby reducing the positive charges and altering structure and function of proteins. Of the five PADs, PAD2 is the dominant isoform in the central nervous system (CNS). Abnormal PC and PAD dysregulation are associated with numerous pathological conditions, including inflammatory diseases and neurodegeneration. Animal model studies have shown therapeutic efficacy from inhibition of PADs, thus suggesting a role of PC in pathogenesis. To determine whether PC contribute to amyotrophic lateral sclerosis (ALS), a deadly neurodegenerative disease characterized by loss of motor neurons, paralysis, and eventual death, we investigated alterations of PC and PAD2 in two different transgenic mouse models of ALS expressing human mutant SOD1G93A and PFN1C71G, respectively. PC and PAD2 expression are altered dynamically in the spinal cord during disease progression in both models. PC and PAD2 increase progressively in astrocytes with the development of reactive astrogliosis, while decreasing in neurons. Importantly, in the spinal cord white matter, PC accumulates in protein aggregates that contain the myelin proteins PLP and MBP. PC also accumulates progressively in insoluble protein fractions during disease progression. Finally, increased PC and PAD2 expression spatially correlate with areas of the CNS with the most severe motor neuron degeneration. These results suggest that altered PC is an integral part of the neurodegenerative process and potential biomarkers for disease progression in ALS. Moreover, increased PC may contribute to disease-associated processes such as myelin protein aggregation, myelin degeneration, and astrogliosis.

Influence of the fractionation method on the protein composition and functional properties

Mild wet fractionation can separate pea proteins into a soluble and a non-soluble fraction. Soluble proteins and solutes are extracted into the soluble protein fraction, insoluble proteins are collected in the non-soluble protein fraction. The protein composition in the respective fractions was investigated by further fractionating the protein fractions with isoelectric precipitation. The molecular mass of the proteins in the fractions was determined by SDS-PAGE and size exclusion chromatography. The protein fractions' solubility was determined in excess water, and the gelation properties were investigated at the same protein concentration. With mild wet fractionation, compared to conventional wet fractionation, both globulins and albumins are extracted. The protein fractions were mixtures of both proteins, although the non-soluble protein fraction contained mainly globulins. Solubility and gelation properties varied with protein type, composition and protein state; isoelectric precipitation decreased the gelation capacity of the protein fractions.

Municipal wastewater treatment and fouling in microalgal-activated sludge membrane bioreactor: Cultivation in raw and treated wastewater

Since the advent of conventional activated sludge biological treatment plants, their low efficiency in nitrogen and phosphorus removal has become a significant concern. Therefore, in this study, the performance of microalgal-activated sludge membrane bioreactor (MAS-MBR) as a self-biological treatment or as a post-treatment for conventional biological treatments was investigated. A continuous MAS-MBR with two ratios of algae/sludge (only microalgae and 5:1) was used to treat raw and treated wastewater. The optimum case was achieved with the cultivation of mixed algae/sludge in raw wastewater (R2 case). In this case, the ammonium and phosphorus removal efficiencies were 94.36 ± 3.5 and 88.37 ± 3 %, respectively. Furthermore, the highest oxygen production rate (OPR) and oxygen uptake rate (OUR) values were obtained in this case (145.58 ± 3 and 68.43 ± 1.5 mgO2/lit.d, respectively). In addition, membrane fouling was adversely increased in R2 compared with other cases, indicating the necessity of membrane cleaning for high-performance bioreactors in nutrient removal. Transmembrane pressure (TMP) jump in R2 was captured after 14 days, while the results supported that the membrane fouling was influenced by raised production of carbohydrate fraction of soluble microbial products (SMPc) and protein fraction of extracellular polymeric substances (EPSp) at first and last stages, respectively. Therefore, although the MAS-MBR is a potential candidate as a self-biological treatment, the membrane fouling is high in this case.

Development and application of criteria for classification of hydrolysed proteins in the framework of feed safety

In the view of a circular economy, there is an increasing need for (re-)using animal by-products that have a wide range of applications and sufficient safety. Hydrolysates of animal proteins (HPs) are frequently used as feed ingredients. Nevertheless, clear criteria for legal use and methods for monitoring feed applications are not available. Here, a range of methods have been used and evaluated for characterizing a set of 26 samples of hydrolysed proteins, ‘hydrolysed’ feather meals and processed animal proteins (PAPs), with verification based on an additional set of eight samples. Methods included determination of ash content, sediment (mineral fraction) content, protein content, species identity, solubility, protein solubility, size exclusion chromatography and polyacrylamide gel electrophoresis (SDS-PAGE). After a comparison of results obtained with water and SDS, water was chosen as the solvent for environmental and occupational reasons. Typical HP samples have a protein content higher than 60%, a solubility exceeding 50% and a virtual absence of a mineral fraction. The first discrimination between HPs and PAPs could be based on the absence or presence, respectively, of a mineral fraction. An approach for HP characterization is designed using a Hydrolysation Index (HI) based on the fraction of peptides smaller than 10 kDa, the solubility of the sample and the fraction of soluble proteins. A simplified version (HIs), exclusively based on the fraction of peptides smaller than 10 kDa and the solubility of the sample, shows a trend among the samples highly comparable to HI. Values for HI and HIs exceeding 60% would characterise HPs. Feather meals, which are heat treated instead of treatment by a chemical process of hydrolysation, range among the PAPs and should not be indicated as “hydrolysed.” The HIs can be used as an easy parameter for classifying HPs and for legal enforcement.

Protein-fat concentrate for enrichment of wheat flour

Wheat flour, especially high grade flour, is significantly impoverished in macro-, micronutrients and other valuable components such as essential amino acids, essential fatty acids, vitamins, minerals, soluble and insoluble dietary fibers. To enrich wheat flour, the authors produced lentil-flax flour (protein-fat concentrate) with the high protein (27.5%) and fat (11.9%) content. The developed technological scheme of the combined grinding of lentil (67%) and flax (33%) seeds included the I–III break systems, sizing system, scratch system, 1–3 reduction systems. When studying physico-chemical and biochemical indicators, it was found that the protein-fat concentrate was close to the corresponding indicators of wheat flour by mechanical characteristics. A significant proportion of the albumin-globulin fraction of soluble proteins (88%), as well as the high content of the most deficient essential α-linolenic acid (6.11% compared to 0.05% in wheat flour with regard to the total fat content) suggest the high nutritional and biological value of the obtained product. Evaluation of an effect of the protein-fat concentrate on the indicators of the finished products (crackers) showed that its introduction in an amount of 15% of the total flour volume not only did not worsen but even improved sensory indices by several criteria. The high total sensory score (32 points) indicates the standard quality of the obtained product. Addition of lentil-flax flour in an amount of 15% of total flour weight in production of bakery products as well as bakery confectionary products will allow extending the assortment of balanced farinaceous products, significantly reducing the deficiency of essential α-linolenic acid in diets upon their consumption according to the physiological norms.

Characterization of soluble and insoluble fractions obtained from a commercial pea protein isolate

In this study, the characteristics of pea protein isolates after aqueous fractionization into water-soluble and water-insoluble fractions by centrifugation, decantation, and lyophilization were studied. Chemical composition and physicochemical properties upon pH changes were determined. The overall protein compositions of both soluble and insoluble pea protein fractions were similar containing albumins, globulins, and lipoxygenases, but amino acid compositions slightly varied. Distinct differences were observed in their charge properties, particle sizes, and voluminosities. The soluble pea protein fraction was free of measurable particles at pH 7, whereas the insoluble proteins contained particles with sizes of > 80 µm. Close to their respective isoelectric points, the soluble (pI = 3.9) and insoluble pea proteins (pI = 4.9) had very similar sizes of 40–50 µm. At pH 3, the particle sizes of soluble proteins did not change, however, the insoluble pea proteins had again sizes of > 80 µm. Voluminosity of the insoluble fraction was pH-dependent and had its highest voluminosity at pH 3 and 7, indicating changes in water binding as a function of pH. In contrast, the voluminosity of the soluble pea protein fraction did not change with pH. Taken together, this study showed that water-soluble and insoluble pea protein fractions of a commercial isolate may differ substantially with respect to their physicochemical properties. Observed inconsistencies in technofunctionality of various commercial preparations could thus be promoted by varying ratios between the two fractions.

Aberrant NOVA1 function disrupts alternative splicing in early stages of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.

Secretion of the disulfide bond generating catalyst QSOX1 from pancreatic tumor cells into the extracellular matrix: association with extracellular vesicles and matrix proteins.

Quiescin sulfhydryl oxidase 1 (QSOX1) is a disulfide bond generating catalyst that is overexpressed in solid tumors. Expression of QSOX1 is linked to cancer cell invasion, tumor grade, and extracellular matrix (ECM) protein deposition. While the secreted version of QSOX1 is known to be present in various fluids and secretory tissues, its presence in the ECM of cancer is less understood. To characterize secreted QSOX1, we separated conditioned media based on size and density. We discovered that the majority of secreted QSOX1 resides in the EV-depleted fraction and in the soluble protein fraction. Very little QSOX1 could be detected in the EVP fraction. We used immunofluorescence to image subpopulations of EVs and found QSOX1 in Golgi-derived vesicles and medium/large vesicles, but in general, most extracellular QSOX1 was not attributed to these vesicles. Next, we quantified QSOX1 co-localization with the EV marker Alix. For the medium/large EVs, ~98% contained QSOX1 when fibronectin was used as a coating. However, on collagen coatings, only ~60% of these vesicles contained QSOX1, suggesting differences in EV cargo based on ECM coated surfaces. About 10% of small EVs co-localized with QSOX1 on every ECM protein surface except for collagen (0.64%). We next investigated adhesion of QSOX1 to ECM proteins in vitro and in situ and found that QSOX1 preferentially adheres to fibronectin, laminins, and Matrigel compared to gelatin and collagen. This mechanism was found to be, in part, mediated by the formation of mixed disulfides between QSOX1 and cysteine-rich ECM proteins. In summary, we found that QSOX1 (1) is in subpopulations of medium/large EVs, (2) seems to interact with small Alix+ EVs, and (3) adheres to cysteine-rich ECM proteins, potentially through the formation of intermediate disulfides. These observations offer significant insight into how enzymes, such as QSOX1, can facilitate matrix remodeling events in solid tumor progression.

Coagulansin-A improves spatial memory in 5xFAD Tg mice by targeting Nrf-2/NF-κB and Bcl-2 pathway

The present study was designed to investigate the underlying molecular signaling of Coagulansin-A (Coag-A) as a therapeutic agent against Alzheimer’s disease (AD). Preliminarily, it exhibited a neuroprotective effect against H2O2-induced oxidative stress in HT-22 cells. The in vivo studies were performed by administering Coag-A (0.1, 1, and 10 mg/kg) intraperitoneally to 5xFAD transgenic (Tg) mouse model. Coag-A (10 mg/kg) significantly attenuated the cognitive decline compared to Tg mice group in the shallow water maze (SWM) and Y-maze test paradigms. The anti-aggregation potential of Coag-A was determined by performing Fourier transform-infrared (FT-IR) spectroscopy and differential scanning calorimeter (DSC) analysis in the prefrontal cortex (PFC) and hippocampal (HC) regions of mice brain. The FT-IR spectra demonstrated the inhibition of amyloid beta (Aβ) through a decrease in β-sheet aggregation, along with the inhibition of changes in the lipids, proteins, and phospholipids. The DSC analysis displayed a low-temperature exotherm associated with the reversible process of aggregation of soluble protein fractions prior to denaturation. Furthermore, Coag-A treatment displayed a regular density of granule cells in H&E stained sections, along with a reduced amyloid load and PAS-positive granules in all the regions of interest in mice brain. The real-time polymerase chain reaction (q-PCR), western blot and immunohistochemical (IHC) analysis demonstrated antioxidant, anti-inflammatory, and anti-apoptotic effect of Coag-A by enhancing the expression of nuclear factor erythroid-2-related factor (Nrf-2) and reducing nuclear factor kappa B (NF-κB) and Bax protein expression. In addition, Coag-A significantly increased the antioxidant enzymes and proteins level, along with a reduced pro-inflammatory cytokines production.

Nutritional and antioxidant changes in lentils and quinoa through fungal solid-state fermentation with Pleurotus ostreatus

Solid-state fermentation (SSF) may be a suitable bioprocess to produce protein-vegetal ingredients with increased nutritional and functional value. This study assessed changes in phenol content, antinutrient content, biomass production and protein production resulting from the metabolic activity of Pleurotus ostreatus, an edible fungus, in lentils and quinoa over 14 days of SSF. The impact of particle size on these parameters was also assessed because the process was conducted in both seeds and flours. Fungus biomass increased during fermentation, reaching 30.0 ± 1.4 mg/g dry basis and 32 ± 3 mg/g dry basis in lentil grain and flour and 52.01 ± 1.08 mg/g dry basis and 45 ± 2 mg/g dry basis in quinoa seeds and flour after 14 days of SSF. Total protein content also increased by 20% to 25% during fermentation, in all cases except lentil flour. However, the soluble protein fraction remained constant. Regarding phytic acid, SSF had a positive impact, with a progressive decrease being higher in flours than in seeds. Regarding antioxidant properties, autoclaving of the substrates promoted the release of polyphenols, together with antioxidant activity (ABTS, DPPH and FRAP), in all substrates. However, these parameters drastically decreased as fermentation progressed. These results provide scientific knowledge for producing lentil- or quinoa-based ingredients with low antinutrient content enriched with protein fungal biomass.Graphical

Interrelating EPS, soluble microbial products and metal solubility in a methanogenic consortium stressed by nickel and cobalt

The relationships between extracellular polymeric substances (EPS), soluble microbial product production, metal solubility, and methanogenic activity were investigated. The individual, and joint, toxic effects of nickel and cobalt on methanogenic consortia fed with glucose as model substrate were studied using biomethane potential assays. Cobalt was found to be less toxic to methanogens than nickel at each concentration tested, and the combined effects of Ni and Co on methane production in the bimetal experiment was higher than the sum of the effects of each metal alone. The protein content of EPS, and extracellular soluble protein fractions, decreased with increasing concentrations of total metals. Meanwhile, no significant change in response to metal stress was apparent for carbohydrate content of EPS or extracellular soluble carbohydrate. Decreasing protein content of EPS was accompanied by reduced methanogenic activity and an increase in the soluble metal fraction. The strong associations observed between these variables could be due to the critical role of EPS in protecting microbial cells against nickel and cobalt stress, possibly by capturing metal cations through their functional groups, thus reducing metal availability to the microbial cells in the methanogenic consortia underpinning the anaerobic digestion process.

Effect of enzyme‐assisted hydrolysis on brewer's spent grain protein solubilization – peptide composition and sensory properties

This study aimed to valorize brewer's spent grain (BSG) from a side-stream into protein ingredients suitable for human consumption. The impact of protease treatments was studied for solubilizing BSG proteins. Treatment with Protamex or simultaneous co-incubation of Protamex and Flavourzyme solubilized up to 60% of the total protein in BSG, whereas co-incubation with Flavourzyme increased the availability of hydrophobic amino acids (Val, Phe, Ala, Leu, Ile) in extracts. The scale-up of protease treatments demonstrated comparable solubilized protein fractions in 0.1 L and 10 L reaction volumes. Thorough sequence-based peptide analysis by liquid chromatography-ion mobility-mass spectrometry resulted in the identification of 479 and 451 water-soluble peptides in the hydrolysates obtained with Protamex or co-incubation of Protamex and Flavourzyme, respectively. Main cutting sites on BSG proteins were identified between Leu-Gln, Tyr-Phe, Pro-X (Protamex), complementing with a variety of cutting sites mainly next to Gln, Pro, Ile, and Phe when combined with Flavourzyme. Uniform protease activity throughout the entire B-hordein sequence and the formation of peptides with varying sequence lengths did not increase the bitterness of the hydrolysates compared to the BSG sample with water extraction. These results support the characterization of enzymatic treatments in plant-based materials and the production of hydrolysates with desired composition.