kDa Cutoff Research Articles

Alternative Strategy To Explore Missing Proteins with Low Molecular Weight.

Identifying more missing proteins (MPs) is an important mission of C-HPP. With the number of identified MPs being attenuated year by year (2,949 to 2,129 MPs from 2016 to 2019), we have realized that the difficulty of exploring the remaining MPs is a challenge in technique. Herein, we propose a comprehensive strategy to effectively enrich, separate, and identify proteins with low molecular weights, aiming at the discovery of MPs. Basically, a protein extract from human placenta was passed through a C18 SPE column, and the bound proteins that were eluted were further separated with an SDS-PAGE gel or a 50 kDa cutoff filter. The separated proteins were subjected to trypsin digestion, and the MS/MS signals were searched against data sets with two different digestion modes (full-trypsin and semitrypsin). The strategy was adopted, resulting in the identification of 4 MPs with 8 unique peptides (≥2 non-nested unique peptides with ≥9 amino acids). Importantly, the identification of 6 out of 8 of the unique peptides derived from the MPs was further supported by parallel reaction monitoring, which confirmed the identification of 3 MPs from human placenta tissues (Q6NT89: TMF-regulated nuclear protein 1; A0A183: late cornified envelope protein 6A; and Q6UWQ7: insulin growth factor-like family member 2, mapped to chromosomes 1, 1, and 19, respectively). The three proteins ranged in length from 80 aa to 227 aa. The study not only establishes a feasible strategy for analyzing proteins with low molecular weights but also fills a small part of a large gap in the list of MPs. The data obtained in this study are available via ProteomeXchange (PXD014083) and PeptideAtlas (PASS01389).

Adsorption kinetics of platinum group elements onto macromolecular organic matter in seawater

Adsorption kinetics of the interaction between Pt, Pd and Rh (defined here as platinum group elements, PGEs) ions and macromolecular organic compounds (MOCs, >10 kDa), including humic acid, carrageenan and bovine serum albumin, and different cutoff fractions of natural organic matter (>1 kDa and >3 kDa) obtained from seawater using centrifugal ultrafiltration devices were investigated. For a given element, all the adsorption kinetics did not reach equilibrium except the interaction between Pt and >1 kDa cutoff, and between Pd and humic acid. For all the tested MOCs, the adsorption kinetics could be divided into two stages, a rapid adsorption process in the first 8 h and the desorption stage after the first 8 h until the equilibrium. The change trend of partition coefficient (log10Kd) values with experiment time was consistent with that of the kinetic curves. However, in the interaction between PGE ions and natural dissolved organic matter (NDOM), an obvious difference in the change trends of log10Kd and kinetic curves was observed. It indicated that the partition behavior of PGE ions interacting with NDOM in seawater was a combined effect of different organic constituents. The adsorption and log10Kd of PGEs in the >1 kDa NDOM fraction were higher and more stable than those in the >3 kDa NDOM fraction. The results also indicated that the 1–3 kDa NDOM may dominate the interaction between PGEs ions and NDOM. Moreover, no kinetic model could perfectly simulate the adsorption process. It indicated that the colloidal struction and morphology of MOCs or NDOM in seawater might be inhomogeneous. Hence, the interaction between PGE ions and organic matter in seawater was a complicated process and needs further research.

Fast purification of graphene oxide solution by continuous counter current hollow fibre dialysis: A step towards large scale production

AbstractContinuous countercurrent hollow fibre based dialysis was used to purify a graphene oxide (GO) solution rapidly. A 12 kDa cutoff membrane was used for this purpose. The deposited layer of GO particles and flakes acted as a dynamic membrane that entrapped electrolytes, to some extent. This method took 17 h to purify 1 L of GO solution compared to several weeks by batch dialysis without compromising purification efficiency. A one‐dimensional steady state model was used to estimate the overall mass transfer coefficient and predict the concentration profiles of feed and dialysate side. Thus, the demonstrated method was a step towards large scale production of GO.

Low-molecular-mass iron complexes in blood plasma of iron-deficient pigs do not originate directly from nutrient iron.

Nutrient iron entering the blood binds transferrin (TFN)d, which delivers iron to cells in the body. In healthy individuals, ∼30% of TFN is iron-bound while the remainder is unbound (apo-TFN). TFN saturates the plasma of individuals with iron-overload diseases such as hereditary hemochromatosis, prompting release of a poorly-defined low-molecular-mass (LMM) iron species called non-transferrin-bound iron (NTBI). An experiment was devised to directly detect NTBI in plasma of iron-deficient pigs and to assess the role of the liver which is known to bind NTBI. Catheters were surgically installed in the portal vein (PV) and either the caudal vena cava or the cranial vena cava. After the animals recovered, 57Fe II ascorbate was injected into the stomach via a feeding tube. Blood was removed through the catheters before and after injection; plasma became 57Fe-enriched after injection. 57Fe-enriched plasma was passed through a 10 kDa cutoff membrane and the flow-through solution (FTS) was subjected to size-exclusion liquid chromatography (LC). The eluent flowed into an ICP-MS where 56Fe and 57Fe were detected. Low-intensity iron peaks with masses of 400-1600 Da were observed, but none became enriched in 57Fe after injection. Rather, the injected 57Fe bound to apo-TFN. Viewed naively, this implies that nutrient-derived 57Fe in healthy mammals passes from the intestines to apo-TFN without first entering the blood as a LMM intermediate. In this case, nutrient iron exported from intestinal enterocytes of healthy individuals may quickly bind apo-TFN such that LMM iron species do not accumulate in blood plasma. Some 57Fe from the FTS may have adsorbed onto the column. In any event, the LMM iron species in plasma that eluted from the column must have originated from iron stored within the body, perhaps in macrophages - not directly from nutrient iron absorption. The liver absorbed and released LMM iron species, but the effect was modest, consistent with its role as a dynamic iron buffer. Passage through the liver also altered the distribution of different forms of TFN present in the PV.

Tunic extract of the host ascidian attracts the causal agent of soft tunic syndrome, Azumiobodo hoyamushi (Kinetoplastea: Neobodonida).

Azumiobodo hoyamushi, a kinetoplastid flagellate, is the causative agent of soft tunic syndrome, an infectious disease of the edible ascidian Halocynthia roretzi. The flagellate is thought to invade the tunic matrix via a damaged area of the tunic on the siphon wall. We hypothesized that the flagellate locates the tunic entry site by a chemotactic response to soluble substances diffused from the host ascidians. To investigate this hypothesis, we examined whether the flagellate shows a chemotactic response to tissue extracts (tunic and other tissues) from the host ascidian H. roretzi. We tested extracts from 5 tissues as well as hemolymph. Only the tunic extract showed significant positive chemotactic activity, and the activity decreased with increasing dilution. Furthermore, autoclaved tunic extract, extracts from diseased individuals, and extract from the styelid ascidian Styela clava also had chemotactic activity, although the activities were lower than that of tunic extract from healthy H. roretzi. Ultrafiltration of the tunic extract through a 3 kDa cutoff membrane completely abrogated the activity; the ultrafiltration retentate still showed activity. Thus, the soluble factors that attract the flagellate are present exclusively in the tunic extract, and the chemotactic factors are larger than 3 kDa. Our experiments also suggested that the tunic extract contains both heat-stable and heat-labile factors. We conclude that the flagellate locates the tunic entry site by chemotaxis toward soluble factors that diffuse from a damaged area of the tunic on the siphon wall.

Acute Inflammatory Biomarker Responses to Diffuse Traumatic Brain Injury in the Rat Monitored by a Novel Microdialysis Technique.

Neuroinflammation is a major contributor to the progressive brain injury process induced by traumatic brain injury (TBI), and may play an important role in the pathophysiology of axonal injury. The immediate neuroinflammatory cascade cannot be characterized in the human setting. Therefore, we used the midline fluid percussion injury model of diffuse TBI in rats and a novel microdialysis (MD) method providing stable diffusion-driven biomarker sampling. Immediately post-injury, bilateral amphiphilic tri-block polymer coated MD probes (100 kDa cut off membrane) were inserted and perfused with Dextran 500 kDa-supplemented artificial cerebrospinal fluid (CSF) to optimize protein capture. Six hourly samples were analyzed for 27 inflammatory biomarkers (9 chemokines, 13 cytokines, and 5 growth factors) using a commercial multiplex biomarker kit. TBI (n = 6) resulted in a significant increase compared with sham-injured controls (n = 6) for five chemokines (eotaxin/CCL11, fractalkine/CX3CL1, LIX/CXCL5, monocyte chemoattractant protein [MCP]1α/CCL2, macrophage inflammatory protein [MIP]1α /CCL3), 10 cytokines (interleukin [IL]-1α, IL-1β, IL-4, IL-6, IL-10, IL-13, IL-17α, IL-18, interferon [IFN]-γ, tumor necrosis factor [TNF]-α), and four growth factors (epidermal growth factor [EGF], granulocyte-macrophage colony-stimulating factor [GM-CSF], leptin, vascular endothelial growth factor [VEGF]). Therefore, diffuse TBI was associated with an increased level of 18 of the 27 inflammatory biomarkers at one through six time points, during the observation period whereas the remaining 9 biomarkers were unaltered. The study shows that diffuse TBI induces an acute increase in a number of inflammatory biomarkers. The novel MD technique provides stable MD sampling suitable for further studies on the early neuroinflammatory cascade in TBI.

Rapid and efficient removal of ionic impurities from graphene oxide through hollow fiber diafiltration

Graphene has tremendous importance due to its potential applications in electronics, catalysis, sensors, etc. On the other hand, graphene oxide (GO) or reduced graphene oxide (rGO) have increasing applications in various fields such as water treatment, catalysis, etc. Major bottleneck of large scale production is purification of GO and it is traditionally conducted in time consuming dialysis in small batches. The present work demonstrates utility and applicability of continuous hollow fiber diafiltration for the removal of ionic impurities from GO solution. 12 kDa cut off hollow fibers demonstrated the best results. Seven stages with 250 ml constant volume of diafiltration and five hours of filtration at 103 kPa transmembrane pressure drop and 10 L/h cross flow rate were found to be suitable conditions of purification. Produced GO had quality comparable to the commercial ones. Thus, continuous diafiltration was found to be an efficient and highly scalable GO purification technique.

ISOLATION AND PAPAIN DIGESTION OF NOVEL ANTIFUNGAL PEPTIDES FROM RED RADISH (RAPHANUS RAPHANISTRUM SUBSP SATIVUS) AND ANALYSIS OF SELECTIVE CYTOTOXICITY FOR CANCER TREATMENT

Treating cancer with chemotherapy is accompanied with side effects and drug resistance by cancer cells which call for alternative anticancer agents such as cationic antimicrobial peptides. In specific, antifungal peptides were chosen for cytotoxicity analysis due to their deteriorating effects on fungi (eukaryotic cells). Novel peptides were isolated and analysed from red radish seeds. Water soaked seeds were ground with phosphate buffer (pH 7) and the antifungal assay was carried out and an inhibition zone of 0.5±0.2 mm was noticed for 50 μl of the extract at 1 mg/ml concentration. The study was extended further, by digestion with papain, trypsin and pepsin with appropriate buffer and pH of which papain digests of red radish seeds produced a zone of inhibition of 1.00±0.2 mm at 500 μg/ml concentration, approximately. This sample was processed with a 3 kDa cut off membrane and the filtrate was confirmed for its antifungal activity (MIC 32 μg/ml). Further, for size confirmation, the sample was subjected to MALDI- TOF-MS analysis, and a peak was obtained at 1680 Da (1.68 kDa). To check the anticancer activity of the antifungal peptides obtained, the sample was tested on HeLa cell lines by method of MTT Assay and significant cell death was obtained at MIC 12.5 μg/ml which increased with higher concentrations of the sample. Thus, it is confirmed that red radish seed extract digested with papain is effective in inhibiting the growth of both fungal and cancer cell lines making them a good therapeutic agent against cancer.

A rapid separation and characterization of mucins from mouse submandibular glands by supported molecular matrix electrophoresis

Mucins are heavily glycosylated proteins with high molecular mass, and are involved in various diseases including infection, inflammation, and cancer. As easy separation method, such as gel electrophoresis, however, does not exist for mucins, due to their large molecular sizes and heterogeneities. In 2009, we published a supported molecular matrix electrophoresis (SMME) method that can be used to characterize mucins. For SMME analysis, mucins have been enriched by ultrafiltration of trypsin digests using a 100 KDa cutoff filter. However, this enrichment results in a loss of protein identification capability using proteomic approaches. In this study, we describe a simple enrichment of mucins without trypsinization for SMME analysis. The enrichment was developed using a porcine submandibular gland and then was applied to study and compare mouse submandibular glands between young and aged mice. From mouse submandibular glands, hyaluronic acid and some mucins were observed by SMME. One of the mucins was identified as MUC10 by proteomic analysis of the band on the SMME membrane and immunostaining using anti-MUC10 antibody. A major O-glycan of MUC10 was determined to be NeuAcα2-3Galβ1-3GalNAc. Furthermore, our experiments revealed that the concentrations of these molecules were lower in aged mice than in young mice, and that an unknown mucin-like molecule was detected only from the aged mouse submandibular gland.

HPLC study of uremic fluids related to optical dialysis adequacy monitoring.

The aim of this study was to investigate uremia-related high-performance liquid chromatography (HPLC) ultraviolet (UV) absorbance profiles of serum and spent dialysate and to study the removal of uremic retention solutes in connection with optical dialysis adequacy monitoring. 10 uremic patients were investigated using online spectrophotometry at a wavelength of 280 nm over the course of 30 hemodialysis treatments. The dialysate and blood samples were taken and analyzed simultaneously using standard biochemical methods and reversed-phase HPLC. Filters with cutoff at 3 kDa and 70 kDa were used for the pre-treatment of the serum. The chromatographic peaks were detected by a UV detector at wavelengths of 254 and 280 nm. This study indicated that the main solute responsible for UV absorbance in the spent dialysate is a low-molecular-weight, water-soluble, non-protein-bound compound uric acid (UA). Three additional uremic retention solutes - creatinine (CR), indoxyl sulphate (IS) and hippuric acid (HA) - were identified from the HPLC profiles. The number of detected HPLC peaks was not significantly different for a serum filtered through the 3 kDa or 70 kDa cutoff filters, and was lower for the spent dialysate, indicating that the molecular weight (MW) of the main UV chromophores in the uremic fluids did not exceed 3 kDa. The reduction ratio (RR) estimated by the total area of HPLC peaks at 254 nm and 280 nm in the serum and by the online UV absorbance at 280 nm was best related to the removal of small water-soluble non-protein bound solutes like urea (UR), CR and UA. The present study contributes new information on the removal of uremic retention solutes during hemodialysis and on the origin of the optical dialysis adequacy monitoring signal.

Low-molecular-mass iron in healthy blood plasma is not predominately ferric citrate.

Blood contains a poorly characterized pool of labile iron called non-transferrin-bound iron (NTBI). In patients with iron-overload diseases such as hemochromatosis, NTBI accumulates in the liver, heart, and other organs. This material is probably nonproteinaceous and low molecular mass (LMM). However, the number, concentration, mass, and chemical composition of NTBI species remain unknown despite decades of effort. Here, solutions of plasma from humans, pigs, horses, and mice were passed through a 10 kDa cutoff membrane, affording flow-through solutions (FTSs) containing ∼1 μM iron. The FTSs were subjected to size-exclusion liquid chromatography at pH 8.5, 6.5, and 4.5. Iron was detected by an online inductively-coupled-plasma mass spectrometer. LC-ICP-MS chromatograms of the FTSs exhibited 2-6 iron-containing species with apparent masses between 400 and 2500 Da. Their approximate concentrations in plasma were 10-8-10-7 M. Not every FTS sample contained every LMM iron species, indicating individual variations. The most reproducible iron species had apparent masses of 400 and 500 Da. Chromatograms of the FTSs from established hemochromatosis patients exhibited no significant differences relative to controls. The peak positions and intensities depended on column pH. Some FTS iron adsorbed onto the column, especially at higher pH. Column-adsorbing-iron coordinated apo-transferrin whereas the more tightly coordinated iron species did not. Ferric citrate standards exhibited LMM iron peaks that were similar to but not the same as those obtained in FTSs. The results indicate that the LMM iron species in healthy blood plasma is not primarily ferric citrate; however, this may be one of many contributing complexes.

Identification of Potent ACE Inhibitory Peptides from Wild Almond Proteins.

This study has shown the potential of wild almond proteins as good sources for producing ACE-inhibitory active peptides. According to this finding, peptides with higher ACE inhibitory activities could be released during the gastrointestinal digestion and contribute to the health- promoting activities of this natural protein source.

A multi-protease, multi-dissociation, bottom-up-to-top-down proteomic view of the Loxosceles intermedia venom

Venoms are a rich source for the discovery of molecules with biotechnological applications, but their analysis is challenging even for state-of-the-art proteomics. Here we report on a large-scale proteomic assessment of the venom of Loxosceles intermedia, the so-called brown spider. Venom was extracted from 200 spiders and fractioned into two aliquots relative to a 10 kDa cutoff mass. Each of these was further fractioned and digested with trypsin (4 h), trypsin (18 h), pepsin (18 h), and chymotrypsin (18 h), then analyzed by MudPIT on an LTQ-Orbitrap XL ETD mass spectrometer fragmenting precursors by CID, HCD, and ETD. Aliquots of undigested samples were also analyzed. Our experimental design allowed us to apply spectral networks, thus enabling us to obtain meta-contig assemblies, and consequently de novo sequencing of practically complete proteins, culminating in a deep proteome assessment of the venom. Data are available via ProteomeXchange, with identifier PXD005523.

Preparation of Intracellular Metabolite Extracts from Liquid Schizosaccharomyces pombe Cultures.

The success of metabolomic analysis relies heavily on the sample preparation protocol. Here we present a protocol for intracellular metabolite extraction from liquid fission yeast cultures based on rapid quenching in pure methanol at -40°C, bead-beating in 50% methanol for cell disruption, and 10 kDa cutoff ultrafiltration for removal of proteins. Samples are concentrated by vacuum evaporation and resuspended in 50% acetonitrile for mass spectrometric analysis. This protocol is optimal for extraction of polar metabolites such as amino acids, organic acids, nucleotides, sugars, or sugar-phosphates. Its implementation requires <6 h and allows preparation of multiple samples in parallel.

The application of ceramic membranes for treating effluent water from closed-circuit fish farming

Abstract The aim of the study was to analyze and assess the possibility of using a two-stage filtration system with ceramic membranes: a 3-tube module with 1.0 kDa cut-off (1st stage) and a one-tube module with 0.45 kDa cut-off (2nd stage) for treating effluent water from a juvenile African catfish aquaculture. The study revealed that during the 1st filtration stage of the effluent water, the highest degrees of retention were obtained with respect to: suspended solids SS (rejection coefficient RI=100%), turbidity (RI=99.40%), total iron (RI=89.20%), BOD5 (RI=76.0%), nitrite nitrogen (RI=62.30%), and CODCr (RI=41.74%). The 2nd filtration stage resulted in a lower reduction degree of the tested indicators in comparison to the 1st filtration stage. At the 2nd stage, the highest values of the rejection coefficient were noted in for the total iron content (RIV=100%), CODCr (RIV=59.52%; RV=64.28%, RVI=63.49%) and turbidity (RIV and RV = 45.0%, RVI=50.0%). The obtained results indicate that ceramic membranes (with 1.0 and 0.45 kDa cut-offs) may be used in recirculation aquaculture systems as one of the stages of effluent water treatment.

Removal of amoxicillin and cefuroxime axetil by advanced membranes technology, activated carbon and micelle–clay complex

Two antibacterials, amoxicillin trihydrate and cefuroxime axetil spiked into wastewater were completely removed by sequential wastewater treatment plant's membranes, which included activated sludge, ultrafiltration (hollow fibre and spiral wound membranes with 100 and 20 kDa cut-offs), activated carbon column and reverse osmosis. Adsorption isotherms in synthetic water which employed activated carbon and micelle–clay complex (octadecyltrimethylammonium–montmorillonite) as adsorbents fitted the Langmuir equation. Qmax of 100 and 90.9 mg g–1, and K values of 0.158 and 0.229 L mg–1 were obtained for amoxicillin trihydrate using activated carbon and micelle–clay complex, respectively. Filtration of antibacterials in the ppm range, which yielded variable degrees of removal depending on the volumes passed and flow rates, was simulated and capacities for the ppb range were estimated. Stability study in pure water and wastewater revealed that amoxicillin was totally stable for one month when kept at 37°C, whereas cefuroxime axetil underwent slow hydrolysis to cefuroxime.

Antibacterial and antifungal activity of water-soluble extracts from Mozzarella, Gouda, Swiss, and Cheddar commercial cheeses produced in Canada

The aim of this study was to evaluate the antibacterial and antifungal activities of water-soluble extracts (WSEs) from different types of cheeses against several food-borne pathogens. A total of five commercial cheeses manufactured in Canada were selected namely Mozzarella, Gouda, Swiss, and old and medium Cheddar. WSEs were ultrafiltrated through 10 kDa cutoff membranes and desalted using Sep-Pak cleanup column. Resulting peptide fractions were subject to physicochemical characterization and assessment for their antimicrobial activity against bacteria (Listeria ivanovii, Listeria monocytogenes, Escherichia coli MC4100, and E. coli O157:H7) and filamentous fungi (Aspergillus, Mucor, Fusarium, and Penicillium). Mozzarella and Gouda WSEs were the most active and inhibited with L. monocytogenes significantly, with respective reductions of 3.83 ± 0.15 and 2.93 ± 0.33 log. After desalting and organic acids removal, Mozzarella and Gouda WSEs produced 3-log reductions of L. ivanovii and E. coli MC4100, with minimal inhibitory concentration (MIC) values ranging 8.5–17 mg.mL−1. At a concentration of 34 mg.mL−1, all cheese peptidic WSEs induced a delay in spore germination. All WSEs were equally active against Fusarium sp., with a minimal concentration of 17 mg.mL−1. Gouda, Mozzarella, and medium Cheddar WSE were the strongest inhibitors in the case of Aspergillus versicolor and Mucor racemosus (17 mg.mL−1), whereas these spores were less sensitive to old Cheddar and Swiss WSE (34 mg.mL−1). This study demonstrates that peptidic WSEs of commercial cheeses manufactured in Canada exhibit antibacterial and antifungal activities, which may offer a promising alternative for purposes of food preservation.

Evaluation of Three Synthetic Membranes as Limiting Barrier for in vitro Drug Release Studies from Hydrogels Containing Polymeric Nanocapsules

In this work we evaluated the suitability of three synthetic membranes for the in vitro assessment of drug release from semisolid dosage forms containing polymeric nanocapsules. The use of cellulose acetate (0.45 µm pore size), polycarbonate (0.05 µm pore size) and dialysis cellulose (12 kDa cut off) membranes was investigated. For this purpose, drug release studies from hydrogels containing clobetasol propionate-loaded lipid-core nanocapsules were carried out. A higher amount of clobetasol propionate reached the release medium using cellulose acetate and polycarbonate membranes, compared to the use of dialysis cellulose membrane. Photon correlation spectroscopy analyses showed an overlap between the particle size distributions of the receptor media from the release studies using cellulose acetate and polycarbonate membranes and the original lipid-core nanocapsules suspension diluted in the receptor medium. The presence of nanoparticles was further confirmed by transmission electron microscopy. On the other hand, particle size distribution observed in the receptor medium of release studies using dialysis cellulose membrane did not show particles at nanoscale. The overall results suggest that the dialysis cellulose membrane ensures that only released drug will reach the receptor compartment, and that it should be the first choice for in vitro drug release studies from semisolid dosage forms containing drug-loaded nanocapsules.

Keggin-lysine hybrid nanostructures in the shape modulation of gold

We show here that L-lysine effectively complexes with phosphomolybdic acid (PMA) and the solution mixture when added to a 10−3 M aqueous solution of HAuCl4 after UV-irradiation for 3 h leads to the slow reduction and consequent formation of gold nanotriangles with a high degree of anisotropy. The same reaction carried out in a 12.5 kDa cutoff dialysis bag where the irradiated PMA-lysine solution was kept inside and stirred in a beaker containing aqueous HAuCl4, did not lead to the formation of gold nanotriangles. This implies that L-lysine plays the role of a shape-modulating agent and hence this study proves an improvement in the understanding of the role of such organic–inorganic hybrid structures in the synthesis and growth of anisotropic nanoparticles.

Antioxidant and Angiotensin I Converting Enzyme Inhibitory Activities of Red Snow Crab Chionoecetes japonicas Shell Hydrolysate by Enzymatic Hydrolysis

We investigated the antioxidant and angiotensin I converting enzyme (ACE) inhibitory activities of red snow crab Chionoecetes japonicas shell (RSCS) hydrolysate by enzymatic hydrolysis and its molecular weight cut-off fractions. The RSCS hydrolysate was fractionated through two ultrafiltration membranes of 3 and 10 kDa cut-offs. Three fractions (<3 kDa, 3-10 kDa, and >10 kDa) were evaluated for total amino acid composition, antioxidant activities using 2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (<TEX>$ABTS^+$</TEX>) radical scavenging and superoxide dismutase (SOD)-like activities and reducing power assays, and ACE inhibitory activity using Hou's method. Although all fractions showed activity, the <3 kDa fraction of RSCS hydrolysate exhibited the greatest <TEX>$ABTS^+$</TEX> radical scavenging, SOD-like and ACE inhibitory activities. However, these fractions exhibited low reducing power. These results suggest that the low-molecular-weight enzymatic hydrolysate of RSCS could be used as a functional ingredient to control oxidative stress and ACE activity.