Simultaneous Purification of Human Interferon Alpha-2b and Serum Albumin Using Bioprivileged Fluorinated Ionic Liquid-Based Aqueous Biphasic Systems.

Interferon alpha-2b (IFN-α2b) is an essential cytokine widely used in the treatment of chronic hepatitis C and hairy cell leukemia, and serum albumin is the most abundant plasma protein with numerous physiological functions. Effective single-step aqueous biphasic system (ABS) extraction for the simultaneous purification of IFN-α2b and BSA (serum albumin protein) was developed in this work. Effects of the ionic liquid (IL)-based ABS functionalization, fluorinated ILs (FILs; [C​2C​1Im][C​4F​9SO​3] and [N​1112(OH)][C​4F​9SO​3]) vs. mere fluoro-containing IL ([C​4C​1Im][CF​3SO​3]), in combination with sucrose or [N​1112(OH)][H​2PO​4] (well-known globular protein stabilizers), or high-charge-density salt K​3PO​4 were investigated. The effects of phase pH, phase water content (%wt), phase composition (%wt), and phase volume ratio were investigated. The phase pH was found to have a significant effect on IFN-α2b and BSA partition. Experimental results show that simultaneous single-step purification was achieved with a high yield (extraction efficiency up to 100%) for both proteins and a purification factor of IFN-α2b high in the enriched IFN-α2b phase (up to 23.22) and low in the BSA-enriched phase (down to 0.00). SDS-PAGE analysis confirmed the purity of both recovered proteins. The stability and structure of IFN-α2b and BSA were preserved or even improved (FIL-rich phase) during the purification step, as evaluated by CD spectroscopy and DSC. Binding studies of IFN-α2b and BSA with the ABS phase-forming components were assessed by MST, showing the strong interaction between FILs aggregates and both proteins. In view of their biocompatibility, customizable properties, and selectivity, FIL-based ABSs are suggested as an improved purification step that could facilitate the development of biologics.

Changes in properties of human milk under different conditions of frozen storage.

Human milk is the best source of nutrition for infants. Lower freezing temperatures and faster freezing rates allow for better preservation of human milk. However, research on the freezing conditions of human milk is limited. This study investigated the effectiveness of quick freezing and suitable freezing conditions for home preservation. Human milk was stored under different freezing conditions (-18 °C, -18 °C quick freezing, -30 °C, -40 °C, -60 °C, and - 80 °C) for 30, 60, and 90 days and then evaluated for changes in the microbial counts, bioactive protein, and lipid. The results showed that the total aerobic bacterial and Bifidobacteria counts in human milk after storage at freezing temperatures of - 30 °C and lower were closer to those of fresh human milk compared to - 18 °C. Furthermore, the lysozyme loss, lipid hydrolysis degree, and volatile organic compound production were lower. However, -18 °C quick freezing storage was not markedly different from -18 °C in maintaining human milk quality. Based on the results, for household and environmental reasons, the recommended temperature for storing human milk is suggested as -30 °C.

Advances in separation and identification of biologically important milk proteins and peptides.

Milk is a rich source of biologically important proteins and peptides. In addition, milk contains a variety of extracellular vesicles (EVs), including exosomes, that carry their own proteome cargo. EVs are essential for cell-cell communication and modulation of biological processes. They act as nature carriers of bioactive proteins/peptides in targeted delivery during various physiological and pathological conditions. Identification of the proteins and protein-derived peptides in milk and EVs and recognition of their biological activities and functions had a tremendous impact on food industry, medicine research, and clinical applications. Advanced separation methods, mass spectrometry (MS)-based proteomic approaches and innovative biostatistical procedures allowed for characterization of milk protein isoforms, genetic/splice variants, posttranslational modifications and their key roles, and contributed to novel discoveries. This review article discusses recently published developments in separation and identification of bioactive proteins/peptides from milk and milk EVs, including MS-based proteomic approaches.

Dual Antifungal and Antiproliferative Activities of a Novel Protein Fraction from a Medicinally Important Herb Trillium govanianum Wall. ex. D. Don.

Antimicrobial resistance of microorganisms and the unwanted side effects of chemoradiation therapy in cancer are major issues in healthcare. In recent times, protein-based drugs have emerged as promising candidates due to their high specificity, less side effects, etc. In this context, the rhizome of Trillium govanianum was first explored for biologically active proteins/peptides. For this, three protein fractions namely Aqueous protein fraction (APF), Hexane-Methanol-treated aqueous protein fraction (HMAPF), and Methanol-treated aqueous protein fraction (MAPF) were prepared and evaluated for antimicrobial and antiproliferative activities. In antifungal activity, HMAPF showed the lowest MIC90 values of 1.56 µg/ml against Candida parapsilosis and Candida glabrata and 3.12 µg/ml against Candida albicans and Candida auris. The antifungal activity was further confirmed by a chitinase assay, a growth kinetics and a proteinase inhibitory assay. Surprisingly, none of the three protein fractions exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus. Moreover, APF exhibited potent antiproliferative and antioxidant activities with IC50 values of 18 µg/ml and 227 µg /ml, respectively. For HMAPF, an IC50 value of 70 µg/ml against the MDA-MB-231 cell line was observed. The present results demonstrate that the protein fractions, particularly HMAPF and APF, might serve as potential sources of a dual antifungal and antiproliferative protein-based drug.

Antioxidant and antimicrobial properties of an extract rich in proteins obtained from Trametes versicolor.

Introduction: Bioactive proteins and peptides generated from fruit, vegetables, meat or fish have great potential as functional food or substitutes for antibiotics. In recent years it has also been demonstrated that the fungus kingdom could be a source of these compounds. The study investigated the bioactivity of an extract of the lignicolous fungus Trametes versicolor and its hydrolysate. Material and Methods: The fungus was collected in a mixed forest in October, extracted and hydrolysed. To inspect the protein and peptide profiles before and after hydrolysis, matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometric analysis was performed. To evaluate the antioxidant properties of the preparations, 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+) radical scavenging assays were used. The activity of the fungus extract and hydrolysate against Aeromonas veronii, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella Typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis was determined by the minimum inhibitory concentration and minimum bactericidal concentration values. Results: The extract and its hydrolysate showed almost 100% ABTS•+ and DPPH• radical scavenging with a low half maximal inhibitory concentration. The water extract and hydrolysate of T. versicolor exhibited antimicrobial activity against two S. aureus strains, E. coli, P. aeruginosa and Salmonella Typhimurium. Conclusion: These results provide compelling evidence that the analysed fungus extract and its hydrolysate hold promise with their antibacterial and antioxidant properties.

Exploring the Proteomic Profile of Soybean Bran: Unlocking the Potential for Improving Protein Quality and Quantity.

Soybean is a rich source of vegetal protein for both animal and human consumption. Despite the high levels of protein in soybean seeds, industrial processing to obtain soybean bran significantly decreases the final protein content of the byproducts. To overcome this problem, cultivars with higher protein contents must be developed. However, selecting the target proteins is difficult because of the lack of information on the proteome profile of soybean bran. Therefore, this study obtained the comparative proteomic profiles of both natural coatless seeds and defatted bran from an elite tropical-soybean cultivar. Thus, their extracts were characterized using LC-MS/MS and a total of 550 proteins were identified. Among these, 526 proteins were detected in coatless seeds and 319 proteins in defatted bran. Moreover, a total of 139 proteins were identified as presenting different levels of content in coatless seeds and defatted bran. Among them, only 46 were retained after the seed processing. These proteins were clustered in several important metabolic pathways, such as amino-acid biosynthesis, sugar biosynthesis, and antioxidant activity, meaning that they could act as targets for bioactive products or genome editing to improve protein quality and quantity in soybean grains. These findings can enhance our understanding regarding protein robustness for both soybean crops and the commercial bran improvement because target proteins must remain intact after processing and must be bioactive when overexpressed. Overall, the soybean bran proteomic profile was explored for the first time, providing a valuable catalogue of target proteins that can tolerate the industrial process.

Functional Bioactivities of Soluble Seed Proteins from Two Leguminous Seeds.

Storage proteins from Sphenostylis stenocarpa and Phaseolus lunatus were fractionated, and their in vitro bioactivities were investigated. Albumin, globulin, prolamin, and glutelin constituents of the respective seeds were successively fractionated using the modified Osborne method. Phenylmethylsulfonyl fluoride (1 mM) was used as a protease inhibitor. The antioxidant, anti-inflammatory, and acetylcholinesterase-inhibitory activities of the protein fractions were evaluated using different appropriate techniques. Globulin was the predominant fraction, with a yield of 43.21±0.01% and 48.19±0.03% for S. stenocarpa and P. lunatus, respectively, whereas prolamin was not detected in both seeds. The protein fraction markedly scavenges hydroxyl radicals, nitric oxide radicals, and 2,2-diphenyl-1-picryldydrazyl radicals with concomitant high free radical-reducing power. Albumin and globulin fractions elicited the highest acetylcholinesterase-inhibitory potential of 48.75% and 49.75%, respectively, indicating their great application potential in managing neurodegenerative diseases. In this study, the albumin, globulin, and glutelin fractions of these underutilized legumes showed great analeptic bioactivities, which could be utilized as health-promoting dietary supplements/products.

Brief report first report of the in vitro ovicidal activity of camel milk and its fractions on zoonotic-liver fluke (Fasciola gigantica) eggs.

Fasciola gigantica is one of the worldwide parasites that cause livestock and human illnesses. Chemotherapy is now the primary therapeutic option for its treatment. Drug abuse has led to the emergence of drug-resistant strains. As a result, there is an urgent need to discover natural and efficient anthelmintics against Fasciola spp. The study aims to evaluate the ovicidal activities of camel milk and its fractions on F. gigantica eggs. In the in vitro assay of F. gigantica eggs were submitted to different concentrations (0.5% and 1%) of camel milk fractions; Camel Milk Whey (CMW), Camel Milk Casein (CMC), and Skimmed Camel Milk (SCM) as well as a positive control (PC) of Nitroxynil (100 mg/ml) and a negative control (NC) with physiological saline. The Egg Hatching Assay (EHA) results showed that camel milk fractions exhibited ovicidal activity, especially CMW, and CMC, which showed 97.58 ± 0.58 and 96.9 ± 1.99 ovicidal activity, respectively, at a concentration of 1% after 15 days of treatment compared to PC, which exhibited 91.75 ± 4.95 ovicidal activity. The egg hatching ratios were 1.67% and 2.33% for CMW and CMC, respectively, compared to 70.17% for the NC and 6% for the PC. The LC50 values for CMW and CMC on the 15th day of treatment were 0.20 and 9.13, respectively. From the results above, we can infer that camel milk and its fractions are promising as a new alternative for fascioliasis control.

Nutritional Quality and Biological Application of Mushroom Protein as a Novel Protein Alternative.

PURPOSE OF REVIEW: Global concerns about population growth, economic, and nutritional transitions and health have led to the search for a low-cost protein alternative to animal origins. This review provides an overview of the viability of exploring mushroom protein as a future protein alternative considering the nutritional value, quality, digestibility, and biological benefits. RECENT FINDINGS: Plant proteins are commonly used as alternatives to animal proteins, but the majority of them are low in quality due to a lack of one or more essential amino acids. Edible mushroom proteins usually have a complete essential amino acid profile, meet dietary requirements, and provide economic advantages over animal and plant sources. Mushroom proteins may provide health advantages by eliciting antioxidant, antitumor, angiotensin-converting enzyme (ACE), inhibitory and antimicrobial properties over animal proteins. Protein concentrates, hydrolysates, and peptides from mushrooms are being used to improve human health. Also, edible mushrooms can be used to fortify traditional food to increase protein value and functional qualities. These characteristics highlight mushroom proteins as inexpensive, high-quality proteins that can be used as a meat alternative, as pharmaceuticals, and as treatments to alleviate malnutrition. Edible mushroom proteins are high in quality, low in cost, widely available, and meet environmental and social requirements, making them suitable as sustainable alternative proteins.

Bovine Colostrum for Veterinary and Human Health Applications: A Critical Review.

Bovine colostrum harbors a diverse array of bioactive components suitable for the development of functional foods, nutraceuticals, and pharmaceuticals with veterinary and human health applications. Bovine colostrum has a strong safety profile with applications across all age groups for health promotion and the amelioration of a variety of disease states. Increased worldwide milk production and novel processing technologies have resulted in substantial growth of the market for colostrum-based products. This review provides a synopsis of the bioactive components in bovine colostrum, the processing techniques used to produce high-value colostrum-based products, and recent studies utilizing bovine colostrum for veterinary and human health.

Chemical Synthesis of Bioactive Proteins.

Nature has developed a plethora of protein machinery to operate and maintain nearly every task of cellular life. These processes are tightly regulated via post-expression modifications-transformations that modulate intracellular protein synthesis, folding, and activation. Methods to prepare homogeneously and precisely modified proteins are essential to probe their function and design new bioactive modalities. Synthetic chemistry has contributed remarkably to protein science by allowing the preparation of novel biomacromolecules that are often challenging or impractical to prepare via common biological means. The ability to chemically build and precisely modify proteins has enabled the production of new molecules with novel physicochemical properties and programmed activity for biomedical research, diagnostic, and therapeutic applications. This minireview summarizes recent developments in chemical protein synthesis to produce bioactive proteins, with emphasis on novel analogs with promising in vitro and in vivo activity.

Recent advances of bioactive proteins/polypeptides in the treatment of breast cancer.

Proteins do not only serve as nutrients to fulfill the demand for food, but also are used as a source of bioactive proteins/polypeptides for regulating physical functions and promoting physical health. Female breast cancer has the highest incidence in the world and is a serious threat to women's health. Bioactive proteins/polypeptides exert strong anti-tumor effects and exhibit inhibition of multiple breast cancer cells. This review discussed the suppressing effects of bioactive proteins/polypeptides on breast cancer in vitro and in vivo, and their mechanisms of migration and invasion inhibition, apoptosis induction, and cell cycle arrest. This may contribute to providing a basis for the development of bioactive proteins/polypeptides for the treatment of breast cancer.

Molecular modeling of lactoferrin for food and nutraceutical applications: insights from in silico techniques.

Lactoferrin is a protein, primarily found in milk that has attracted the interest of the food industries due to its health properties. Nevertheless, the instability of lactoferrin has limited its commercial application. Recent studies have focused on encapsulation to enhance the stability of lactoferrin. However, the molecular insights underlying the changes of structural properties of lactoferrin and the interaction with protectants remain poorly understood. Computational approaches have proven useful in understanding the structural properties of molecules and the key binding with other constituents. In this review, comprehensive information on the structure and function of lactoferrin and the binding with various molecules for food purposes are reviewed, with a special emphasis on the use of molecular dynamics simulations. The results demonstrate the application of modeling and simulations to determine key residues of lactoferrin responsible for its stability and interactions with other biomolecular components under various conditions, which are also associated with its functional benefits. These have also been extended into the potential creation of enhanced lactoferrin for commercial purposes. This review provides valuable strategies in designing novel nutraceuticals for food science practitioners and those who have interests in acquiring familiarity with the application of computational modeling for food and health purposes.

The Health-promoting Potential of Edible Mushroom Proteins.

Edible mushrooms have been classified as "next-generation food" due to their high nutritional value coupled with their biological and functional potential. The most extensively studied and reported mushroom macromolecules are polysaccharides. However, macrofungi proteins and peptides are also a representative and significant bioactive group. Several factors such as species, substrate composition and harvest time significantly impact the mushroom protein content, typically ranging between 19 and 35% on a dry weight basis. Proteins work based on their shape and structure. Numerous extraction methods, including chemical and non-conventional, and their implications on protein yield and stability will be discussed. Beyond their biological potential, a great advantage of mushroom proteins is their uniqueness, as they often differ from animal, vegetable, and microbial proteins. According to recently published reports, the most relevant mushroom bioactive proteins and peptides include lectins, fungal immunomodulatory proteins, ubiquitin-like proteins, and proteins possessing enzymatic activity such as ribonucleases laccases, and other enzymes and ergothioneine. These are reported as antioxidant, antiviral, antifungal, antibacterial, antihypertensive, immunomodulatory, antitumour, antihypercholesterolemic or antihyperlipidemic, antidiabetic and anti-inflammatory properties, which improved proteins and peptides research interest and contributed to the increase of mushroom market value. This review provides an overview of the most relevant biochemical and biological properties of the main protein groups in edible mushrooms, explicitly focusing on their biomedical potential. Although mushrooms are a rich source of various proteins, many of these molecules have yet to be identified and characterised. Accordingly, it is crucial to identify and characterise new macromolecules of macrofungi origin, which opens an opportunity for further investigation to identify new bioactives for food, nutraceutical, or medicinal applications.

Nanosecond pulsed electric field treatment of human milk: Effects on microbiological inactivation, whey proteome and bioactive protein.

In the current study, nanosecond pulsed electric field (nsPEF) was investigated at lab-scale to optimise processing conditions of donor human milk to reduce bacterial counts, and to evaluate its effect on the bioactive proteins in human milk. Response surface methodology was utilized to optimise critical processing parameters. Two optimal nsPEF processing conditions were validated: 15 kV voltage, 6000 pulses at 20 Hz frequency, and 15 kV voltage, 6000 pulses at 50 Hz frequency. Compared to raw human milk, nsPEF processed milk had over 60 % retention of lysozyme, lactoperoxidase and lactoferrin, and 100 % retention of xanthine oxidase and immunoglobulin A. The contents of the five proteins were significantly higher after nsPEF processing when compared with Holder pasteurization. Liquid chromatography-mass spectrometry analysis showed that loss of milk proteins was smaller for samples treated with nsPEF than Holder pasteurization. These results indicated that nsPEF is a promising novel pasteurization method.

Physicochemical Properties and Whey Proteomes of Camel Milk Powders Produced by Different Concentration and Dehydration Processes.

Camel milk powder production is an alternative to preserve the perishable milk for later-date consumption. However, the impacts of dehydration processes on bioactive compounds in camel milk are largely unknown. Hence, the present study attempted to compare the physicochemical properties and protein profiles of camel milk powders produced by different concentration and dehydration processes. Six camel milk powders were produced by freeze- and spray-drying methods in conjunction with two liquid concentration techniques, namely spray dewatering and reverse osmosis. The results of proteomic analysis showed that direct freeze-dried camel milk powder had the least changes in protein profile, followed by direct spray-dried powder. The camel milk powders that underwent concentration processes had more profound changes in their protein profiles. Among the bioactive proteins identified, lactotransferrin and oxidase/peroxidase had the most significant decreases in concentration following processing. On the contrary, glycosylation-dependent cell adhesion molecule 1, peptidoglycan recognition protein 1, and osteopontin increased in concentration. The results revealed that direct freeze drying was the most ideal method for preserving the bioactive proteins during camel milk powder production. However, the freeze-drying technique has cost and scalability constraints, and the current spray-drying technique needs improvement to better retain the bioactivity of camel milk during powder processing.

Antibacterial action mechanisms and mode of trypsin inhibitors: a systematic review.

This systematic review (SR) aimed to gather studies describing the antibacterial action mechanisms and mode of trypsin inhibitors. The review protocol was registered (PROSPERO: CRD42020189069). Original articles resulting from studies in animal models, in bacterial culture, and using cells that describe antibacterial action of trypsin inhibitor-type peptides or proteins were selected in PubMed, Science Direct, Scopus, Web of Science, BVS, and EMBASE. The methodological quality assessment was performed using the PRISMA and OHAT tool. 2382 articles were retrieved, 17 of which were eligible. Four studies demonstrated the action mechanism directly on the bacterial membrane, and the fifth study on endogenous proteases extracted from the bacteria themselves. The antibacterial action mode was presented in the other studies, which can generate bacteriostatic or bactericidal effects without describing the mechanisms. This study generated information to enable new preclinical or clinical studies with molecules contributing to public health.

The Impact of Homogenization on Donor Human Milk and Human Milk-Based Fortifiers and Implications for Preterm Infant Health.

An exclusive human milk diet (EHMD) has been shown to reduce health complications of prematurity in infants born weighing ≤1250 g compared with cow milk-based diets. Accordingly, the number of available human milk (HM)-based nutritional products continues to increase. Newly available products, and those reportedly soon to enter the market, include homogenized donor HM and homogenized HM-based fortifiers. Existing literature demonstrating the benefits of an EHMD, however, is limited to non-homogenized HM-based products. Herein, we summarize existing evidence on the impact of homogenization on HM, with a particular focus on changes to the macromolecular structure of the milk fat globule and the subsequent impact on digestion kinetics. We use these published data to create a conceptual framework for the potential implications of homogenized HM-based nutritional products on preterm infant health. Importantly, we underscore that the safety and efficacy of homogenized HM-based products warrant investigation.

Comparing the effects of hydrostatic high-pressure processing vs holder pasteurisation on the microbial, biochemical and digestion properties of donor human milk.

In this study, hydrostatic high-pressure processing (HHP), a non-thermal pasteurisation method, was used to achieve the microbiological safety of donor human milk. After HHP, no bacteria were detected in human milk processed at 400 MPa for 5 min. Activities of a selection of bioactive components, including lysozyme, xanthine oxidase, lactoperoxidase, immunoglobulin A, lactoferrin, lipoprotein lipase and bile salt-stimulated lipase, did not decrease significantly. This study further investigated the gastrointestinal digestion kinetics of HoP and HHP milk compared with raw human milk, using an in vitro static infant digestion model. After 60 min of 'gastric digestion', the microstructure and protein profile of HHP milk samples were more similar to raw milk samples than HoP milk samples. Overall, HPP showed a better retention in milk nutrients and closer digestion behavior than that of HoP.

Cytotoxic Lactalbumin-Oleic Acid Complexes in the Human Milk Diet of Preterm Infants.

Frozen storage is necessary to preserve expressed human milk for critically ill and very preterm infants. Milk pasteurization is essential for donor milk given to this special population. Due to these storage and processing conditions, subtle changes occur in milk nutrients. These changes may have clinical implications. Potentially, bioactive complexes of unknown significance could be found in human milk given to preterm infants. One such complex, a cytotoxic α-lactalbumin-oleic acid complex named "HAMLET," (Human Alpha-Lactalbumin Made Lethal to Tumor cells) is a folding variant of alpha-lactalbumin that is bound to oleic acid. This complex, isolated from human milk casein, has specific toxicity to both carcinogenic cell lines and immature non-transformed cells. Both HAMLET and free oleic acid trigger similar apoptotic mechanisms in tissue and stimulate inflammation via the NF-κB and MAPK p38 signaling pathways. This protein-lipid complex could potentially trigger various inflammatory pathways with unknown consequences, especially in immature intestinal tissues. The very preterm population is dependent on human milk as a medicinal and broadly bioactive nutriment. Therefore, HAMLET's possible presence and bioactive role in milk should be addressed in neonatal research. Through a pediatric lens, HAMLET's discovery, formation and bioactive benefits will be reviewed.