Architecture of ß-lactoglobulin coating modulates bioinspired alginate dialdehyde-gelatine/polydopamine scaffolds for subchondral bone regeneration.

In this study, we developed polydopamine (PDA)-functionalized alginate dialdehyde-gelatine (ADA-GEL) scaffolds for subchondral bone regeneration. These polymeric scaffolds were then coated with ß-Lactoglobulin (ß-LG) at concentrations of 1 mg/ml and 2 mg/ml. Morphological analysis indicated a homogeneous coating of the ß-LG layer on the surface of network-like scaffolds. The ß-LG-coated scaffolds exhibited improved swelling capacity as a function of the ß-LG concentration. Compared to ADA-GEL/PDA scaffolds, the ß-LG-coated scaffolds demonstrated delayed degradation and enhanced biomineralization. Here, a lower concentration of ß-LG showed long-lasting stability and superior biomimetic hydroxyapatite mineralization. According to the theoretical findings, the single-state, representing the low concentration of ß-LG, exhibited a homogeneous distribution on the surface of the PDA, while the dimer-state (high concentration) displayed a high likelihood of uncontrolled interactions. ß-LG-coated ADA-GEL/PDA scaffolds with a lower concentration of ß-LG provided a biocompatible substrate that supported adhesion, proliferation, and alkaline phosphatase (ALP) secretion of sheep bone marrow mesenchymal stem cells, as well as increased expression of osteopontin (SPP1) and collagen type 1 (COL1A1) in human osteoblasts. These findings indicate the potential of protein-coated scaffolds for subchondral bone tissue regeneration. STATEMENT OF SIGNIFICANCE: This study addresses a crucial aspect of osteochondral defect repair, emphasizing the pivotal role of subchondral bone regeneration. The development of polydopamine-functionalized alginate dialdehyde-gelatine (ADA-GEL) scaffolds, coated with ß-Lactoglobulin (ß-LG), represents a novel approach to potentially enhance subchondral bone repair. ß-LG, a milk protein rich in essential amino acids and bioactive peptides, is investigated for its potential to promote subchondral bone regeneration. This research explores computationally and experimentally the influence of protein concentration on the ordered or irregular deposition, unravelling the interplay between coating structure, scaffold properties, and in-vitro performance. This work contributes to advancing ordered protein coating strategies for subchondral bone regeneration, providing a biocompatible solution with potential implications for supporting subsequent cartilage repair.

Chemically Modified Bovine ß-Lactoglobulin as a Broad-Spectrum Influenza Virus Entry Inhibitor with the Potential to Combat Influenza Outbreaks.

Frequent outbreaks of the highly pathogenic influenza A virus (AIV) infection, together with the lack of broad-spectrum influenza vaccines, call for the development of broad-spectrum prophylactic agents. Previously, 3-hydroxyphthalic anhydride-modified bovine ß-lactoglobulin (3HP-ß-LG) was proven to be effective against human immunodeficiency virus (HIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and it has also been used in the clinical control of cervical human papillomavirus (HPV) infections. Here, we show its efficacy in potently inhibiting infection by divergent influenza A and B viruses. Mechanistic studies suggest that 3HP-ß-LG binds, possibly through its negatively charged residues, to the receptor-binding domain in the hemagglutinin 1 (HA1) subunit in the HA of the influenza virus, thus inhibiting the attachment of the HA to sialic acid on host cells. The intranasal administration of 3HP-ß-LG led to the protection of mice against challenges by influenza A(H1N1)/PR8, A(H3N2), and A(H7N9) viruses. Furthermore, 3HP-ß-LG is highly stable when stored at 50 °C for 30 days and it shows excellent safety in vitro and in vivo. Collectively, our findings suggest that 3HP-ß-LG could be successfully repurposed as an intranasal prophylactic agent to prevent influenza virus infections during influenza outbreaks.

Probing the conjugation of epigallocatechin gallate with ß-lactoglobulin and its in vivo desensitization efficiency.

Epigallocatechin gallate (EGCG) and ß-lactoglobulin (ßLg) were conjugated by covalent bonds to form EGCG-ßLg conjugates. This conjugation causes structural and bioactivity changes in ßLg, which in turn can be used as a possible approach for desensitization to allergens. In this study, the desensitization mechanism was investigated by monitoring ßLg secondary structure and immunoglobulin E (IgE) combining capacity changes on the basis of the conjugation mechanism. Furthermore, the desensitization efficiency in vivo was evaluated through animal experiments. The results show that temperature influenced the conjugation by decreasing the binding affinities (Ka) and binding numbers (n) of EGCG. The conjugation of EGCG decreased ßLg's IgE combining capacity by decreasing the ß-sheet component and imparted antioxidant properties by the introduction of hydroxyl groups. In addition, animal experiment results indicated that ßLg induced significant changes in the levels of IgE and inflammatory cytokines, and the relative abundance of small intestinal flora, linked to the inflammatory lesions and anaphylaxis symptoms. EGCG-ßLg conjugates can suppress the allergic response, attenuating serum IgE and relieving the anaphylaxis symptoms.

Anabolic effects of oral leucine-rich protein with and without ß-hydroxybutyrate on muscle protein metabolism in a novel clinical model of systemic inflammation-a randomized crossover trial.

BACKGROUND: ß-lactoglobulin (BLG) stimulates muscle protein synthesis and ß-hydroxybutyrate (BHB) inhibits muscle breakdown. Whether combining the 2 can additively attenuate disease-induced muscle loss is unknown. OBJECTIVE: Based on previous observations of anticatabolic effects of protein and ketone bodies during inflammation, and using a novel model combining ongoing systemic inflammation, fasting, and immobilization, we tested whether the anticatabolic muscle response to oral amino acids is altered compared with control conditions, as well as whether coadministration of oral BHB and BLG further improves the muscle anabolic response. Muscle net balance (NBphe) was the primary outcome and intramyocellular signals were assessed. METHODS: In a randomized crossover design, 8 young men underwent either preconditioning with LPS (prestudy day: 1 ng/kg, study day: 0.5 ng/kg) combined with a 36-h fast and bed rest to mimic catabolic inflammatory disease (CAT) or an overnight fast (control [CTR]) prior to isocaloric nutritional interventions on 3 occasions separated by ∼6 wk (range 42 to 83 d). RESULTS: NBphe increased similarly upon all conditions (interaction P = 0.65). From comparable baseline rates, both Rdphe [muscle synthesis, median ratio (95% CI): 0.44 (0.23, 0.86) P = 0.017] and Raphe [muscle breakdown, median ratio (95% CI): 0.46 (0.27, 0.78) P = 0.005] decreased following BHB + BLG compared with BLG. BLG increased Rdphe more under CAT conditions compared with CTR (interaction P = 0.02). CAT increased inflammation, energy expenditure, and lipid oxidation and decreased Rdphe and anabolic signaling [mammalian target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E-binding protein 1 (4EPB1) phosphorylation]. CONCLUSION: In contrast to our initial hypothesis, NBphe increased similarly following BLG during CAT and CTR conditions; CAT however, specifically stimulated the BLG-mediated increase in protein synthesis, whereas BHB coadministration did not affect NBphe, but distinctly dampened the BLG-induced increase in muscle amino acid fluxes thereby liberating circulating amino acids for anabolic actions elsewhere.

The effects of denatured major bovine whey proteins on the digestive tract, assessed by Caco-2 cell differentiation and on viability of suckling mice.

Alpha-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG) are contained in bovine milk whey. Chemical and physical treatments are known to alter the conformation of these proteins and we have previously reported that α-LA denatured with trifluoroethanol (TFE) and isolated from sterilized market milk inhibited the growth of rat crypt IEC-6 cells. In the present study, we aimed to evaluate the effects of TFE-treated α-LA and ß-LG on cell growth using cultured intestinal cells and on their safety using a suckling mouse model. First, we investigated the effect of the TFE-treated whey proteins on human colonic Caco-2 cells at various differentiation stages. In the undifferentiated stage, we assessed cell growth by a water-soluble tetrazolium-1 method. The native whey proteins enhanced cell proliferation, whereas the TFE-treated whey proteins strongly inhibited cell growth. We investigated cell barrier function in the post-differentiated stage by measuring transepithelial electrical resistance (TER). Not only native but also the TFE-treated whey proteins increased TER. Next, we evaluated whether the TFE-treated α-LA and ß-LG have adverse effects on healthy suckling mice. No mice given by the TFE-treated samples showed any adverse symptoms. We also performed a safety test using a human rotavirus infected gastrointestinal disease suckling mice model. Even the TFE-treated whey proteins appeared to prevent the development of diarrheal symptoms without any adverse effects. Although we cannot know the effect of long-term ingestion of denatured whey proteins, these results suggest that they have no adverse effects on differentiated intestinal cells and digestive tract, at least in short-term ingestion.

Cow's milk protein ß-lactoglobulin confers resilience against allergy by targeting complexed iron into immune cells.

BACKGROUND: Beta-lactoglobulin (BLG) is a bovine lipocalin in milk with an innate defense function. The circumstances under which BLG is associated with tolerance of or allergy to milk are not understood. OBJECTIVE: Our aims were to assess the capacity of ligand-free apoBLG versus loaded BLG (holoBLG) to protect mice against allergy by using an iron-quercetin complex as an exemplary ligand and to study the molecular mechanisms of this protection. METHODS: Binding of iron-quercetin to BLG was modeled and confirmed by spectroscopy and docking calculations. Serum IgE binding to apoBLG and holoBLG in children allergic to milk and children tolerant of milk was assessed. Mice were intranasally treated with apoBLG versus holoBLG and analyzed immunologically after systemic challenge. Aryl hydrocarbon receptor (AhR) activation was evaluated with reporter cells and Cyp1A1 expression. Treated human PBMCs and human mast cells were assessed by fluorescence-activated cell sorting and degranulation, respectively. RESULTS: Modeling predicted masking of major IgE and T-cell epitopes of BLG by ligand binding. In line with this modeling, IgE binding in children allergic to milk was reduced toward holoBLG, which also impaired degranulation of mast cells. In mice, only treatments with holoBLG prevented allergic sensitization and anaphylaxis, while sustaining regulatory T cells. BLG facilitated quercetin-dependent AhR activation and, downstream of AhR, lung Cyp1A1 expression. HoloBLG shuttled iron into monocytic cells and impaired their antigen presentation. CONCLUSION: The cargo of holoBLG is decisive in preventing allergy in vivo. BLG without cargo acted as an allergen in vivo and further primed human mast cells for degranulation in an antigen-independent fashion. Our data provide a mechanistic explanation why the same proteins can act either as tolerogens or as allergens.

Investigation of beta-lactoglobulin derived bioactive peptides against SARS-CoV-2 (COVID-19): In silico analysis.

The coronavirus disease of 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which started in late 2019 in Wuhan, China spread to the whole world in a short period of time, and thousands of people have died due to this epidemic. Although scientists have been searching for methods to manage SARS-CoV-2, there is no specific medication against COVID-19 as of yet. Two main approaches should be followed in the treatment of SARS-CoV-2; one of which is to neutralize the virus, and the other is to inhibit the host cell membrane receptors, where SARS-CoV-2 will bind. In this study, peptides derived from beta-lactoglobulin, which inactivates both the virus and its receptors in the host cell, were identified using computer-based in silico analysis. The beta-lactoglobulin derived peptides used in this study were obtained by the treatment of goat milk whey fraction with trypsin. The structure of the peptides was characterized by the liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS), and six beta-lactoglobulin derived peptides were selected as candidate peptides. Subsequently, the effects of peptides on SARS-CoV-2 and host cells were identified using virtual screening. According to the results of this in silico analysis, Ala-Leu-Pro-Met-His-Ile-Arg (ALMPHIR) and Ile-Pro-Ala-Val-Phe-Lys (IPAVFK) peptides were evaluated as potential candidates to be used in the treatment of SARS-CoV-2 after the future in vitro and in vivo studies.

Controlled release and antioxidant activity of chitosan and ß-lactoglobulin complex nanoparticles loaded with epigallocatechin gallate.

In order to investigate the influence of different embedding methods on the properties and function of polyphenols, the Epigallocatechin gallate (EGCG) loaded chitosan nanoparticles prepared with or without ß-lactoglobulin (ß-Lg) were obtained by ionic cross linking method. The average particle sizes of EGCG loaded chitosan nanoparticles (EGCG-CS NPs) decreased from 190 nm to 157 nm after adding with ß-Lg, whereas the encapsulation efficiency (EE) increased from 59.79 % to 76.29 %. The results of transmission electron microscopy (TEM) showed that the obtained nanoparticles had obvious core-shell structure. The results of simulated gastrointestinal digestion showed that the release rate of EGCG in CS/ß-Lg NPs was much lower than that of CS-NPs. Compared with free EGCG, the DPPH and FRAP assay showed that EGCG-CS NPs and EGCG-CS/ß-Lg NPs had slow-controlled antioxidant activity. Meanwhile, the study of cellular antioxidant activity (CAA) showed that the EC50 values of EGCG-CS NPs and EGCG-CS/ß-Lg NPs were decreased by 8.56 % and 18.35 %, respectively.

ß-Lactoglobulin Heptapeptide Reduces Oxidative Stress in Intestinal Epithelial Cells and Angiotensin II-Induced Vasoconstriction on Mouse Mesenteric Arteries by Induction of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) Translocation.

Peptides derived from buffalo dairy products possess multiple healthy properties that cannot be exerted as long as they are encrypted in parent proteins. To evaluate the biological activities of encrypted peptide sequences from buffalo ricotta cheese, we performed a simulated gastrointestinal (GI) digestion. Chemical and pharmacological characterization of the digest led to the identification of a novel peptide endowed with antioxidant and antihypertensive action. The GI digest was fractionated by Semiprep-HPLC, and fractions were tested against reactive oxygen species (ROS) release in an H2O2-treated intestinal epithelial cell line. UHPLC-PDA-MS/MS analysis revealed the presence of an abundant ß-lactoglobulin peptide (BRP2) in the most active fraction. Pharmacological characterization of BRP2 highlighted its antioxidant activity, involving ROS reduction, nuclear factor erythroid 2-related factor 2 (Nrf2) activation, and cytoprotective enzyme expression. The bioavailability of BRP2 was evaluated in intestinal transport studies through a Caco-2 cell monolayer. Equal bidirectional transport and linear permeability indicate that BRP2 was absorbed mainly through passive diffusion. In addition to its local effects, the BRP2 administration on mouse mesenteric arteries was able to reduce the angiotensin II-induced vasoconstriction by the Nrf2 nuclear translocation, the reduction of the active form of Ras-related C3 botulinum toxin substrate 1 (Rac1), and the NADPH oxidase activity. These data further highlight the role of buffalo ricotta cheese-derived peptides against oxidative stress-related diseases and suggest their health-promoting potential.

Alpha-Casein and Beta-Lactoglobulin from Cow Milk Exhibit Antioxidant Activity: A Plausible Link to Antiaging Effects.

Studies on the discovery and function of antioxidants are consistently being performed because oxidative stress can cause various diseases. Many compounds and natural products have antioxidant activity in vitro; however, it is often difficult to reproduce their effects in vivo. Additionally, methods to measure antioxidant activities in cells are also scarce. Here, we investigated the antioxidant activity of milk proteins by observing the formation of arsenite-induced stress granules as a tool to evaluate antioxidant activity in cells. Milk proteins not only decreased the formation of stress granules in several cell types but also scavenged 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cations in vitro. In addition, milk proteins inhibited cellular senescence based on an SA-ß-galactosidase assay, and increased differentiation to myotubes from myoblasts isolated from the skeletal muscles of mouse pups. Taken together, our results demonstrate that milk proteins have an antiaging effect, especially prevention of skeletal muscle loss, through their antioxidant activities. PRACTICAL APPLICATION: Our results provide that antioxidant effects of milk proteins containing α-caseins, ß-caseins, and ß-lactoglobulin can mitigate aging-related damage induced by oxidative stress through showing inhibition of cellular senescence and increase of differentiation and maturation of myoblast. Therefore, we suggest that milk proteins could be potent health supplements to prevent aging-associated diseases, especially sarcopenia.

Investigation and comparison of the anti-tumor activities of lactoferrin, α-lactalbumin, and ß-lactoglobulin in A549, HT29, HepG2, and MDA231-LM2 tumor models.

To investigate the anti-tumor activities of lactoferrin, α-lactalbumin, and ß-lactoglobulin, 4 types of human tumor cells (lung tumor cell A549, intestinal epithelial tumor cell HT29, hepatocellular cell HepG2, and breast cancer cell MDA231-LM2) were exposed to 3 proteins, respectively. The effects on cell proliferation, migration, and apoptosis were detected in vitro, and nude mice bearing tumors were administered the 3 proteins in vivo. Results showed that the 3 proteins (20 g/L) inhibited viability and migration, as well as induced apoptosis, in 4 tumor cells to different degrees (compared with the control). In vivo, tumor weights in the HT29 group (0.84 ± 0.22 g vs. control 2.05 ± 0.49 g) and MDA231-LM2 group (1.11 ± 0.25 g vs. control 2.49 ± 0.57 g) were significantly reduced by lactoferrin; tumor weights in the A549 group (1.07 ± 0.19 g vs. control 3.11 ± 0.73 g) and HepG2 group (2.32 ± 0.46 g vs. control 3.50 ± 0.74 g) were significantly reduced by α-lactalbumin. Moreover, the roles of lactoferrin, α-lactalbumin, and ß-lactoglobulin in regulating apoptotic proteins were validated. In summary, lactoferrin, α-lactalbumin, and ß-lactoglobulin were proven to inhibit growth and development of A549, HT29, HepG2, and MDA231-LM2 tumors to different degrees via induction of cell apoptosis.

Seleno-ß-lactoglobulin (Se-ß-Lg) induces mitochondria-dependant apoptosis in HepG2 cells.

Selenium compounds have been widely investigated as novel anticancer agents due to high efficacy and selectivity against cancer cells in recent years. This study aimed to research the potential inhibitory effects of seleno-ß-lactoglobulin (Se-ß-Lg) on HepG2 cells in vitro. MTT results demonstrated that the synthetized Se-ß-Lg exhibited strong antitumor activity on HepG2 cells with few side effects on human normal cells (LO2) and relatively weaker cytotoxic effects compared to inorganic selenium (SeO2). Scanning electron microscope (SEM), hoechst 33342/PI double staining, annexin V-FITC/PI staining and cell cycle detection results showed that Se-ß-Lg could induce the apoptosis of HepG2 cells via arresting them in S and G2/M phases and lead to the obvious morphological changes (loss of adhesion, cell shrinkage, and membrane blebbing, membrane permeabilities and DNA fragmentation). Besides, JC-1 staining, western blotting (WB) and polymerase chain reaction (PCR) results showed that Se-ß-Lg could gradually destroy the mitochondrial membrane potential of HepG2 cells, and finally resulting in the mitochondria-dependant apoptosis via up-regulation of Bax, Cytochrome c, Caspase-3 and down-regulation of Bcl-2. Our data could provide a theoretical basis for practical application of Se-ß-Lg in food and drug industries.

Antitumor effects of seleno-ß-lactoglobulin on human breast cancer MCF-7 and MDA-MB-231 cells in vitro.

Seleno-ß-lactoglobulin (Se-ß-Lg) was synthesized using seleninic acid, an organoselenium compound, and ß-lactoglobulin (ß-Lg), an important component of milk. Previously, we have studied the effects of Se-ß-Lg on hepatocellular carcinoma and gastric cancer cells. In this study, we investigated the antitumor effects of Se-ß-Lg and its potential mechanisms of action against human breast cancer cells (MCF-7 and MDA-MB-231). The results showed that the half-maximal inhibitory concentrations (IC50) of Se-ß-Lg were 40.84 µg/mL for MCF-7 cells and 46.04 µg/mL for MDA-MB-231 cells at 24 h, while the compound showed no cytotoxicity to normal breast cells. The involvement of reactive oxygen species (ROS) in the activation of the apoptotic signaling pathway by Se-ß-Lg was demonstrated by the incubation of cells with 80 µg/mL Se-ß-Lg and determination of the rates of apoptosis and intracellular ROS levels after the addition of 10 mM N-acetyl-l-cysteine, a ROS inhibitor. Our findings revealed highly potent anticancer activities of Se-ß-Lg against breast cancer cells and suggested that the compound may be used as a chemopreventive agent for breast cancer. Furthermore, we thoroughly elucidated the antitumor mechanism of Se-ß-Lg.

Antitumor activity of selenium modification of the bovine milk component ß-Lg (Se-ß-Lg) on H22 cells.

In this study, the apoptosis induction and antitumor activity of a novel complex, seleno-ß-lactoglobulin (Se-ß-Lg), on H22 cells were explored. In in vitro experiments, the MTT assay showed that Se-ß-Lg was cytotoxic to H22 cells in a concentration- and time-dependent manner and displayed few proliferation inhibition effects on normal liver L02 cells. Annexin V-FITC/PI and PI staining assays showed that Se-ß-Lg induced apoptosis changes of H22 cells from early to late apoptosis and led to S phase cell cycle arrest. Western blot and Z-VAD-FMK inhibitor assays showed that Se-ß-Lg triggered the Fas/FasL-mediated caspase 8-dependent extrinsic death receptor pathway in H22 cells. In in vivo experiments, Se-ß-Lg effectively repressed the growth of transplanted H22 solid tumors in a dose-dependent manner and exhibited few toxic effects on the host animals. H&E and PI staining of tumor tissues showed that Se-ß-Lg caused the occurrence of typical apoptosis morphology features and dose-dependently increased the proportion of apoptosis peaks (Sub-G1 peak) in H22 solid tumors. These results suggest that Se-ß-Lg has the capacity to induce H22 tumor cell apoptosis in vitro and in vivo and support that Se-ß-Lg can be applied as a functional complex in food.

Antitumor effects of seleno-ß-lactoglobulin (Se-ß-Lg) against human gastric cancer MGC-803 cells.

Seleno-ß-lactoglobulin (Se-ß-Lg) is a selenium conjugating protein synthesized by binding ß-lactoglobulin (ß-Lg) with inorganic selenium. The present study was designed to investigate the antitumor mechanism of Se-ß-Lg on human gastric cancer MGC-803 cells. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), scanning electron microscope (SEM), Annexin V-FITC/PI double staining and cell cycle detection suggested that Se-ß-Lg exhibited a significant inhibitory effect on the proliferation of MGC-803 cells with typical morphological characteristics of apoptosis by inducing cell cycle arrested at G2/M phase. Additionally, Se-ß-Lg could induce the disruption of mitochondrial membrane potential (MMP), improve the levels of intracellular reactive oxygen species and Bax, and down-regulate the Bcl-2 expression, further resulting in the release of cytochrome c from mitochondria into cytoplasm, the activation of caspase-9/-3, and the cleavage of poly-ADP-ribose polymerase (PARP). Taken together, these data clearly indicated that Se-ß-Lg had significantly cytotoxic effects on MGC-803 cells by inducing the caspase-dependent cell apoptosis and triggering the Bax- and Bcl-2-mediated mitochondria apoptosis pathway.

ß-Lactoglobulin Peptide Fragments Conjugated with Caffeic Acid Displaying Dual Activities for Tyrosinase Inhibition and Antioxidant Effect.

The regulation of tyrosinase activity and reactive oxygen species is of great importance for the prevention of dermatological disorders in the fields of medicine and cosmetics. Herein, we report a strategy based on solid-phase peptide chemistry for the synthesis of ß-lactoglobulin peptide fragment/caffeic acid (CA) conjugates (CA-Peps) with dual activities of tyrosinase inhibition and antioxidation. The purity of the prepared conjugates, CA-MHIR, CA-HIRL, and CA-HIR, significantly increased to 99%, as acetonide-protected CA was employed in solid-phase coupling reactions on Rink amide resins. The tyrosinase inhibitory activities of all CA-Pep derivatives were higher than the activity of kojic acid, and CA-MHIR exhibited the highest tyrosinase inhibition activity (IC50 = 47.9 µM). Moreover, CA-Pep derivatives displayed significantly enhanced antioxidant activities in the peroxidation of linoleic acid as compared to the pristine peptide fragments. All CA-Pep derivatives showed no cytotoxicity against B16-F1 melanoma cells.

Changes in structure and antioxidant activity of ß-lactoglobulin by ultrasound and enzymatic treatment.

Effects of ultrasound (20-40% amplitudes at 45-55 °C) and enzymatic (pepsin and trypsin) treatment on structure and antioxidant activity of ß-lactoglobulin were studied. Changes in structure of ß-lactoglobulin were investigated using spectroscopy techniques and changes in antioxidant activity were measured by chemical and cellular-based assays. Ultrasound treatment had considerable impact on the structure of ß-lactoglobulin and increased the susceptibility of ß-lactoglobulin to both pepsin and trypsin proteolysis. Intrinsic fluorescence intensity of ß-lactoglobulin was increased by ultrasound and then decreased after following enzymatic treatment. Compared with control, the ß-lactoglobulin after ultrasound and enzymatic treatments showed significantly higher oxygen scavenging activities in Caco-2 cells models, ABTS (2, 2'-Azinobis-3-ethylbenzthiazoline-6-sulphonate) radical scavenging activity and oxygen radical absorbance capacity (p < 0.05). Results indicated that ultrasound treatment increased the proteolysis of ß-lactoglobulin by both pepsin and trypsin and improved the antioxidant activity of the protein and its proteolytic products.

Probing the interaction of two chemotherapeutic drugs of oxali-palladium and 5-fluorouracil simultaneously with milk carrier protein of ß-lactoglobulin.

ß-Lactoglobulin (ßLG) is a basic element of globular carrier protein, which is the major protein in the whey of ruminant milk and is of main interest in the dairy industry. In the present study, the simultaneous effects of both of the important anticancer drugs of 5-fluorouracil (5-FU) and oxali-palladium, on the structure of ßLG were investigated using different spectroscopic methods of fluorescence and circular dichroism (CD) in combination with a molecular docking at two temperatures of 25 and 37°C. The resulted data from intrinsic fluorescence spectra of protein indicated that 5-FU and oxalli-palladium can quench the fluorescence intensity of ßLG in dose-dependent manner via static mechanism of fluorescence quenching. Analysis of fluorescence quenching data in agreement with theoretical results have represented that there are I binding sites on ßLG for binding of oxali-palladium and also II binding sites for 5-FU, at both temperatures of 25 and 37°C. Also, competitive binding results showed that the number of binding sites on protein for each of the drug when the protein incubated with one of the drug did not show any changes. The values of thermodynamic parameters of ΔH°, ΔS° and ΔG° illustrate that van der Waals and hydrogen-bond interactions have the main role in the binding of oxali-palladium and 5-FU to ßLG, respectively. The analysis of circular dichroism spectra indicated reduction in stability of the protein and alteration in the secondary structure of protein with reduction of α-helical structure and increasing of ß-sheet structure in the presence of increasing concentration of oxali-palladium and 5-FU. Also, the transition temperature (Tm) value of ßLG indicated the significant decreasing in the presence of 5-FU and oxali-palladium. As a result, it can be concluded that both of the chemotherapeutic drugs of oxali-palladium and 5-FU can bind to independent binding sites on carrier protein of ßLG, which can be used in design and simultaneous delivery of both drugs.

Trypsin-Treated ß-Lactoglobulin Improves Glucose Tolerance in C57BL/6 Mice by Enhancing AMPK Activation and Glucose Uptake in Hepatocytes.

It was reported that trypsin-treated ß-lactoglobulin (ß-LG) had a glucose-lowering effect in the oral glucose tolerance test (OGTT) in mice and a dipeptidyl peptidase-4 (DPP-4) inhibition activity in vitro. However, whether trypsin-treated ß-LG improves glucose tolerance by inhibiting DPP-4 in vivo has not yet been examined, and the mechanism of the glucose-lowering effect of trypsin-treated ß-LG is thus unclear. Here we investigated the detailed mechanism underlying the glucose tolerance effect of trypsin-treated ß-LG. The oral administration of trypsin-treated ß-LG significantly decreased the blood glucose concentrations in both the OGTT and an intraperitoneal glucose tolerance test (IPGTT). However, trypsin-treated ß-LG did not increase the insulin secretion after glucose loading. Trypsin-treated ß-LG potently increased the level of phosphorylated AMP-activated protein kinase (AMPK) in human hepatocellular carcinoma (HepG2) cells and in mice hepatocytes. Moreover, trypsin-treated ß-LG significantly enhanced glucose uptake into the HepG2 cells. These results indicate that trypsin-treated ß-LG decreases blood glucose levels after glucose loading by upregulating AMPK activation and glucose uptake in the liver, which could contribute to the reduction of postprandial hyperglycemia.

Peptide composition and dipeptidyl peptidase IV inhibitory properties of ß-lactoglobulin hydrolysates having similar extents of hydrolysis while generated using different enzyme-to-substrate ratios.

ß-Lactoglobulin hydrolysates (ßlgHs) were generated using elastase at enzyme-to-substrate ratios (E:S) of 0.5, 1.0 and 1.5% in order to reach target degree of hydrolysis (DH) values of 9 and 13%. The impact of different E:S during manufacture on hydrolysates having similar DHs was assessed. Samples with similar DHs generated with different E:S showed comparable molecular mass distribution profiles and in vitro dipeptidyl peptidase IV (DPP-IV) inhibitory activities (p>0.05). Liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis showed that 62 and 84% of the peptides identified were common within hydrolysates having a similar DH of 9 or 13%, respectively. Differences in the peptides identified within hydrolysates having similar DHs may be due to E:S dependent modifications in specificity and enzyme kinetics. Overall, this study showed that reduction in E:S while targeting the development of a similar DH for ßlgHs may be employed to reduce the cost of hydrolysate production without having an adverse impact on the bioactivity and physicochemical properties studied herein.