Maolin Tu

Hydroxyapatite nanorod and microsphere functionalized with bioactive lactoferrin as a new biomaterial for enhancement bone regeneration

Lactoferrin (LF) has been recently recognized as a promising new novel bone growth factor for the beneficial effects on bone cells and promotion of bone growth. Currently, it has been attracted wide attention in bone regeneration as functional food additives or a potential bioactive protein in bone tissue engineering. The present study investigated the possibility that hydroxyapatite (HAP) particles, a widely used bone substitute material for high biocompatibility and osteoconductivity, functionalized with lactoferrin as a composite material are applied to bone tissue engineering. Two kinds of hydroxyapatite samples with different sizes, including nanorods and microspheres particles, were functionalized with lactoferrin molecules, respectively. A detailed characterization of as-prepared HAP-LF complex is presented, combining thermal gravimetric analysis (TGA) and Fourier Transform Infrared Spectroscopy (FT-IR). Zeta potential and the analysis of electrostatic surface potential of lactoferrin were carried to reveal the mechanism of adsorption. The effects of HAP-LF complex on MC3T3-E1 osteoblast proliferation and morphology were systematically evaluated at different culture time. Interestingly, results showed that cell viability of HAP-LF group was significantly higher than HAP group indicating that the HAP-LF can improve the biocompatibility of HAP, which mainly originated from a combination of HAP-LF interaction. These results indicated that hydroxyapatite particles can work as a controlled releasing carrier of lactoferrin successfully, and lactoferrin showed better potentiality on using in the field of bone regeneration by coupling with hydroxyapatite. This study would provide a new biomaterial and might offer a new insight for enhancement of bone regeneration.

Lactoferrin promotes MC3T3-E1 osteoblast cells proliferation via MAPK signaling pathways

Lactoferrin has attracted great attention as a potential functional factor to prevent osteoporosis due to its various bioactivities. However, the molecular mechanism underlining the osteogenic activity of lactoferrin is unclear. In this study, effect of lactoferrin on MC3T3-E1 osteoblast cells proliferation was determined using MTT assay, while MAPK signaling pathways related to proliferation of MC3T3-E1 osteoblast cells were investigated based on mRNA and protein expressions. The distribution of cells at different cell cycle stages was evaluated by flow cytometry. Our findings indicated that lactoferrin enhanced MC3T3-E1 osteoblast cells proliferation in a dose-dependent manner; namely, it increased the proportion of cells in S and G2/M phases. Furthermore, we also found that lactoferrin could stimulate ERK, JNK and p38 MAPK. The mRNA expression of MAPK were significantly enhanced after treatment of lactoferrin. Lactoferrin significantly promoted the activation-associated phosphorylation of ERK and p38 MAPK and prevented the activation of JNK. Additionally, lactoferrin could enhance c-Fos and c-Jun expression by 3 times and 26 times, respectively. These results indicated that lactoferrin induced MC3T3-E1 osteoblast cells proliferation through c-Fos and c-Jun by stimulating ERK, JNK and p38, elucidating the molecular basis of the osteogenic activity of lactoferrin on MC3T3-E1 osteoblast cells.

Identification of a novel ACE-inhibitory peptide from casein and evaluation of the inhibitory mechanisms

Various bioactive peptides are continuously being identified from casein hydrolysates. In this work, a novel angiotensin I-converting enzyme (ACE)-inhibitory (ACEI) peptide, NMAINPSKENLCSTFCK, derived from the αs2-casein fragment residues 25-41, was screened and identified by UPLC-ESI-Q-TOF-MS/MS from tryptic casein hydrolysate. The IC50 value of the peptide, determined by an HPLC method, was 129.07 μM. The Lineweaver-Burk plot showed that this peptide acted as a mixed-type inhibitor against ACE, which might be attributed to the peptide being susceptible to degradation by ACE, indicating that the mixed-type inhibition could partly be a result of newly generated peptide fragments. The physicochemical characteristics and the secondary structure were evaluated by circular dichroism analysis and online prediction software (Expasy, PepDraw, and ProtParam) to identify the basic characteristics of this peptide. Moreover, molecular docking was simulated by Discovery Studio 2017 R2 software to provide the potential mechanisms underlying the ACEI activity of the peptides.

Isolation and Characterization of Lactoferrin Peptides with Stimulatory Effect on Osteoblast Proliferation

Lactoferrin is reported to be a potential food protein with osteogenic activity. However, the activity of lactoferrin peptides is questionable. In the present study, we isolated and characterized peptides from lactoferrin with stimulatory effect on osteoblast proliferation. Peptides from the lactoferrin pepsin hydrolysate were purified using cation-exchange and gel-filtration chromatography. Effects of different hydrolysates and peptides on the proliferation of osteoblast MC3T3-E1 cells were compared by MTT assay. Results showed that fraction P5-a from Superdex Peptide 10/300 GL gel chromatography showed better activity. Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis and high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry confirmed that two peptides components of P5-a corresponded to fractions of 20-78 and 191-277 amino acids in Bos taurus lactoferrin molecule (GI: 221706349). These results will provide some theoretical and practical data for the preparation and application of osteogenic peptides in functional food industry.

Identification and Antithrombotic Activity of Peptides from Blue Mussel (Mytilus edulis) Protein

The blue mussel (Mytilus edulis) reportedly contains many bioactive components of nutritional value. Water-, salt- and acid-soluble M. edulis protein fractions were obtained and the proteins were trypsinized. The resultant peptides were analyzed by ultra-performance liquid chromatography quadrupole time of flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). 387 unique peptides were identified that matched 81 precursor proteins. Molecular mass distributions of the proteins and peptides were analyzed by sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE). The differences between the three protein samples were studied by Venn diagram of peptide and protein compositions. Toxicity, allergic and antithrombotic activity of peptides was predicted using database website and molecular docking respectively. The antithrombotic activity of enzymatic hydrolysate from water-, salt- and acid-soluble M. edulis protein were 40.17%, 85.74%, 82.00% at 5 mg/mL, respectively. Active mechanism of antithrombotic peptide (ELEDSLDSER) was also research about amino acid binding sites and interaction, simultaneously.

Structure, Function, and Nutrition of Lactoferrin

Lactoferrin (Lf) is a polyfunctional protein from varied secretions of organisms. To date, about 20 different physiological roles of Lf have been reported. More and more studies on the structure and functions of Lf as well as the relationship of them have been intensively reported. In order to understand the Lf bioactivities and the mechanisms of different Lf functions, especially on the relationship between structure and functions, the research advancements are described in this study. In particular, the structure, bioactive site, thermal stability, several kinds of bioactive mechanisms such as antimicrobial, osteogenic, immunomodulatory, antitumor, antioxidant, and enzymic activities, and so forth are involved. Altogether, these are expected to provide some new ideas for the interesting topics about Lf, not only helpful for scientific research, but also for practical application in the medical and food industries, respectively.

Identification and mechanism evaluation of a novel osteogenesis promoting peptide from Tubulin Alpha-1C chain in Crassostrea gigas

Marine shellfish provides a series of biofunctionality account of its high-protein level. In this study, the osteogenic effect of a novel peptide, YRGDVVPK, from Crassostrea gigas protein hydrolysates on preosteoblast MC3T3-E1 proliferation was examined. Synthetic peptide with 100 nM significantly promoted the proliferation of MC3T3-E1 cells for a treatment of 72 h assayed by MTT method, and which was confirmed by the increase of alkaline phosphatase (ALP) activity. The peptide, YRGDVVPK, was docked with integrin α5β1 (PDB ID: 3VI4), which is a surface receptor of MC3T3-E1. The interaction of the peptide with integrin α5β1 (PDB ID: 3VI4) was analyzed by the molecular modeling algorithm of CDOCKER, which showed a more stable combination than the original ligand. The results suggested the novel peptide could promote the preosteoblast MC3T3-E1 proliferation probably by activating the signaling pathway of MAPK, which is induced through binding with peptide YRGDVVPK.

Bioactive hydrolysates from casein: Generation, identification, and in silico toxicity and allergenicity prediction of peptides

BACKGROUND Bioactive casein peptides have attracted considerable attention for their applications in industry. However, there is little clarity regarding mass spectrometric profiles for peptides in enzymatic hydrolysates of casein produced under varying conditions. In this study, the compositions of the peptides from casein hydrolysates were compared for different enzyme/substrate ratio (E/S) and hydrolysis times. The toxicity, allergenicity and bioactivity of the identified peptides were assessed in silico. RESULTS A total of 70 unique peptides were identified, and there were 28, 21, 13 and 8 peptides from αs1 -casein, αs2 -casein, β-casein and κ-casein respectively. The peptide number decreased with the increase in E/S and hydrolysis time. Moreover, peptides with relative molecular mass Mr ranging from 1000 to 1500 Da occupied the highest proportion of 31.43%, and almost all of the peptides showed Mr less than 5000 Da. In silico analysis showed that all of the peptides were non-toxic and non-allergenic, and several of them were assessed by PeptideRanker as having a relatively high likelihood of being bioactive peptides. CONCLUSIONS Composition of the peptides in the casein hydrolysates varied with the enzymolysis conditions. This studys results may facilitate the production of target bioactive peptides by controlling E/S and hydrolysis time, which is beneficial for the application of casein peptides in the functional food industry.

Complementation of UPLC-Q-TOF-MS and CESI-Q-TOF-MS on identification and determination of peptides from bovine lactoferrin

Digested peptides of bovine lactoferrin as the functional hydrolysates were identified by the Q-TOF tandem mass spectrometry (Q-TOF-MS) coupled with ultra performance liquid chromatograph (UPLC) and capillary electrophoresis (CE). The former (UPLC-Q-TOF-MS) identified 106 peptides while the latter (CE-Q-TOF-MS) characterized 102 peptides after comparison of peptides in terms of their molecular weight (MW), mass-to-charge ratio (m/z), and isoelectric point (pI). In addition, the hydrophilic value, net charge (q), and molecular radius (r) of the peptides were calculated, and a correlation analysis of the two methods was conducted between the retention time (RT) and r/q ratio of the peptides in order to elucidate the different separation principles of the unique peptides. It was shown that the peptides with larger hydrophilic value were beneficial to be separated by UPLC, while the peptides with larger r/q ratio were beneficial to be separated by CE. Combination of the above mentioned two complementary techniques have confidently improved the sequence coverage of lactoferrin and enhanced the identification of peptides, which makes it up to 65.8% in this study.

Analysis and Evaluation of the Inhibitory Mechanism of a Novel Angiotensin-I-Converting Enzyme Inhibitory Peptide Derived from Casein Hydrolysate

Casein hydrolysates exert various biological activities, and the responsible functional peptides are being identified from them continuously. In this study, the tryptic casein hydrolysate was fractionated by an ultrafiltration membrane (3 kDa), and the peptides were identified by capillary electrophoresis-quadrupole-time-of-flight-tandem mass spectrometry. Meanwhile, in silico methods were used to analyze the toxicity, solubility, stability, and affinity between the peptides and angiotensin-I-converting enzyme (ACE). Finally, a new angiotensin-I-converting enzyme inhibitory (ACEI) peptide, EKVNELSK, derived from αs1-casein (fragment 35-42) was screened. The half maximal inhibitory concentration value of the peptide is 5.998 mM, which was determined by a high-performance liquid chromatography method. The Lineweaver-Burk plot indicated that this peptide is a mixed-type inhibitor against ACE. Moreover, Discovery Studio 2017 R2 software was adopted to perform molecular docking to propose the potential mechanisms underlying the ACEI activity of the peptide. These results indicated that EKVNELSK is a new ACEI peptide identified from casein hydrolysate.