Cuiping Yu

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.

Antioxidant and ACE Inhibitory Activity of Enzymatic Hydrolysates from Ruditapes philippinarum

Ruditapes philippinarum proteins were hydrolyzed by trypsin, neutrase, and pepsin. The antioxidant activities and ACE inhibitory activity of hydrolysates were analyzed and the antioxidant activities were related to their molecular weight distribution and amino acid compositions. Results indicated the hydrolysis of proteins led to an increase in small peptides and free amino acids. The antioxidant activities of Ruditapes philippinarum hydrolysates against DPPH radical scavenging, inhibition on linoleic acid peroxidation, and reducing power showed that the neutrase hydrolysate exhibited the strongest antioxidant activity. In addition, an ACE inhibition assay revealed that the pepsin hydrolysate had the highest ACE inhibitory ability. Ruditapes philippinarum protein hydrolysates could be a promising source of natural antioxidant and ACE inhibitory.

Effect of Ball Mill Treatment on the Physicochemical Properties and Digestibility of Protein Extracts Generated from Scallops (Chlamys farreri)

The effects of ball mill treatment (0, 2, 4, 6, 8, and 10 min) on the physicochemical and digestible properties of scallops (Chlamys farreri) protein (CFP) were investigated. The CFP particle size decreased with increasing ball-milling time. The content of free sulfhydryl (SH) of CFP increased from 13.08 ± 0.25 μmol/g protein to 18.85 ± 0.24 μmol/g protein when the ball-milling time increased from 0 min to 10 min. A sharp increase of the surface hydrophobicity index (H0) from 48.53 ± 0.27 to 239.59 ± 0.37 was found when the ball-milling time increased from 0 min to 4 min. Furthermore, the foaming capacity increased from 46.08 ± 6.12% to 65.11 ± 1.05% with increasing ball-milling time from 0 min to 6 min, after which it reached a plateau. SDS-PAGE results showed that ball mill treatment did not change the primary structure of CFP. Digestible properties of BMCFP simulated gastrointestinal digestion as a function of ball mill treatment were analyzed by Tricine-SDS-PAGE and nitrogen recovery index. After 60 min of simulated human gastro digestion, nitrogen recovery index of CFP had a significant rise from 42.01 ± 0.31% to 58.78 ± 3.37% as the ball-milling time increased from 0 min to 6 min. Peptides from hydrolysates of Chlamys farreri protein (CFP) were identified by ultraperformance liquidchromatographysystem coupled to a Synapt Mass Quadrupole Time-of-Flight Mass Spectrometer (UPLC-Q-TOF-MS). After 2 h and 4 h of simulated human duodenal digestion, the number of peptides with 7–10 amino acids length increased apparently with the ball-milling time increased. This study presents an approach to investigating the effect of the ball-milling process on the physicochemical and digestible properties of CFP, which may provide valuable information on the application of CFP as an ingredient in food products.

Effects of ball-milling treatment on mussel (Mytilus edulis) protein: structure, functional properties and in vitro digestibility

Summary nIn this study, the effects of ball-milling treatment on structural properties and functional properties of mussel protein were investigated. In vitro protein digestibility and free amino acid contents were also evaluated. Ball-milling treatment did not change the primary structure of mussel protein, but caused changes of secondary structure. The tertiary and quaternary structure also changed. After 20xa0min of ball-milling treatment, the whiteness and oil-binding capability of mussel protein significantly improved from 13.67 to 26.62 and 43.76% to 196.00%, while the protein solubility and water-holding capability of mussel protein significantly decreased from 74.89% to 53.10% and 215.67% to 90.91%. Ball-milling treatment increased the inxa0vitro digestibility significantly. These results provide theoretical basis for the utilisation of mussel protein in food industry.

Effects of high-pressure homogenization on functional properties and structure of mussel (Mytilus edulis) myofibrillar proteins

Mussel myofibrillar proteins (MMP) suspensions (10.6%u202f±u202f0.5%, w/v) were treated by high-pressure homogenization (HPH) at 0 (control), 20, 40, 60, 80 or 100u202fMPa for 3u202fcycles. Particle size distribution, zeta potential, solubility, water and oil holding capacity, emulsifying, foaming properties, secondary structure, free sulfhydryl and surface hydrophobicity of the obtained suspensions were analyzed. The results showed that functional properties of MMP significantly (Pu202f<u202f0.05) improved after HPH treatment. Absolute zeta potential, emulsifying activity index, emulsion stability index, foaming ability and foaming stability increased by 23.64u202fmV, 14.99u202fm2/g, 4.3u202fmin, 17.3% and 29.7% at 80u202fMPa, protein solubility and oil holding capacity increased by 7.4% and 1300% at 100u202fMPa. However, HPH treatment significantly (Pu202f<u202f0.05) decreased particle size and water holding capacity. HPH treatment altered secondary structure, tertiary and quaternary structure. Functionality improvements mainly resulted from changes in structure and decrease in particle size. The results showed that HPH has potential for improving functional properties of MMP, thus expand its application in food industry.

Effects of Limited Hydrolysis and High-Pressure Homogenization on Functional Properties of Oyster Protein Isolates

In this study, the effects of limited hydrolysis and/or high-pressure homogenization (HPH) treatment in acid conditions on the functional properties of oyster protein isolates (OPI) were studied. Protein solubility, surface hydrophobicity, particle size distribution, zeta potential, foaming, and emulsifying properties were evaluated. The results showed that acid treatment led to the dissociation and unfolding of OPI. Subsequent treatment such as limited proteolysis, HPH, and their combination remarkably improved the functional properties of OPI. Acid treatment produced flexible aggregates, as well as reduced particle size and solubility. On the contrary, limited hydrolysis increased the solubility of OPI. Furthermore, HPH enhanced the effectiveness of the above treatments. The emulsifying and foaming properties of acid- or hydrolysis-treated OPI significantly improved. In conclusion, a combination of acid treatment, limited proteolysis, and HPH improved the functional properties of OPI. The improvements in the functional properties of OPI could potentiate the use of oyster protein and its hydrolysates in the food industry.

Characterization of natural hydroxyapatite originated from fish bone and its biocompatibility with osteoblasts

Hydroxyapatite (HAP) was very attractive for using as bone implant material for a long period due to the close similarity with natural bone in composition and osteoconductive properties. In this study, three kinds of natural HAP (nHAP) derived from rainbow trout (Onchorynchus mkiss), cod (Gadus) and salmon (Oncorhynchus keta) bones were prepared using thermal calcination method for the first time. Resultant nHAPs were characterized by fourier transform infrared spectroscopy (FT-IR), x-ray diffraction analysis (XRD), field-emission scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analysis. Biocompatibility of calcined nHAP was evaluated through MTT cell viability assay and alkaline phosphatase activity experiment using mouse preosteoblast MC3T3-E1. Results of cell experiment indicated that the nHAP originated from rainbow trout and salmon bones showed better biological compatibility compared with the nHAP originated from cod bone and chemical synthetic HAP (cHAP). This is most likely attributed to the different element composition in nHAP, i.e., the nHAP derived from rainbow trout and salmon bones showed the presence of CO32- and Mg2+. Therefore, the nHAP originated from rainbow trout and salmon bones have a great potential for application as implant material substitute in bone tissue engineering and the natural waste fish bone product can be used for hydroxyapatite synthesis as a part of bio-waste management.

Identification and In Silico Prediction of Anticoagulant Peptides from the Enzymatic Hydrolysates of Mytilus edulis Proteins

Mytilus edulis is a typical marine bivalve mollusk. Many kinds of bioactive components with nutritional and pharmaceutical activities in Mytilus edulis were reported. In this study, eight different parts of Mytilus edulis tissues, i.e., the foot, byssus, pedal retractor muscle, mantle, gill, adductor muscle, viscera, and other parts, were separated and the proteins from these tissues were prepared. A total of 277 unique peptides from the hydrolysates of different proteins were identified by UPLC-Q-TOF-MS/MS, and the molecular weight distribution of the peptides in different tissues was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bioactivity of the peptides was predicted through the Peptide Ranker database and molecular docking. Moreover, the peptides from the adductor muscle were chosen to do the active validation of anticoagulant activity. The active mechanism of three peptides from the adductor muscle, VQQELEDAEERADSAEGSLQK, RMEADIAAMQSDLDDALNGQR, and AAFLLGVNSNDLLK, were analyzed by Discovery Studio 2017, which also explained the anticoagulant activity of the hydrolysates of proteins from adductor muscle. This study optimized a screening and identification method of bioactive peptides from enzymatic hydrolysates of different tissues in Mytilus edulis.

Effects of High-Pressure Homogenization at Different Pressures on Structure and Functional Properties of Oyster Protein Isolates

Abstract Oyster protein isolate (OPI) suspensions (6.19u2006% ± 0.82u2006%, w/v) were treated by high-pressure homogenization (HPH) at 0 (control), 20, 40, 60, 80 or 100 MPa for three cycles. Protein profiles, secondary structure, free sulfhydryl, surface hydrophobicity, particle size distribution, zeta-potential, solubility, water and oil holding capacity (OHC), emulsifying and foaming properties of the obtained suspensions were analyzed. The results showed that HPH treatment did not cause changes in protein profiles of OPI, but caused changes in secondary structure, content of α-helix decreased but content of β-turn and random coil increased significantly (P < 0.05). Free sulfhydryl and surface hydrophobicity all increased significantly (P < 0.05) after HPH treatment, indicating that tertiary and quaternary structures changed. Functional properties of OPI significantly (P < 0.05) improved after HPH treatment, such as zeta-potential (from −12.67 to −33.57 mV), solubility (from 20.24u2006% to 57.99u2006%), OHC (from 981.77u2006% to 1229.40u2006%), foaming ability (from 17.50u2006% to 35.00u2006%), foaming stability (from 44.49u2006% to 66.60u2006%), emulsifying activity index (from 8.87 to 17.06 m2/g) and emulsion stability index (from 14.65 to 41.68 min). At 60 MPa and 80 MPa, the improvements were more remarkable. However, HPH treatment significantly (P < 0.05) decreased particle size (from 200–500 nm to 0–200 nm) and water holding capacity (from 341.15u2006% to 216.96u2006%). These improvements were closely related to structural changes and reduction of particle size. Application of different pressures affected functional properties of OPI. These results could provide information for determining HPH applying condition in OPI modification.