| Tuna head is rich in protein,fat,mineral elements,eicosapentaenoic acid（EPA）,docosahexaenoic acid（DHA）and other polyunsaturated fatty acids,which have extremely high nutritional value.Soup is one of the most popular diets in the world due to its remark able advantages such as easy processing,delicious taste and easy absorption by the human body.The soup dissolves and retains most of the nutrients in the raw materials,which is especially suitable for the elderly,children,infants and the infirm.Tuna head soup cooking is a process in which various nutrients and flavor substances in the raw materials are continuously migrated into the soup,which is also an effective method for tuna head utilization.This research is to reveal the nutritional mechanism of tuna head soup by proving their migration and changes with the cooking time through the analysis of various nutrients,including fat,protein,carbohydrate,etc.in tuna head soup.The tuna head undergoes a series of chemical and physical reactions during boiling,and the chemical composition in the soup includes not only the original ingredients migrated from the tuna head but also the micro/nano-sized colloidal particles formed by self-assembling during boiling.New compositions such as micro/nano-sized colloidal particles（MNPs）,the chemical localization,structural stability and potential antioxidant effects of such MNPs on human health need to be further studied.This paper takes tuna head as the research object.Firstly,the changing rule of protein,lipid and other nutrient in tuna head soup with boiling time was analyzed.The nutrient composition of MNPs nutrition in soup,the distribution site in the three-dimensional structure and the change rule during the formation process,and the effect of MNPs in the soup on H2O2 induced apoptosis of HUVECs in vitro were also analyzed.In addition,the hydroxyapatite and chitosan complex was prepared from the residual fish bones in tuna head soup,and the effects on the movement function repairment after spinal cord injury in rat model were studied.This study aims to explore the self-assembly mode and formation mechanism of MNPs in fish-head soup,and provide theoretical innovation to their biological activity characteristics.The results of this study would provide theoretical basis and development directions to facilitate high-quality utilization of aquatic product processing and the industrial production of instant soup.（1）The migration rules of proteins,lipids,free amino acids,fatty acids,mineral elements and other substances in tuna head soup were studied.The results showed that various nutrients in raw materials of tuna head were continuously dissolved into the soup system during the continuous heating and boiling of soup.A total of 18 kinds of amino acids were detected in tuna head soup,including 8 kinds of essential amino acids and 4 kinds of umami amino acids.Twenty-two kinds of fatty acids were detected,of which SFA,MUFA and PUFA were 9,6 and 7 kinds,respectively,Unsaturated fatty acids accounted for 66.6%of total fatty acids,and EPA+DHA accounted for 31.5%of total fatty acids.Tuna head soup was found rich in EPA and DHA and various essential amino acids.Fifteen kinds of mineral elements were detected,among which the content of K was the highest in the macro element,followed by Na,Ca and Mg.The content of Zn in the trace element was the highest,the content of Fe was the second,and the content of other elements was low.The amount of lead,arsenic,mercury,cadmium and chromium did not exceed the national limit standard.Results also showed that protein,lipids,minerals and other nutrients in tuna head soup increased gradually as boiling time increased,and the rising rate increased significantly in 30～90 min,and then flattened in 120～150 min.The amount of free amino acids and fatty acids in the soup increased slowly in 0～7.5 min,relatively increased in 9～30 min,and increased most rapidly in 30～90 min,and then increased slowly after 90 min,some of which even decreased slightly.The contents of all fatty acids reached the maximum at 150 min.The rising rates of Ca,Na,K,Mg,Zn and Fe increased significantly with the heating and boiling of the soup.The migration rates of As,Pb and Cu were relatively low,and the peak contents are all less than 2 mg/100 m L at 150 min.The changes of B,Cr,Se,Mn,Cd and Hg were insignificant,and the contents were between 0.1 and 0.5 mg/100m L.（2）The formation rule of micro/nano colloidal particles self-assembly was analyzed.Using optical microscopy,Malvern particle size analyzer,confocal laser microscopy,transmission electron microscopy and infrared spectroscopy,it was found that the nutrients migrated into the soup self-assembled into MNPs,with triglycerides distributed in the core of the colloidal particles,sodium ions,phospholipids,proteins and sugar-based molecules in the periphery of the colloidal particles,and chloride ions in the core and periphery of the colloidal particles.Therefore,MNPs are core-shell colloidal spherical structures,similar to droplet structures in emulsions.The core part may be hydrophobic,consisting of TG and Cl-,and the shell layer may be hydrophilic,consisting of Cl-,Na+,phospholipids,proteins,and glycosyl molecules.Within 0～150min of the cooking time,the MNPs in the soup began to appear gradually.The geometry of particles increased and then gradually decreased until stablized.When boiling for 5min,colloidal particles with a diameter of 0.5～100μm appeared in the tuna head soup.Within 0～10 min,the colloidal particles in the soup began to enter a relatively active period of decomposition and polymerization.The variation of particle size of the colloidal particles is the largest in this period and the overall trend of sizeis gradually decreasing.After 10～30 min,the variation range of particle size of the colloidal particles become relatively small,and the colloidal particles with aparticle size larger than 30μm disappeared.Within 30～60 min,the soup system experienced drastic changes to relatively stable changes.With the continuous boiling,new substances in the tuna head continuously moved into the soup.Therefore,both new colloidal particles appeared and old ones disappeared.During the boiling period of 60～120 min,the soup compositions and colloid system tended to be stable as a whole,the particle size distribution was further narrowed and the average particle size was reduced accordingly.The particle size distribution of the colloidal particles was mainly concentrated at 8μm and 12μm.When boiled for 150 min,three peaks appeared in the particle size distribution,which may be caused by the decomposition and merge of colloidal particles.Coalescence occurs when two or more colloidal particles approach and fuse together to form one large colloidal particle.When the attractive force between the two particles is greater than the repulsion force,the interface layer between the two particles fractures and fuses.The LSCM images of MNPs after boiling for 150 min showed that no hydrophilic substances（such as proteins,phospholipids,glycosyl molecules,sodium ions,chloride ions,etc.）were distributed in the core of MNPs.In addition,during the boiling process,water was sufficiently agitated and actively entered into the MNPs or passively participated in the aggregation of colloidal particles.（3）The effect of MNPs on H2O2-induced apoptosis of HUVECs was analyzed.The cell viability was measured with CCK-8 method.The results showed that H2O2treatment could induce the apoptosis of HUVECs cell significantly compared with the control group,which proved that H2O2-induced apoptosis of HUVECs cell was a suitable cell model for studying the antioxidant properties of some new substances.Different concentrations of MNPs could all significantly reduce H2O2-induced apoptosis,and the medium dose（10μg/m L MNPs+50μM H2O2）and high dose（100μg/m L MNPs+50μM H2O2）groups had no significant difference compared with the control group.The difference（P<0.05）indicated that the medium and high dose of MNPs could both effectively inhibit the oxidative damage induced by H2O2,which indicated that MNPs in the soup had significant inhibitory effect on the apoptosis of HUVECs cells induced by H2O2.In the meantime,real-Time PCR was used to detect the m RNA expression levels of GRP78,CHOP and ATF4 of HUVECs protected by 10μg/m L MNPs against H2O2-induced apoptosis,and H2O2 treatment significantly up-regulated the m RNA levels compared with the control group.It was confirmed that H2O2-induced apoptosis of HUVECs was involved in the enhancement of endoplasmic reticulum stress.While MNPs treatment could significantly down-regulate the three m RNA levels,MNPs treatment was not able to reduce the m RNA levels to the level of the control group.Finally,flow cytometry was used to detect the H2O2-induced apoptosis of HUVECs protected by MNPs.Compared with the control group（5.5±1.0%）,H2O2treatment significantly enhanced the proportion of HUVECs apoptosis（33.1±2.4%）.It was confirmed that H2O2 treatment could significantly induce HUVECs apoptosis.Compared with the H2O2 group,MNPs treatment could significantly reduce the proportion of apoptosis（16.8±2.4%）.However,MNPs treatment could not completely inhibit the apoptosis of HUVECs cells.This result was in consistent with the results of the m RNA experiment.（4）The repair effect of hydroxyapatite and chitosan complexes prepared from residual fish bones in tuna head soup on movement function after spinal cord injury in a rat model was verified.Nano-hydroxyapatite was obtained by calcining and grinding the bone slices from tuna head soup.The nano-hydroxyapatite was then fused with chitosan for 24 h under room temperature magnetic stirring for preparing chitosan-hydroxyapatite nanoparticles.The particle size of chitosan-hydroxyapatite nanoparticles was measured as 105 nm,the Zeta potential was 9 m V,and the stability could be maintained for 4 weeks at low temperature.The particle size of chitosan nanoparticles used was 90 nm,and the Zeta potential was close to 11 m V after 4 weeks storage at4℃.Comparative analysis showed that hydroxyapatite could be successfully attached to the chitosan nanoparticles.Spectral analysis of chitosan-hydroxyapatite nanoparticles showed a significant characteristic absorption peak at 3500 cm-1,which was caused by the joint stretching vibration of hydroxyl and N-H groups.The characteristic peak of amide group appeared at 1700 cm-1,C-H stretching was observed at 1400 cm-1,and C-O-C stretching was observed at 1150 cm-1 and 1050 cm-1.The peaks near 1070 cm-1and 570 cm-1 represented PO43-stretching group,and the peaks near 1450 cm-1represented CO32-stretching vibration,which were the characteristic absorption bands of hydroxyapatite,indicating that hydroxyapatite were successfully encapsulated by chitosan nanoparticles.A rat model of spinal cord injury was established,treated with chitosan-hydroxyapatiteand its functional recovery after spinal cord injury was evaluated.Compared with the SCI control group,the score of 7d BBB after chitosan-hydroxyapatite treatment increased significantly.On the other hand,chitosan treatment alone did not significantly enhance the BBB score at different time compared with the control group.In order to investigate the effect of chitosan-hydroxyapatite on tissue recovery after spinal cord injury,HE staining was performed 28 days after injury.The results showed that chitosan-hydroxyapatite treatment could significantly reduce the volume of the lesion cavity,and the chitosan treatment did not significantly improve the volume of the lesion cavity compared to SCI group.In addition,as compared with the SCI and CN treatment groups,the dispersed structure of the rat spinal cord tissue slices in the chitosan-hydroxyapatite treatment group was significantly improved. |
