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Studies on Separation of Immune Activity Peptide from Tuna Head Hydrolysates and Its Effect on Mouse

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Tutor: YangPing
School: Guangdong Ocean University
Course: Aquatic Products Processing and Storage Engineering
Keywords: tuna head,immunomodulatory peptides,sephadex,lymphocyte,macrophage
CLC: S917.4
Type: Master's thesis
Year:  2012
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The tuna is a kind of valuable marine fish, which has been recognized as the idealhealthy food for its rich nutrition and delicious taste. Tuna head is the main component ofscraps in tuna industrial process. For its high protein content, reasonable amino acidcomposition and rich bioactive components, the tuna head protein can therefore serve as anadditional source of protein to product activity peptides. According to the results of ourprevious experiments, the10-5KDa portions of enzyme hydrolysate of Yellowfin Tuna(Thunnus albacares) head protein could markedly increase the mice immune function. Inthis study, the nutrition assessment and physicochemical properties on the10-5KDaportions of tuna head protein hydrolysate were analysed. The optimum conditions toseparate the immune activity peptide from10-5KDa portions using the gel-filtrationchromatograph were studied. The effects of the active component on the mice peritonealmacrophages were studied. The main findings are as follows:The results showed that the10-5KDa portions (TIP) from the tuna head enzymatichydrolysate by ultrafiltration had a high crude protein content up to86.1%, the fat contentwas0.86%,containing essential mineral elements such as potassium, sodium, magnesium,calcium, phosphorus and trace elements like iron, zinc, copper, selenium. its amino acidcomposition is reasonable. The total amino acid content was5.15g/100mL while theessential amino acids accounted for36.28%and flavor amino acids accounted for48.12%.The molecular weight distribution result showed that TIP is a mixture of multiplemolecular weight fragments, in which the10-5KDa constituent accounted for27.49%, the3.6-1.4KDa constituent accounted for40.93%and whose molecular weight less than1KDa accounted for18.20%. TIP had an excellent solubility, no matter pH ranged from2to12, or temperature ranged from30to85℃, nitrogen soluble index (NSI) all reached above0.9. The emulsifying properties of TIP were average, but its foaming properties wereobserved well. The highest foaming value reached1.60when the protein concentration was3%, while the foaming stability was generally low, but at the value of pH10, it significantlyenhanced.Sephadex G-25was used for the preliminary separation of TIP. The optimumconditions were determined as: column size was2.6×60cm; flow rate was0.5mL/min,sample volume was20mg/mL x5mL.Under this condition,TIP could be seperated to five fractions which were TIP1, TIP2TIP3, TIP4, and TIP5. TIP3could significantly promotemice spleen lymphocyte proliferation (P<0.05) in middle-dose (0.2mg/mL), and inlow-dose (0.01mg/mL). TIP3was separated by Sephadex G-15further and three elutionpeaks were obtained with the main components TIP3B and TIP3C. The results of aminoacid analysis and electrospray ionization mass spectrometry analysis showed that peptidecontent of TIP3B accounted for86%, with molecular weight mainly distributed between200and1200Da and peptide content of TIP3C was about74%with molecular weightmainly distributed between200and800Da. TIP3C significantly promoted the proliferationof mouse spleen lymphocytes (P<0.05), showing a high level of immune activity at aconcentration of0.05mg/mL.The effect of TIP3C on mice peritoneal macrophages was studied by MTTcolorimetric assay, phagocytosis of neutral red, Griess and ELISA assay. The resultsindicted that TIP3C can significantly enhance metabolic activity of mice peritonealmacrophages (P <0.05) in every experimental designed dose (0.04mg/mL,0.02mg/mL and0.01mg/mL) and the effect was close to that of the positive control group (LPS,0.02mg/ml).The low-dose group TIP3C (0.01mg/mL)could significantly enhance themouse peritoneal macrophage phagocytosis (P <0.05) and the effect was even higher thanthat of the positive control group(LPS,0.02mg/ml). The amount of NO, TNF-α,IL-6thatreleased by mouse adominal macrophage which stimulated by TIP3C was larger theamount of negative control group, but there were no significant differences (P>0.05).
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