Study On Structure Identification And Biological Activity Of Enzymolyzed Hairtail And Ferrous Chelated Small Peptides

Hairtail is one of the economically important commercial fish resources in our country, and accountsfor much market share as leader in fish production. The total hairtail caught in2010amounted to1,100,000tones which occupied70%-80%of the total product. It is a kind of aquatic product with high nutritionalvalue, tender fat and delicious meat, and so loved by the people. But the process will produce a largeamount of waste, its weight is approximately40%-50%of raw fish. Such kind of waste is very rich inprotein resources. If it is not treated effectively, this producing process will not only cause environmentalpollution, but also waste the resources. Now, the aquatic product leftovers utilization focuses on theproduction of feed, fish sauce, hydrolyzed protein. It is a waste of high quality protein resources, and theprocess is complicated with high cost.Iron is an essential nutrient, plays an important role in the metabolism of human body. The iron in foodmainly trivalent form, cannot be directly absorbed by organism. As to used current supplements-ironabsorption and utilization rate is low, and leads to certain side effects. Natural protein can hydrolyse togenerate peptides, the latter with trace elements combine to form a complex can be directly absorbed by thesmall intestine. Therefore, preparation of antibacterial and antioxidant efficacy of functional foods or foodingredients using hairtail waste protein,it make developing and utilizating low value marine fish resourcespossible,and open up new way to achieving the comprehensive utilization of resources.Small hairtail captured in Zhoushan sea area (9-10month) is used raw material, to prepareantibacterial activity of ferrous chelate peptide using hairtail waste protein disposed proteasome and ferrouschloride is studied, and also the structure of ferrous chelating peptides, antibacterial activity andmechanism is studied.The main research contents and results are as follows:1,With DH and iron chelating rate as the index, an enzymatic hydrolysis screening experiment was done tothe alkali protease, papain, trypsin, neutral protease and pepsin, respectively; then, the compound enzymehydrolysis experiment was implemented. With a with DH and Iron chelating rate as the index, the quadraticrotation-orthogonal combination design was applied to study the effect of the category of enzymepreparation, proportion of the compound enzyme, method to add enzyme, total volume of the enzyme, PHvalue, time of enzymolysis and temperature on the craft of preparation of ferrous chelate polypeptides. Dueto the comprehensive consideration of DH and iron chelating, the following condition was determined as the optimal craft to produce the ferrous chelate polypeptide with compound enzyme hydrolysis hairtailprotein: total volume of added enzyme is22000U/g while the ratio between alkali protease and papainwhich were added successively, was4:6; the time of enzyme hydrolysis of alkali protease was9h and thatof papain was8h; PH value of alkali protease was8.0and that of papain was6.0; the temperature ofenzyme hydrolysis was45℃. In this condition, DH and iron chelating rate of the hairtail protein ferrouschelate polypeptide were52.22%and82.31%, respectively.2,In order to study the antibacterial antioxidant activity of the peptide in which the molecular weightdistributes unevenly, the ultra-filtration membranes with the molecular weights of10kDa,5kDa and3kDawere used to separate the ferrous chelates of the hairtail protease solution in hierarchy, and the peptidecontent and the antibacterial antioxidant activity of the hairtail protein ferrous chelate peptide at allhierarchies were compared; in addition, the membrane separating condition was optimized with theorthogonal test. The finding indicates that the antibacterial antioxidant activity of the peptide solutionfiltered by the3kDa membrane is the highest, and the best membrane separation condition is as follows:when the content of chelating peptide is30%, PH value is6.5and the temperature is35℃, the scavengingactivity of DPPH free radical of the hairtail protease hydrolysis fluid ferrous chelates percolate is81.2%(2mg/ml), and its inhibition ratio of staphylococcus aureus is91.3%. Therefore, membrane with themolecular weight being3KD is the best to separate the hairtail protein ferrous chelating peptide with highantibacterial antioxidant activities.3,In order to study the factor affecting the antibacterial stability of hairtail protein ferrous chelating peptide,Plackett-Burman in Minitab was used to design the experiment, selecting several factors which haveobvious effect on the antibacterial antioxidant of hairtail protein ferrous chelating peptide from the relevantfactors which are PH value, temperature, light, air, ultrasonic, density of chelating peptide solution,pasteurization processing and magnesium ion (magnesium sulfate). Finally, PH value, density of chelatingpeptide (mg/ml) and magnesium ion were chosen and their importance was sequenced as follows: PH value> density of chelating peptide> magnesium ion.4,The antibacterial peptide of the hairtail protein ferrous chelate with the molecular weight being less than3KD was separated and purified with the gel filtration chromatography (GFC) G-25and reversed phasehigh performance liquid chromatography (RP-HPLC), and a single component of F1-2with antibacterialcapacity was acquired. The component was sequenced with MALDI-TOF/TOF MS/MS, and the amino acid sequence was His-Tyr-Asp; besides, the nuclear magnetic resonance and infrared spectrum of the structureindicated that the chelating mechanism of tripeptide and ferrous ion was as follows: two-NH-and-NH2onthe tripeptide were coupled with Fe²⁺, while-C=O and-OH on-COOH of tripeptide were coupled withFe2+, thus forming an unstable structure. This obtained substance was named Small peptides ferrouschelates of Hairtail protein (Fe(â…¡)-SHPH).5,The staphylococcus aureus was taken as the indicative bacteria in the study of antibacterial mechanism.The minimum inhibitory concentration (MIC) of Fe(â…¡)-SHPH was0.20mg/ml and minimum bactericidalconcentration was0.26mg/ml. The antibacterial characteristics indicated that Fe(â…¡)-SHPH principallyimpeded the cell division of staphylococcus aureus in the exponential phase; besides, Fe(â…¡)-SHPH alsohad a significant effect on the permeability of the membrane of staphylococcus aureus. When theconcentration was high (1/2MIC), relative conductivity of the solution gradually rose as the response timepassed; after12h, its relative conductivity rose to19.4%. After8h since the effect of Fe(â…¡)-SHPH onstaphylococcus aureus was detected by the flow cytometer (FCM), it could be seen that the number of thecell increased in G0/G1period, but decreased in S period, which indicated that Fe(â…¡)-SHPH has a certaininhibiting effect on the division and reproduction of staphylococcus aureus.6,During the study on the inhibiting effect of the ferrous chelates of hairtail small peptide on the virulencefactor of staphylococcus aureus, it was found that the ferrous chelates of hairtail small peptide had aninhibiting effect of the formation of BBF of staphylococcus, but the effect was not very obvious. WesternBlot analysed the effect of ferrous chelates of hairtail small peptide of different concentrations on thesecretion of enterotoxin A and enterotoxin B of staphylococcus, and the result indicated that the inhibitingeffect on the of the high-concentration (1/2MIC) of the ferrous chelates of hairtail small peptide on thesecretion A was higher than that of the low-concentration (1/16MIC) ferrous chelates of hairtail smallpeptide. However, their effects on the expression of B showed no obvious differences. However, thehigh-concentration (1/2MIC) ferrous chelates of hairtail small peptide obviously impeded the expression ofstaphylococcus aureus α-hemolysin protein.