| Tuna is a kind of fish of high yield,which is consumer's favorite food because of its delicate taste and eutrophy.While during the process,a lot of tuna waste,about50%~70% of the total weigh,would be left.Most of the waste are made into fodder or bait,which has low economic value.The tuna skin is rich in collagen and could be good source of preparation of ACE inhibitory peptides.Collagen has a special stable structure,tripe helix,which could not be hydrolyzed by common enzyme but enzymes belonging to the matrix metallo proteinases(MMPs)family.So the material need to be pretreated to loose tripe helix to explore enzymatic site.The traditional pretreatments include acid and alkali treat methods,but these methods take a lot of time,and alkali pretreat could produce something are toxic.Ultrosonic is a green energy and could be used to pretreat tuna skin.The article aims to optimize the ultrasonic pretreatment condition to release peptide from tripe helix,the ultrasonic effects on collagen structure,the gastrointestinal digestion of ACE inhibitory peptides and the structural characteristics of digestion products.To optimize the pretreatment condition to release peptide from trip helix,tuna skin as material,DH(degree of hydrolysate)and Hyp contents as evaluation index,the different ultrasonic power,ultrasonic time,ultrasonic liquid / liquid ratio are tested.The results showed that the better pretreatment conditions as follows,pretreatment time is20 min,ultrasonic power is 30%(195 W),the ratio of material to liquid is 1:15.And theDH is 14.89%,and the Hyp contents is 0.046 g at these conditons.Compared with the untreated group,the DH and Hyp of the ultrasound treatment group were significantly improved.Which indicated that ultrasonic pretreatment could promote the release peptides from trip helix and the enzymatic process.The small molecule peptides were absorbed easily and has functional activity.The ultrasonic pretreatment group contains88.67% small molecule,while the untreated group contains only 46.39%.The FTIR results showed that ultrasonic pretreatment could damage the structure of collagen and thus could promote the release peptides from trip helix and the enzymatic process.In order to study the release mechanism and the effect of ultrasonic pretreatment on ACE inhibitory peptide release,the DH and ACE inhibitory activity of hydrolysate by different ultrasonic time and enzyme time are determined,and DSC and FTIR are detected.The DH and FTIR results showed that ultrasonic could damage collagen change the mode of enzyme,so that enzymatic for 5min could release the peptide with high ACE inhibitory activity compared with un-pretreatment.So ultrasonic could promote ACE inhibitory peptide from triple helix.The SDS-PAGE and HPLC results showed that the content of large weight molecular increased repeatedly,and ultrasonic could accelerate the regularity and release ACE inhibitory peptide efficiently.The DSC results showed that after 1,5,20 min ultrasonic pretreatment,the thermal denaturation temperature of collagen reduced,and the results in accordance with the hydrolysate has high ACE inhibitory activity.Because the ultrasonic reduce the degree of polymerization of collagen.While when the pretreatment time prolonged,the thermal denaturation temperature of collagen by ultrasonic 40 min was increased.Because long-term ultrasound made the remaining collagen more stable.The FTIR results showed ultrasonic could affect the balance of hydrogen bond in collagen,and the hydrogen bond of triple helix and N-H decreased.The results also showed that ?-helix and ?-fold contents of collagen in tuna skin decreased after the ultrasonic pretreatment,while random coil contents increased and ?-turn contents increased significantly,so that ultrasonic made the the structure of the collagen to the disorder.These changes could explore enzymatic site in triple helix and accelerate the release of ACE inhibitory peptide.To verify ACE inhibitory peptides could perform ACE inhibitory activity,the vitro gastrointestinal digestion of hydrolysate was tested,and the digestible products with high activity were isolated and purified.The results showed that peptide from tuna skin keep high ACE inhibitory activity after digested,and digestible products has manySmall molecule polypeptides(under 3 kDa).To ensure the condition of separation,the digested product of E5U20 was separated by Sephadex G-15 firstly,and the results showed that chromatographic column was 1 m × 10 mm,the elution rate was 0.5mL/min,the eluant was water.The peak F2 of E5U20 digestible products was best,which IC50 value was 0.393 mg/mL.The F2 was further separated by semi-high performance liquid chromatographic.There are 8 peaks after separated.The IC50 value of peak 3 was 153.31 ?mol/L.And then the peak 3 was identified by LC-MS/MS,the results show that peak 3 peptide sequence was GPSGPPGP,this sequence derive from842-849 of collagen ?1 chain.And this amino acids were consistent with the structure-activity characteristics of ACE inhibitory peptides,and the sequences were not reported by now. |
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