Study Of DPP-Ⅳ Inhibitory Peptide And Antioxidant Peptide From Defatted Antarctic Krill Powder And Pilot Scale Preparation

Diabetes mellitus is a metabolic disease whose main characteristic is hyperglycemia.Diabetes may cause a number of complications,most of which are caused by elevated levels of reactive oxygen radicals in the patient’s body.Antioxidants can prevent diabetes and its complications by scavenging free radicals in the body to reduce oxidative damage.DPP-Ⅳ（dipeptidyl peptidase 4）is an enzyme involved in blood glucose regulation in the body,and its inhibitors are now commonly used in clinical hypoglycemic therapy.The commonly used inhibitors include selegiline and saxagliptin,but long-term use can cause a series of side effects.It is reported that active peptides are non-toxic,mild and easily absorbed,and some antioxidant peptides,besides having antioxidant functional activity,can also delay the absorption of glucose in the body to achieve hypoglycemia by inhibiting the activity of DPP-Ⅳ.This makes the extraction of source DPP-Ⅳ inhibitory peptides and antioxidant peptides from natural proteins a hot research topic.Defatted Antarctic krill meal is a by-product of shrimp oil processing of Antarctic krill（Euphausia superba）,which has a protein content of more than 65%and is characterized by high protein content and low-fat content,and is a high-quality raw material for the extraction of bioactive peptides.Therefore,in this study,bioactive peptides were extracted by enzymatic digestion using defatted Antarctic krill meal as raw material,and single-factor and response surface process optimization was performed with raw material utilization as the index.The peptides were purified by ultrafiltration and gel chromatography using ABTS and DPPH radical scavenging ability as well as DPP-Ⅳ inhibition ability as criteria.Finally,the active peptide was structurally characterized and the inhibition mechanism of DPP-Ⅳ inhibitory peptide and the potential inhibitory activity of MPO（myeloperoxidase）were investigated by molecular docking.Finally,pilot scale preparation was performed.In order to provide a theoretical basis for the high-value utilization of defatted Antarctic phosphorus powder and the development of hypoglycemic and antioxidant drugs.The specific research contents are as follows.1.In this paper,defatted Antarctic krill powder was used as the raw material,and the raw material utilization rate was used as the optimization index,and the enzymatic process was optimized by single-factor experiment and response surface experiment.The optimum enzymatic conditions for defatted krill powder were obtained as follows:enzymatic digestion time of 4h19min,material-to-liquid ratio of 1:7.78,and enzyme addition of 1.66%.Under this enzymatic digestion process,the raw material utilization rate was 80.68%±0.32%,and the relative molecular weight of the lyophilized powder was98%,the DPP-Ⅳ inhibition rate was 80.06±6.6%,the DPPH radical scavenging rate was43.50±1.2%,and the ABTS radical scavenging rate was 34.35±1.3%,which had good biological activity.2.The effects of different enzymatic digestion times on the hydrolysis degree,DPP-Ⅳ inhibition activity,DPPH radical scavenging rate,ABTS radical scavenging rate and molecular weight distribution of the hydrolysate were investigated.It was found that the hydrolysis degree gradually increased with the increase of enzymatic digestion time and leveled off at 4 h.At this time,the scavenging rate of ABTS radicals was 34.35±1.3%,the scavenging rate of DPPH radicals was 43.50±1.2%,and the DPP-Ⅳ inhibitory activity was 78.06±3.6%.The longer the enzymatic digestion time the smaller the percentage of small molecular weight of the hydrolysate,and the hydrophobic amino acid content of the peptides accounted for 44.16%of the total amino acid content,which is an important factor affecting the biological activity.Then the enzymatic digest was ultrafiltered to obtain four fractions,≥10 ku,10 ku>MW≥5 ku,5 ku>MW≥3 ku and<3ku,in which the fraction<3 ku had higher hypoglycemic activity and antioxidant activity,DPP-Ⅳ inhibition rate 83.12±0.77%,DPPH radical scavenging rate 55.40±0.45%,ABTS radical scavenging rate of 40.25±1.11%.The fractions<3k Da in the ultrafiltration fraction were separated into six fractions（M1,M2,M3,M4,M5 and M6）by Sephadex G-15 chromatography,and the M3 fraction showed the highest activity with 77.99±1.22%DPPH radical scavenging activity,84.60±0.20%ABTS radical scavenging activity,and DPP-Ⅳ inhibiting activity of 57.23%±0.48%（5 mg/m L）.The M3 fraction was analyzed by mass spectrometry for peptide sequences,and 128 peptides were identified.Combining the mass spectrometry score,Peptide Ranker score,i DPPⅣ-SCM score and antioxidant score,six peptides with potentially high antioxidant and hypoglycemic activities were screened:WPPLSPFRCPR,NRPIPPWI,IPDWFLNRQ.PFGLYVHHSWF,FLWLKKTPLPL,and DTVPWFPR.3.The activities of the six peptides were verified after solid-phase synthesis.All six peptides showed good antioxidant activities,and four of them showed good DPP-Ⅳ inhibitory activities:WPPLSPFRCPR,IC₅₀=2.121±0.31 mmol/L;IPDWFLNRQ,IC₅₀=0.6163±0.097 mmol/L;FLWLKKTPLPL,IC₅₀=1.158±0.71 mmol/L;DTVPWFPR,IC₅₀=4.923±2.0 mmol/L.The molecular docking results showed that these four peptides docked with DPP-Ⅳ molecules in the active pocket;all six peptides exhibited low docking scores with MPO（myeloperoxidase）,further suggesting their potential as antioxidant peptides.The stability of the activity of the defatted Antarctic krill powder peptides was investigated by using an in vitro simulated gastrointestinal digestion model.The results showed that the antioxidant activity decreased and the DPP-Ⅳ inhibitory activity increased slightly after simulated gastrointestinal digestion,but still remained at a relatively high level,indicating that its defatted Antarctic krill powder active peptide could also maintain good activity in vivo.4.By expanding the preparation process of defatted Antarctic krill powder active peptide to 100L pilot scale,the obtained peptide powder with molecular weight<5000Da accounted for 98.28%,peptide content was 82%,total nitrogen was 14.1.The peptide powder concentration was 10mg/ml,hydroxyl radical scavenging rate was 63.11%,total reducing power was 22.51%,superoxide anion radical scavenging rate was 30.86%,α-amylase inhibitory activity was 54.21%,α-glucosidase inhibitory activity was 65.10%.30.86%,α-amylase inhibitory activity 54.21%,α-glucosidase inhibitory activity 65.10%,showing good hypoglycemic activity and antioxidant activity,which provided a useful reference for the industrial production of bioactive peptides from defatted Antarctic krill powder.