Utrasound-assisted Subcritical Water Extraction Of Peptides From Spirulina Platensis And Their Hypoglycemic Activities

Spirulina platensis,which contains nutritional value and medicinal value.The biological activities of Spirulina platensis and Spirulina protein,such as anti-oxidant,anti-tumor,anti-bacterial and so on,have been extensively studied at home and abroad.In this paper,the hypoglycemic activity of Spirulina polypeptide was studied,which extends the medicinal value of Spirulina further.In this study,utrasound-assisted subcritical water extraction of peptides from Spirulina platensis was investigated compared with the pretreatment methods of coalition of multigelation,homogeneity and ultrasonic-extraction.Then The factors affected on the extraction rate,including temperature,ultrasonic power,pressure and time were systematically discussed using single factor level analysis,and response surface methodology was applied.The antioxidant and hypoglycemic activity of crude protein/peptide extracted with different temperatures was determined to screen for better active components.Analytical HPLC was used to determine its molecular weight distribution and it was separated and purified by preparative HPLC.The obtained proteolytic were analyzed and identified the sequence by the mass spectrometry method.The biological activity of peptides was predicted by Peptide Ranker and pepATTRACT to Screen synthesis of the top three peptide sequences and verify its hypoglycemic activity.The results as follows:1)The extractive reactor was redesigned to increase the contact area of the raw material and subcritical water,and the extraction rate of crude protein is improved significantly from10.30%to 40.20%（p<0.01）;The four factors’order is temperature>ultrasonic power>extraction time>pressure;Establishing a responds surface model.The factors including temperature,ultrasonic power and extraction were investigated in terms of the extraction rate.and the best extraction rate of 74.02%was achieved under the following extraction conditions:temperature of 153°C,ultrasonic power of 221W,extraction time of 64min and pressure of10MPa.The extraction rate of 45.76%was achieved by the pretreatment methods of coalition of multigelation,homogeneity and ultrasonic-extraction.So ultrasound coupling subcritical water extraction of crude protein/peptide from Spirulina platensis was better.2)The antioxidant activity of crude protein/peptide extracted with different temperatures was determined by measuring its reducing power and clearance ability on DPPH and ABST+and OH-radicals in vitro,and the results show that protein/peptide extraction exhibits an excellent antioxidant activity better than the protein/peptide extracted by the pretreatment methods of coalition of multigelation,homogeneity and ultrasonic in a dose-dependent manner at concentrations of 0¹0 mg/mL.3)The hypoglycemic ability of Spirulina protein/peptide was evaluated by measuring its inhibitory effects onα-amylase,α-glucosidase,and DPP4 in vitro.Results of inhibition of the crude protein/peptide suggest that the best inhibitory effect of these three enzymes was 120°C.In combination with antioxidation experiments,The Spirulina protein/peptide extracted at120°C and 130°C was selected for further verification combined antioxidant text.4)The proteins/peptides obtained from the Spirulina obtained by 120°C and 130°C,ultrasonic homogenization and repeated freezing and thawing were used as the test substances,and the hypoglycemic effect was evaluated by establishing a cell model with HepG 2 cells by MTT assay.The results showed that the Spirulina protein/peptide obtained by ultrasound coupling subcritical water method under 120°C had better hypoglycemic activity.5)The molecular weight protein/peptide of Spirulina platensis by ultrasound coupling subcritical water was discussed.The results show that extract were peptides because it’s molecular weight≤5000 Da.6)Using preparative HPLC,the Spirulina peptide obtained under extraction conditions at120°C was isolated and purified to obtain two components,labeled as P1 and P2.The results of the study showed that both components had a hypoglycemic effect.7)Peptide of Spirulina at 120°C was identified,then,11 peptide sequences were identified as follows:LCQLCAGKGMDK、GVPMPNK、RNPFVFAPTLLTVAAR、NTRSILFLGK、LRSELAAWSR、RPLTVCKIL、ALAEEAEPALGVEKEAHGR、LALSSAGR、GIEILNK、EGGQTAPASTR、HAEELKAK.The 11 peptide sequences were predicted for biological activity by Peptide Ranker and pepATTRACT.Results showed that RNPFVFAPTLL TVAAR（RR）;LRSEL AAWSR（LR）;GVPMPNK（GK）;possessed strong hypoglycemic activity.8)The corresponding synthetic peptide RR,LR,GK had good inhibitory effects onα-amylase.Their IC₅₀0 value were 1077.59μg/m L,313.61μg/mL,236.23μg/mL.The corresponding synthetic peptide RR,LR,GK had good inhibitory effects onα-glucosidase.Their IC₅₀0 value were 164.49μg/mL,134.23μg/mL,151.46μg/mL.RR,LR,GK had good inhibitory effects on DPP4,their IC₅₀0 value were 181.16μg/mL,167.3μg/mL,192.31μg/mL.