Preparation Of Peptides From Seabuckthorn Seed Protein And Its Anti-obesity Activity

The number of obese patients was increasing recently.At present,the common anti-obesity products in the market were less effective and parts of drugs even caused side effects.Therefore,it was of great significance to explore food-derived anti-obesity product.Seabuckthorn seed meal,as a by-product of processing,has rich nutritional value and high protein content(about 20-30%),which can be used as resource of plant-based peptides.Seabuckthorn seed protein(SSP)was prepared by different methods and the enzymatic hydrolysis was optimized to obtain the hydrolysates.The peptides were purifited by ultrafiltration,macroporous resin separation and solid phase extraction,combining with autodock and ultra-high pressure liquid chromatography mass spectrometry to identify and screen.Then the anti-obesity ability and mechanisim of synthetic peptides were investigated.In addition,the digestion stability and transport absorption characteristics of peptides were explored.The research contents and results are as follows:(1)Compared with the color,structure and functional properties of SSP by different extraction methods,phytase assisted extraction was choosen as the optimal.SSP possessed higher protein solubility,digestibility,oil retention and foamability by using this method.In addition,it also had the characteristics of high brightness(L*64.05),low redness(a*9.99)and low yellowness(b*14.52).Phytase treatment could significantly reduce the contents of phytic acid,proanthocyanidins(TP),polyphenols(TPC)and flavonoids(TFC)in SSP.Correlation analysis showed that L*was significantly negatively correlated with TP,TPC and TF(p<0.001),while a*and b*were significantly positively correlated with them(p<0.001).In addition,polyphenols played a key role on protein color.In a word,phytase could separate phytate-metal ions from proteins,and inositol could provide a strong polar environment for substances such as polyphenols,which destroyed the interaction between polyphenols and proteins,thus weakening the color.(2)The enzymatic hydrolysis of SSP was optimized with the inhibitory rate of porcine pancreatic lipase(PPL)as the index.Results showed that the optimal conditions for the preparation of seabuckthorn seed protein hydrolysate by Alcalase(SSPA)were:initial p H10.0,temperature 50°C,enzyme dosage 0.25 g/100 g protein and 8 h of hydrolysis time.Under these conditions,SSPA inhibited PPL activity by 35.12%at 0.8 mg/m L.Inhibiting kinetics showed that SSPA inhibited PPL in a non-competitive manner by forming PPL-substrate-SSPA or PPL-SSPA-substrate complex.The content of peptide with molecular weight less than 3 k Da in SSPA was 60.54%,which was rich in Glu,Asp and Arg.SSPA exhibited the activity on scavenging DPPH and ABTS free radicals,chelating ferrous ions,and could effectively reduce the damage of hydrogen peroxide to yeast cells(the survival rate was 68.49%,15%higher than that of the control group).In addition,SSPA showed higher thermal stability,p H stability and gastrointestinal digestion stability.Moreover,the PPL inhibitory rate of SSPA could be significantly improved by adding appropriate amount of Na⁺and Mg²⁺,and which couldn't be influenced by exposure to air during short-term storage.(3)The component with molecular weight less than 3 k Da was obtained by ultrafiltration,then it was separated combined with dynamic adsorption by HPD826 resin and the elution with Oasis Max microcolumn.UPLC-MS/MS analysis revealed 31 peptides in SSPA,and six peptides with higher peptides content were predicted via molecular docking as anti-obesity peptides.The peptides(<3 k Da peptides part)content was as follows:VR(2.90%),FR(7.40%),RDR(1.10%),APYR(1.50%),NLLHR(1.40%)and EEAASLR(1.10%),respectively.The IC₅₀value of PPL inhibition rate was as follows:VR(371.07?g/m L),FR(243.07?g/m L),RDR(250.50?g/m L),APYR(350.41?g/m L),NLLHR(220.70?g/m L),EEAASLR(510.55?g/m L),respectively.The PPL-inhibiting actions of these peptides involved hydrophilic and hydrophobic interactions,hydrogen bonding,?-?stacking and Van der Waals interactions.These interactive forces stabilized the peptide-PPL complex while affecting PPL conformational change for accommodating the substrate.Pearson correlation analysis showed that there was a positive correlation between molecular binding energy and PPL inhibition rate of peptides from seabuckthorn seed protein(r=0.865,p<0.05).(4)In the range of 0.2-1.0 mg/m L,all the peptides(VR,FR,RDR,APYR,NLLHR and EEAASLR)could effectively inhibit the proliferation of 3T3-L1 cells by 20%to 70%,and APYR had the best inhibitory effect(IC₅₀=0.35 mg/m L).Flow cytometry showed that APYR could block the G2-M phase of 3T3-L1 proliferation and reduce the number of cells in the G0-G1 phase.High dose of the synthesic peptides(0.2 mg/m L)could effectively inhibit the differentiation of 3T3-L1 cells,also decreased the accumulation of triglyceride(TG)content by 60%-98%.APYR expressed the best effect on inhibiting the accumulation of TG by76.24%.After 8 d of differentiation,the glycerol release in APYR treatment group was the highest(109.44?M),which was about 2.06 times of that in model control group.The glycerol release in other treatment groups was about 1.50-1.80 times of that in model control group.Results of RT-q PCR and WB showed that high-dose peptides could activate the c GMP-PKG pathway and phosphorylate HSL and AMPK.On the one hand,HSL plays the role of lipopolysis and reduces the number of lipid droplets in cells.On the other hand,phosphorylated-AMPK could inhibit the expression of PPAR?,C/EBP?,and Fas,so as to inhibit the differentiation of adipocytes and reduce the synthesis of fatty acids.Phosphorylated-AMPK could activate the expressions of CPT-1 and PGC1?,in return promoted the?-oxidation of fatty acids in mitochondria.(5)After gastrointestinal digestion,the anti-proliferation and anti-differentiation ability of the peptides from SSP was decreased significantly,except APYR.There was 86.56%of APYR remained after gastrointestinal digestion and 49.10%of APYR could be transported to blood through intestinal epithelial cells.The P_appof APYR was 3.03×10^-6cm/s showing that its easily absorption characteristic.The retention and transport rates of the other five peptides were significantly lower than those of APYR.It could be seen that the molecular weight and charge of peptides had significant influence on transshipment and absorption,while the influence of hydrophobic effect was hidden with the increment of molecular weight.In addition,the existence of Val and Arg could promote the transportation through membrane,and the existence of Pro could improve the absorption capacity by reducing the hydrolysis by peptidase.