Preparation And Functional Properties Of Chlorella Protein And Its Hydrolyzed Peptide-calcium Chelate

Chlorella is a type of single-celled green algae,rich in nutrients,of which the protein content is about 60%,which is a potential high-quality protein resource.At present,Chlorella is widely used in oil extraction and the preparation of biomass resources.After oil extraction,Chlorella residues are mostly used as animal feed or directly treated as waste,causing serious waste of resources and environmental pollution.Based on this,in this study,the extraction of Chlorella protein by synchronous dual-frequency ultrasound assisted alkali was investigated,and its structure and functional properties were characterized;on this basis,the Chlorella proteolytic product was chelated with calcium to prepare peptide-calcium chelate,and the antioxidant activity,absorption and transport of this chelate were studied.It is expected to lay a theoretical foundation and scientific basis for the comprehensive development and utilization of Chlorella protein and its hydrolytic peptide chelated calcium.The main research contents and results of this thesis are as follows:1.Study on the ultrasonic-assisted extraction,structure and functional properties of chlorella protein(1)Synchronous dual-frequency ultrasonic-assisted alkaline extraction of Chlorella protein through single factor and response surface optimization.The optimal process parameters are:dual-frequency 28/40 k Hz,ultrasonic power 360 W,extraction temperature 45?,alkali concentration 3%,and ultrasound time 20 min.Under these conditions,the extraction rate of chlorella protein reached 52.36%,which was increased by 24.24%compared with traditional alkaline extraction method.The results showed that the simultaneous dual-frequency ultrasound-assisted alkaline extraction can significantly improve the extraction efficiency of chlorella protein,and it is a green and efficient new method for extracting chlorella protein.(2)Chlorella protein was characterized by chemical methods combined with chromatographic analysis technology.Compared with alkaline extraction method,the chlorella protein extracted by synchronous dual-frequency ultrasound has obvious differences in secondary structure and tertiary structure,and the contents of?-helix and?-turn are 13.47%and 21.55%respectively,which are significantly lower than those of alkaline extraction method(17.02%and 27.14%,respectively,p<0.05).The contents of?-sheets and random coils are significantly increased(p<0.05).The intensity of the absorption peak and the fluorescence absorption peak were both significantly enhanced.The hydrophobic amino acid content was 28.1%,which was significantly higher than that of alkaline extraction method(10.0%,p<0.05).The sulfhydryl content increases,the protein particle size decreased,the molecular surface became smooth,and the molecules became dispersed.It showed that the chlorella protein molecules changed from order to disorder after ultrasonic treatment.(3)The functional properties of chlorella protein were determined by chemical methods.As a result,the solubility of the protein obtained by simultaneous dual-frequency ultrasound method(62.45%)was better than that of the alkali method(32.11%,p<0.05),and the foamability and foaming stability,emulsification and emulsification stability were all significantly improved(p<0.05,both compared with alkali extraction method),however,no significant differences were found in water holding capacity and oil holding capacity(p>0.05).2.Preparation and characteristic study of chlorella proteolytic peptide-calcium chelates(1)Chlorella protein was hydrolyzed by alkaline protease,the degree of hydrolysis was 11.34%,and the yield of polypeptide was 52.23%;the chlorella proteolytic solution was subjected to ultrafiltration and fractionation,and four fractions of polypeptide solution were obtained:Mw<1 k Da(F1),1 k Da<Mw<3 k Da(F2),3 k Da<Mw<5 k Da(F3),Mw>5 k Da(F4),the proportions of which are 30.12%,36.25%,13.55%,and 20.08%,respectively,of which small peptides with Mw<3k Da account for 66.37%,indicating chlorella protein hydrolysate is mainly composed of peptides with smaller molecular weight.(2)Different peptide fragments were chelated with calcium,iron,and zinc ions,and the best metal ion was selected as calcium ion,based on the chelation rate and the metal ion content in the chelate.The peptide fragments are F2 and F3.The chelating ability of calcium ions is strong,and two peptide-calcium chelates,F2-Ca and F3-Ca,were prepared.The chlorella peptide and its calcium chelate were characterized by ultraviolet spectroscopy,Fourier infrared spectroscopy,scanning electron microscopy,and Zeta potential.The results showed that the amino and carboxyl groups of chlorella peptide are involved in the calcium ion chelation process.At the same concentration,after chlorella peptide was chelated with calcium ions,its ability to scavenge free radicals was enhanced,namely F2-Ca>F2,F3-Ca>F3,and can significantly increase H₂O₂-induced CAT and SOD in RAW 264.7 cells,showing that peptide chelated calcium has strong antioxidant capacity,that is F2-Ca>F3-Ca.3.Study on the in vitro bioavailability of chlorella peptide-calcium chelatesCaco-2 cells were used as an uptake and transport model to determine the calcium bioavailability of chlorella protein peptide-calcium chelate.As a result,in the range of calcium content of 0.125-1.0 mg/m L,the bioavailability of chlorella peptide calcium chelates were significantly higher than that of calcium chloride,and it was concentration-and time-dependent.When 1.0 mg/m L was digested for 120 min,the bioavailability of F2-Ca and F3-Ca were 58.83%and 54.74%in decibels,which were significantly higher than Ca Cl₂(31.27%,p<0.05),indicating that chlorella peptide can effectively promote the absorption of calcium in the human intestine.