Study On The Preparation And Properties Of Nannochloropsis Calcium-chelating Peptide

In this study,Green Nannochloropsis oceanica was mainly studied for its proteins,lipids and it’s a good source of proteins for various uses.Nannochloropsis oceanica protein is rich in source,easily available and lower in price.There are many techniques to extract proteins from the sample,but ultrasound–assisted extraction have been extensively studied in recent year.Moderate ultrasonic treatment was used to enhance the extraction as well as increase the quality of the products with less consumption of solvent and time.Extraction was done by optimization of different extraction factor using single factor.Naturally prepared calcium binding peptides are effective in replacement of synthetic supplements.Nannochloropsis proteins can be an excellent source of calcium chelated peptides.Present study explains the Calcium binding Nannochloropsis peptides process i.e interaction between Ca ²⁺ions and peptides/amino acids.Firstly the optimization of conditions were studied using single factor method that includes temperature,ultrasonic power,ultrasonic time,material-liquid ratio,and concentration of Na OH,measuring index were Ca ²⁺chelation activity,Ca ²⁺chelation rate and chelate yield.The preliminary identification of Ca ²⁺chelated peptides was done using ninhydrin color test method.Stability of Ca-peptides were studied under PBS buffer,p H,temperature and simulated gastrointestinal digestion in vitro.Finally,characterization of chelates was done by using FTIR,UV-VIS,FLS,Zeta potential,TG,DSC and X-ray differential.Morphological analysis of peptides and Ca-binding peptides was done by using SEM and AFM.The amino acid and molecular weight distribution of calcium chelated peptide were analyzed.The results are as follows:（1）Screening of optimal extraction condition,the optimal conditions for the protein extraction rate,purification and yield were:extraction temperature 40℃,time 60 mins,ultrasonic power 800W,material to liquid ratio 1:50 g/m L,Na OH concentration 0.4M.Resulting in protein yield 51.69±0.0121%,protein extraction rate 94.81±0.151%and protein purity was 76.86±0.0191%.Howeve,protein extraction rate is only 60.02±0.01%by traditional extraction for 3 hours.It can be seen that the extraction rate of protein by ultrasonic extraction was increased by 34.79%,and the extraction time was shortened by two-thirds.protein extraction rate is too low,only 60.02±0.01%.Therefore,ultrasound-assisted extraction is an effective method for Nannochloropsis protein.（2）Physiochemical properties of extracted proteins such as solubility,FC,FS,WAC and OAC shown increasing trend when p H was increased.It can be concluded that p H is an important factor that can effect properties of proteins significantly.All the physiochemical properties studied were minimum at isoelectric point.Precisely functional properties of Nannocholoropsis are directly related to their physiochemical characteristics.Therefore Nannocholoropsis proteins can be used in food industry if extracted using optimum conditions and potentially replace many synthetic products.（3）The optimal conditions for Ca-chelates formation:chosen parameters are material-liquid ratio 9:1,chelating time 15mins,chelating p H 9,temperature 40^o C and ultrasonic power 1000W resulted in Calcium chelation rate 61.89±0.121%,Calcium chelation activity 9.08±0.151%and Calcium chelation yield 65.57±0.190%.（4）Stability of Calcium chelates in different concentrations of PBS buffer,p H,temperature and simulated gastrointestinal digestion in vitro,explained that the Ca-peptide retention rate was 81.93±0.00761%at 30mins of pepsin digestion,trypsin digestion further lead to lower the retention rate,Ca-chelated peptides were stable at higher p H.Ca chelated peptides were not affected by temperature change and at phosphate buffer lower concentration（10mmol/L）,the chelates were stable.（5）FTIR confirms the chelates formation and UV-VIS and FLS spectrum scanning,zeta potential,Intrinsic fluorescence,particle size,X-ray,TG,DSC and amino acid analysis not only confirmed the production of new complexes as chelates,but also explained that different spectral and signal analysis indicated that metal ions combined with peptides with amino,carboxyl and other active chelating groups in Nannochloropsis peptides to form metal chelates.（6）Furthermore morphological analysis by SEM and AFM images showed the dense structure of the chelated peptides.The analysis results showed that the Nannochloropsis polypeptide-metal ion chelate should be a new substance produced by chelation.The results of this study put emphasis on the development of functional food from Nannochloropsis peptides.