Study On The Qualitative And Quantitative Detection Methods Of CPPs Derived From Enzymatic Hydrolysates Of Casein And Its Application

The structural properties of Casein Phosphopep tides(CPPs)determine the diversity of its types.Different companies use different enzymes and enzymatic conditions,the final access to the CPPs sequence there will be some differences,there is no uniform qualitative and quantitative methods to evaluate and detect CPPs.In order to solve this problem,the casein was enzymatically hydrolyzed,its hydrolyzate was qualitatively analyzed,and the calcium-active peptide was isolated.The main components were isolated and identified.The pretreatment methods of samples containing CPPs were screened.Meanwhile,the method of high performance liquid chromatography(HPLC)for CPPs was established and applied.The main research contents and results were as follows:(1)CPPs were screened from enzymatic hydrolysa tes of casein,and five monomer components were isolated and the structural sequence of the active monomer was identified.The sequence of the maximum number of phosphorylated serine monomer was RELEELNVPGEIVESLSSSEESITR.(2)The pretreatment methods of liquid milk,milk powder and calcium tablet were screened and optimized.Milk using trichloroacetic acid(TCA)method combined with thermal denaturation,yogurt with the first dilution,adjusting p H and then TCA method,milk powder using isoelectric point me thod,calcium tablets using dissolved ultrasonic extraction method,the test results showed that the analysis is better.(3)HPLC analysis conditions of CPPs such as column,mobile phase,column temperature and flow rate were optimized.The optimized conditions were as follows: the CPPs are separated by Welch C18 column with the mixture of 1 ‰ trifluoroacetic acid(TFA)aqueous solution and 1 ‰TFA acetonitrile solution as mobile phase,the column temperature was 35 ?,under the binary high pressure gradient elution mode,the flow rate was 0.8 mL/min and detection was performed at 215 nm.(4)The HPLC method for the quantitative detection of CPPs in milk powder was established.The gradient elution conditions were as follows: 10 %~20 % and 20 %~26 % acetonitrile(1 ‰ TFA)elution for 15 min and 30 min.The injection volume was 20 ?L.The detection limit of this method was 15.34 mg/kg and the limit of quantification was 51.14 mg/kg.The results showed that CPPs can be well separated from impurities,and the recovery rate of CPPs in milk powder samples was between 97.59 % and 100.00 %,which was closer to the real addition.At the same time,the CPPs content of milk powder from different manufacturers were determined,which were close to the nominal value of the manufacturers.This method was proved to be with good applicability.(5)The HPLC method for the quantitative detection of CPPs in milk was established.The gradient elution conditions were as follows: 5 %~35 % acetonitrile(1 ‰ TFA)elution for 45 min.The injection volume was 20 ?L.The detection limit of this method was 6.00 mg/kg and the limit of quantification was 20.00 mg/kg.The results showed that CPPs can be well separated from impurities,and the recovery rate of CPPs in milk samples was between 89.36 % and 94.94 %,which was closer to the real addition.(6)The HPLC method for the quantitative detection of CPPs in yoghourt was established.The gradient elution conditions were as follows: 10 %~20 % and 20 %~26 % acetonitrile(1 ‰ TFA)elution for 15 min and 30 min.The injection volume was 20 ?L.The detection limit of this method was 3.07 mg/kg and the limit of quantification was 10.23 mg/kg.The results showed that CPPs can be well separated from impurities,and the recovery rate of CPPs in the yoghurt samples was between 97.73 % and 102.28 %,which was closer to the real addition.(7)The HPLC method for the quantitative detection of CPPs in calcium tablet was established.The gradient elution conditions were as follows: 5 %~35 % acetonitrile(1 ‰ TFA)elution for 45 min.The injection volume was 20 ?L.The detection limit of this method was 13.62 mg/kg and the limit of quantification was 45.40 mg/kg.The results showed that CPPs can be well separated from impurities,and the recovery rate of CPPs in calcium tablet samples was between 95.51 % and 102.26 %,which was closer to the real addition.At the same time,the CPPs content of different calcium tablet were determined.The results were close to the nominal content of the manufacturers.This method was proved to be with good applicability.The CPPs rapid calcium salt intuitive evaluation method of demonstration experiment was established,combined with atomic absorption spectrometry to detect calcium content,the reliability of this method was confirmed,and based on the results we can see that the addition of CPPs in calcium,calcium ions remain soluble longer than the others.