Study On The Functional Properties Of Casein Peptide And Its Bitter Peptides

Casein peptide has attracted much attention because of its bioactivities. But bitter taste is one of the most serious problems which limited the wide use of casein peptide. Bitter peptides, containing hydrophobic amino acids, lead to the bitter taste of casein peptide. There are some debittering methods of removing or decomposing the bitter peptides. But some bitter peptides also have bioactivities. So it is necessary to take account of the bitter peptides while removing them. In this paper, bitter peptides from Sodium caseinate (NaCN) hydrolysised by Alcalase were studied on hydrophobicity, molecular weight distribution, amino acid composition and sequence. Simultaneously, the functional properties of casein peptide, bitterless peptides (aqueous phase peptides) and bitter peptides (secondary butyl alcohol phase peptides) were compared. Based on it, casein short peptide with low bitter taste and high degree of hydrolysis was produced by compound enzymes and then the functional properties of different molecular weight distribution were studied. The studies above provide theory foundation for bitter peptide effective and reasonable application.Screening suitable proteases and optimum hydrolysis technologies were studied. NaCN was hydrolyzed by Pangbo Papain, Pangbo neutrase, Neutrase, Alcalase, Protamex and Trypsin. Alcalase had the highest TCA-SNI, DH and lower bitter taste. Based on the study of protease dosage, substrate concentration, pH, temperature and hydrolysis time, the regression models was established between the TCA-SNI and the four factors that affect the preparation of casein peptide by homogeneous design. The factors were optimized: time 199min, protease dosage 4240 U/g pro.,pH values 10.38, temperature 50℃. In these conditions, DH was 24.27%, TCA-SNI was 79.85%, PCL was 4.12, average molecular weight was 471.23, and bitter value was 2.53.The methods of bitter peptide extracted with secondary butyl alcohol (SBA) were studied and the compositions of different DH bitter peptide were analyzed. Casein peptides of DH 10%, 15%, 20% were produced respectively in the conditions of protease dosage 4240 U/g pro.,pH values 10.38, temperature 50℃by Alcalase. The study on different conditions of bitter peptide extracted with SBA showed bitter peptide can be well extracted in the conditions of casein peptide concentration 20%, SBA amount 50% and naturally pH. And then three kinds of DH bitter peptides (CNH-A-10,CNH-A-15 and CNH-A-20) were produced. Study on molecular weight distribution of three DH bitter peptides showed they were the compound of wide scope and short peptides and a small quantity of free amino acid. Compared with NaCN, bitter peptide had more hidtidine, tyrosine, valine, methionine, phenylalanine, leucine and praline. The content of essential, hydrophobic, aromatic and branched-chained amino acid was increased. Free amino acid in bitter peptide was less than 1% of the total amino acid amount. The content of Proline, leucine and phenylalanine was high while methionine and lysine was low. Essential, hydrophobic amino acid had a high content in free amino acid and basic, aromatic amino acid was low. CNH-A-10 and CNH-A-20 were analyzed by MALDI-TOF-MS and RP-HPLC-ESI-MS. Because the bitter peptide was a compound of all kinds of peptide and amino acid, the result analyzed by MALDI-TOF-MS was not good. While the result analyzed by RP-HPLC-ESI-MS was satisfied. Statistical results of bitter peptide sequence showed: Bitter peptide was the short peptide of less than 20 amino acid and molecular weight lower than 3000Da. With the hydrolysis, the position of hydrophobic amino acid constantly changed from middle to two ends and released to free amino acid, so the bitter taste became less.Functional properties were compared among casein peptide and two samples from SBA and aqueous phases respectively. NaCN was hydrolyzed by Alcalase in the optimized conditions and then extracted with SBA. Casein peptide (CNH), aqueous phase peptide (CNH-H) and SBA phase peptide (CNH-A) were studied. The analysis of RP-HPLC showed: CNH was the compound of hydrophilic and hydrophobic peptides. Most of the hydrophilic peptides were in CNH-H and hydrophobic peptides were in CNH-A. The three samples all had a good solubility between the pH 2~11. Emulsifying activity, emulsifying stability, foaming forming and foaming stability were greatly lower than NaCN (p<0.01). CNH had antioxidative activity. CNH-H had a high eliminating activity on O2- and ?OH. CNH-A had a high eliminating activity on DPPH? and antioxidative effect in linoleic acid. The three samples also had antibacterial activity. CNH-H had a strong antibacterial effect on Pseudomonas, Staphylococcus aureus, Escherichia Coli and Micrococcus luteus. CNH-A had a strong antibacterial effect on Bacillus subtilis. The three samples all had ACEI activity, among them, CNH-H was the highest. Becauseβ-Casomorphin-5,7 had high average hydrophobic value, they were greatly in CNH-A and less in CNH-H.In order to reduce the bitter taste and improve the DH of hydrolysates, the technology of compound enzymes hydrolysis was used. Pangbo Papain, Pangbo neutrase, Neutrase and Protamex were tested to go on hydrolysis the Alcalase hydrolysates. Pangbo Papain was selected because of its hydrolysates had the highest TCA-SNI, DH and the lowest bitter taste. Then the single factors and homogeneous design were tested and the optimum conditions were obtained: protease dosage 2461 U/g pro.,pH values 5.94, temperature 57.4℃. In these conditions, TCA-SNI was 89.75%, DH was 27.31%, PCL was 3.66, average molecular weight was 420.6, and bitter value was 1.01. Three parts of hydrolysates were separated by different molecular weight distribution membranes and the functional properties were studied too. Casein peptide was separated by the 3500 kD and 1000 kD) to acquire three parts: larger than 3000Da (A), between 3500Da and 1000Da (B), lower than 1000Da (C). Analyzed by RP-HPLC, A and B were both composed of hydrophilic and hydrophobic peptides while C had shorter retention time and had more hydrophilic peptides. The bitter value of three samples was below 2. A had good solubility; EC and ES were higher than NaCN; FC and FS were lower than NaCN. B, C had good solubility, EC, ES, FC and FS were greatly lower than NaCN (p<0.01). Biology activity tests showed: The antioxidant activity was B> C> A. Compared with the highest activity of Alcalase hydrolysates, B had a higher elimination rate on O2-, DPPH? and antioxidative effect in linoleic acid, while it reduced 4.04% on eliminating ?OH. Different molecular weight of casein short peptide had antibacterial effect on Pseudomonas, Staphylococcus aureus, Escherichia Coli, Micrococcus luteus and Bacillus subtilis, especially well for Pseudomonas, Staphylococcus aureus, Micrococcus luteus. Generally speaking, the antibacterial activity was B> A> C. Three samples all had ACEI activity. With the decrease of molecular weight, the activity of ACEI increased. C had the best activity of 96.01%. There were lessβ-Casomorphin-5 andβ-Caso morphin-7 in casein short peptide.