| Milk proteins exert a wide of nutritional, functional, and biological activities. Many milk proteins posses specific biological properties that make these components potential ingredients of health-promoting foods. Moreover, milk proteins are precursors of many different biologically active peptides which are inactive within the sequence of the precursors protein but can be released by enzymatic proteolysis or ferment. Bioactive peptides usually contain 2-20 amino acid residues per molecule and exhibit various activities, such as antioxidative, antihypertensive, antimicrobial, cytomodulatory, immunoregulatory, opioid or mineral-carrying. Many milk-derived peptides reveal multi-functional properties. Studies on new function of peptides are developing rapidly. Lactobacillus helveticus is uses in the manufacture of dairy products because it is a homofermentative bacteria that is a strong acidifier of milk and has good acid tolerance and strong proteolytic activity. So, production of casein-derived peptides with Lactobacillus helveticus cells and their biological activities were studied in this paper.(1) Lactobacillus (L1, L2, L3 and L4) isolated from yogurt and identified based on physiological-biochemical characteristics. L1 and L2, L3, L4 belong to Lactobacillus helveticus, Lactobacillus casei and Lactobacillus acidophilus respectively. Based on 16S rRNA sequence, L1 and L2 were identified as Lactobacillus helveticus and were named as L.helveticus 9 and L.helveticus 6 respectively.(2) The proteolytic activities of two Lactobacillus helveticus strains were compared by proteolytic zone and degree of casein hydrolysis.The degree of casein hydrolysis and the concentration of peptides of Lactobacillus helveticus 9 hydrolyzates were evaluated during different time.The hydrolyzates were determined by SDS-PAGE and the elution patterns of SephadexG-25 gel fitration.The results indicated that the proteolytic activity of Lactobacillus helveticus 9 was more than that of Lactobacillus helveticus 6. The strain of L. helveticus 9 showed relative proteolytic activity.(3) Effects of inoculated level, incubation period, temperature and substrate concentration on the peptides production from casein-degrading by the proteolytic activity of Lactobacillus helveticus 9 was investigated based on the degree of hydrolysis.The results showed that the optimum fermented conditions were 1×108cfu/mL inoculated level, 5.0% casein, 37℃incubated temperature and 48h incubation, the degree of hydrolysis attained to 17.33% and the concentration of peptides was 0.62mg/mL respectively.(4) Casein hydrolysate was purified by ultrafiltration, SephadexG-25 ,SephadexG-15 and high speed counter current chromatography gradually. Then, the purifed fractions were further identified by ESI-MS. The ESI-MS results showed that F3-1-1 were peptides mixture with molecular weight from 600Da to 1300Da, F3-1-2 mainly were amino acid mixed with low amount of bi-peptide and tri-peptide, F3-2-1 almost were amino acids. The amino acid sequence of F3-1-1-1 was Glu-Pro-Val-Leu-Gly-Pro-Val–Arg–Gly–Pro.(5) Casein hydrolysates exhibited a certain extent ACE inhibitory activities. Moreover, ACE inhibitory activity increased with purification degree. IC50 of F3-1-1-1 showing highest ACE inhibitory activity was 3.125mg/mL.The amino acid sequence of peptide was Glu-Pro-Val-Leu-Gly-Pro-Val–Arg–Gly–Pro (β-CN, f210-219) and molecular weight was 1022.00Da. It shared several features in common with other ACE inhibitory peptides reported. But it is necessary to further test its antihypertensive in vivo.(6) Casein hydrolysates showed a ceitain extent antioxidant activity, such as scavenging activity on DPPH, superoxide radical, and Hydroxyl radical. F3(﹤5000Da) exhibited the highest antioxidant activity. The purified peptide (F3-1-1-1) scavenged 47.01% on superoxide radical at 256.78μM using ultra-weak luminescence analyzer. The amino acid sequence of purified peptide was Glu-Pro-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro (β-CN, f210-219) and molecular weight was 1022.00Da. In addition, it shared several common features of antioxidant peptides reported. It exhibited multi-functional properties which not only showed antioxidant activity, but also ACE inhibitory activity.(7) Casein hydrolysates showed antiproliferative effects and apoptosis induction on the intestinal cells. Moreover, the effects were in a dose and purification dependant manner. F3(﹤5000Da) exhibited the highest cytotoxicity. The growth inhibitory activities of F3 on Caco-2 cells and IEC-6 cells were 33.72% and 8.91% respectively at 5mg/mL. The growth inhibitory activities of F3-1-1 on Caco-2 cells and IEC-6 cells were 41.12%% and 9.59% respectively at 5mg/mL. In TUNEL assay and flow cytometry tests, apoptosis rate of Caco-2 induced by F3-1-1 was up to 14.93%, no apoptosis induction effect on IEC-6 cells was observed by F3-1-1-1, showing good candidate for developing apoptosis specific antitumour agents with low toxicity.(8) Production of casein-derived peptides with L.helveticus cells differed from that producted through enzyme hydrolysation common used nowdays. The peptides showing some physiological effects in the successive results indicated it was feasible to gain bioactive peptides by this way.
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