Release of Short and Proline-Rich Antihypertensive Peptides from Casein Hydrolysate with an Aspergillus oryzae Protease

Abstract

Angiotensin-I converting enzyme inhibitory activities were measured after hydrolysis of casein by 9 different commercially available proteolytic enzymes. Among these enzymes, a protease isolated from Aspergillus oryzae showed the highest angiotensin-I converting enzyme inhibitory activity per peptide. The A. oryzae peptide also showed the highest antihypertensive effect in spontaneously hypertensive rats when the systolic blood pressure was measured 5 h after oral administration of 32 mg/kg of various enzymatic hydrolysates. Significant antihypertensive effects were observed with dosages of 9.6, 32, and 96 mg of the A. oryzae peptide/kg of body weight (BW), and the effects were dependent on these peptide dosages. Analysis of peptide length showed the A. oryzae hydrolysate was the shortest of all tested casein hydrolysates; the peptide mixture had an average value of 1.4 amino acids (AA) in the sequence. To further characterize the A. oryzae hydrolysate, we analyzed the AA sequence of the whole peptide mixture. Various AA were detected at the first AA position, however, an increased number of Pro residues were observed at the second and third position of the A. oryzae hydrolysate. No strong signals were detected after the fourth AA position of the A. oryzae hydrolysate. These results suggest that the casein hydrolysate of A. oryzae, which expressed potent antihypertensive effects in spontaneously hypertensive rats, mainly contain short peptides of X-Pro and X-Pro-Pro sequences.

Effects of a Casein Hydrolysate Prepared fromAspergillus oryzaeProtease on Adjuvant Arthritis in Rats

“…Furthermore, these -casein (1-28) or casein hydrolysates prepared with gastrointestinal proteases, which were almost completely composed of endoproteases, would have consisted of larger peptides than that with A. oryzae protease which mainly contained a large amount of oligopeptides with 2 to 3 amino acid residues and free amino acids. 16) In the present study, CH might have included novel short peptides which were able to suppress inflammation and/or immunostimulation.…”

“…17,18) Therefore, novel biological and physiological functions would be observed by the application of peptides prepared by digestion with microbial enzymes. In this study, we prepared CH as a protein hydrolysate from casein, 16) and administered CH orally to adjuvant arthritis rats to evaluate the effects on the arthritic inflammation. CH contains amino acids and more than 100 kinds of peptides derived from casein (data not shown).…”

“…CH was prepared by Calpis Co., Ltd., using A. oryzae protease as described previously. 16) Sodium caseinate (CNa) prepared from cow's milk was dissolved in hot water (80 C) to an 8% (wt/vol) solution, and the pH value was adjusted to 7.0 by adding 1N NaOH. After cooling to <20 C, the protease isolated from A. oryzae was added directly to the CNa solution at a final concentration of 0.32% (wt/vol), and the mixture incubated at 50 C for 14 h. The reaction was stopped by heating the solution at 100 C for 5 min to inactivate the enzyme, and the hydrolysate was powdered with a spray drier before use.…”

“…In the present study, we prepared a casein hydrolysate (CH) from casein prepared with the proteolytic enzyme from Aspergillus oryzae (A. oryzae) which contained a large amount of oligopeptides with 2 to 3 amino acid residues and free amino acids. 16) It has been demonstrated that such peptides as L-valyl-L-prolyl-Lproline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP) had potent antihypertensive activities by their ACEinhibitory effects. 17,18) These active peptides would not usually be produced by digestion with endogenous gastrointestinal enzymes, when casein is taken as a food.…”

“…17,18) These active peptides would not usually be produced by digestion with endogenous gastrointestinal enzymes, when casein is taken as a food. 16) Therefore, novel physiological functions would be observed for the administration of peptides prepared by digestion with microbial enzymes.…”