博士 / 國立中興大學 / 食品科學系 / 92 / ABSTRACT
For determining the inhibition of angiotensin I-converting enzyme (ACE) activity, protein hydrolysates were prepared from tuna cooking juice (contain 4% water-soluble protein) with various commercial proteases. Tuna cooking juice presented little inhibitory activity against ACE. Orientase hydrolysate exhibited the most potent inhibitory activity on ACE. The best hydrolysis conditions with respect to inhibition of ACE were 3h of incubation at 50℃ and pH 6.5, and a ratio of enzyme (orientase) to substrate (tuna cooking juice) of 1/250. Preparation [orientase hydrolysate (OAH)] under these conditions yielded potent activity (IC50 12.52 ± 0.02 mg/mL).
Fractionation of the OAH on a Sephadex G-25 gel filtration chromatograph resulted in more potent inhibition of ACE. Four major fractions, OA1 (Mw>2573), OA2 (926>Mw>565), OA3 (Mw<565) and OA4 (Mw<565) were collected. The active fractions were peptides with lower molecular weights (< 1,000 Da). OA1, with higher molecular weight, did not affect ACE. The IC50 values for OA2 and OA4 were 25.26 ± 0.02 and 0.52 ± 0.03 mg/mL, respectively. The most active fraction OA3 had an IC50 value of 0.21 ± 0.01 mg/mL. This fraction was apparently rich in basic amino acids and aromatic amino acids.
Five ACE inhibitory peptides were isolated from the hydrolyzates (mixed OA2, OA3 and OA4 fractions) by reversed-phase high performance liquid chromatography (HPLC). The peptides comprised the residues of 2-5 amino acids in the sequences, including Pro-Thr-His-Ile-Lys, His-Ile-Lys, Pro-Ala-Gly-Tyr, Leu-Tyr, Pro-Lys-Ala.
OAH inhibits ACE apparently. Systolic blood pressure (SBP) reduced 18 mmHg after 2 h for 9 week-old spontaneously hypertensive rats (SHR) orally administered the hydrolysate at 5 ml hydrolysate /kg body weight (≒0.25 g/kg), and reduced, 22 and 30 mmHg respectively, for those administered with lyophilized OAH at 0.5 and 1.0 g/kg body weight. This showed that the reduction effect on SBP was dose-dependent. OA3 exhibited strongest effect on SBP in SHR. The next was OA4. On the contrary, OA1 and OA2 showed no significant effect on SBP in SHR. Furthermore, 30 week-old SHR were applied to the antihypertensive test. Results showed ACE inhibitors also exhibited effect on SBP in 30 week-old SHR.
Commercial diets mixed with 0.25, 1.25 or 2.50% (w/w) lyophilized OAH were fed to 9 weeks-old SHR to investigate long-term (8 weeks) administration effect on SBP in SHR. SBP of SHR fed a normal diet gradually increased, while it decreased, then increased slower for those fed diets containing OAH. The more OAH in diets the lower increase in SBP. These findings imply that OAH participates in suppressing or delaying the development of increases in blood pressure.
OAH was applied to imitative digestion tests with enzymes from different segments of porcine small intestine or with gastrointestinal enzymes to investigate the effects of digestive enzymes on ACE-inhibiting activity from OAH. Enhanced ACE inhibitory activity (decreased in IC50 value) was observed when OAH was digested with pepsin at 37℃, pH 3.0. However, after peptic predigestion followed by hydrolysis with enzymes from different intestine segments, all OAH preparations exhibited evident reduction in ACE inhibitory activity. Moreover, the longer digestive time showed the lower ACE inhibitory activity. Among these digestions, enzymes from the first intestine segment strongly reduced ACE inhibitory activity. OAH remained only 62 % of the original activity, and IC50 values increased from 9.65 to 17.15 mg/mL, after digested by the first intestine segment for 12h. The effect of lowering ACE inhibitory activity along the gut decreased from the first through the fifth segment. These results suggest that the ACE-inhibiting effect of OAH increased by the digestive enzymes from stomach, and declined by intestinal enzymes. Additionally, hydrolysis performed with trypsin or chymotrypsin will conduct the increase ACE-inhibiting effect in OAH.
Protection and absorption-enhancing treatments were applied to enhance antihypertensive activity of OAH and OA3 on SHR. Results showed that 30% egg yolk emulsion did not exhibit greater effect on lowering SBP in SHR. On the contrary, 30% lecithin emulsion, 30% arabic gum encapsulation, liposome entrapment significantly increased antihypertensive effect of OAH and OA3. Liposome entrapment exhibited best enhancing-effect in lowering SBP in SHR.
For preparing more active ACE inhibitor, two-step hydrolysis method was used in producing protein hydrolysates from tuna cooking juice. Results indicted that a two-step hydrolysis using orientase or alcalase with flavorzyme was unable to obtain more active ACE inhibitory hydrolysate. Oppositely, orientase with alcalase acquire more active ACE inhibitor.
In conclusion, retrieve and utilization of tuna cooking juice will achieve value-added benefit.
| Identifer | oai:union.ndltd.org:TW/092NCHU0253015 |
| Date | January 2004 |
| Creators | Jyh-Sheng Hwang, 黃至盛 |
| Contributors | Wen-Ching Ko, 柯文慶 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 164 |
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