Antihypertensive effect of heshiko, a fermented mackerel product, on spontaneously hypertensive rats

ABSTRACT:   During the processing of heshiko , a fermented mackerel product, a rapid increase in peptide content in the extract and a remarkable decrease in the IC₅₀ (the inhibitor concentration to inhibit 50% of enzyme activity) as an index of the angiotensin I-converting enzyme (ACE) inhibitory activity were observed. Systolic blood pressure (SBP) in spontaneously hypertensive rats (SHR) decreased between 2 and 4 h after single oral administration of heshiko extract at a dose of 10 mg/kg as a peptide, and SBP recovered its initial level by 8 h. For single doses of extract at three different levels (5, 10 and 50 mg/kg), SBP similarly decreased after between 2 and 4 h. The decreased SBP at 50 mg/kg was almost equal to that at 10 mg/kg, indicating a low dose dependency for heshiko extract. Through successive administration of heshiko extract or its desalted extract at 10 mg/kg for 10 days, SBP decreased 7 days after the start of administration and it recovered its initial level 5 days after stopping administration. During these periods, the change in ACE activity in blood plasma from SHR administered the extract roughly corresponded to that of SBP, suggesting that ACE inhibition was related to a decrease in SBP. For long-term administration of the extract to 5-week-old SHR for 70 days, SBP decreased 28 days after the start of administration. The decreased SBP remained low for 28 days after stopping administration, whereas the decreased ACE activity recovered its initial level. These results suggest that heshiko extract influences not only ACE inhibition, but also other systems that regulate blood pressure.     

Antihypertensive effect of Narezushi, a fermented mackerel product, on spontaneously hypertensive rats

ABSTRACT:   During the fermentation of mackerel to narezushi , the concentration of peptides required to inhibit 50% of the ACE activity in the assay media (IC₅₀), as an index of the angiotensin  I-converting enzyme (ACE) inhibitory activity, was remarkably decreased with a rapid increase in peptide contents. Systolic blood pressure (SBP) in spontaneously hypertensive rats (SHR) decreased between 2 and 4 h after the single oral administration of greater than 10 mg peptide/kg narezushi extract, and recovered to the initial level by 8 h thereafter. The SBP decreased at seven successive daily doses of 10 mg/kg of narezushi extract and then recovered to the initial level 5 days after stopping a total of 10 daily administrations. The extract was administered to five-week-old SHR rats for 70 days and SBP decreased 21 days after starting and continued for 28 days after the end of administration. The peptide-rich fraction from narezushi extract had a powerful antihypertensive effect, whereas the other fraction had a similar, but weak effect.     

Identification of oyster-derived hypotensive peptide acting as angiotensin-I-converting enzyme inhibitor

Angiotensin-I-converting enzyme (ACE) plays a crucial role in the crisis of hypertension. Some peptides that originate from protease hydrolysates are known to suppress ACE activity in vitro and in vivo. Here, we investigated whether trypsin hydrolysate of oyster Crassostrea gigas showed hypotensive activity and ACE inhibition. The hydrolysate significantly suppressed systolic blood pressure and ACE activity in spontaneously hypertensive rats following a one-shot oral administration and a long-term feeding experiment lasting 9 weeks. Each hydrolysate from oyster tissue showed ACE inhibitory activity, indicating the hypotensive effect was due to synergism. One potent ACE inhibitory peptide, Asp-Leu-Thr-Asp-Tyr, was identified from the hydrolysate of the striate muscle, and the peptide exhibited hypotensive activity in vivo. Protease digestion analysis suggested that Asp-Tyr could be the real effector of this penta-peptide in vivo.     

Angiotensin-converting enzyme I inhibitory activity of phlorotannins from Ecklonia stolonifera

ABSTRACT:   As part of our study of the isolation of antihypertensive agents derived from natural marine products, the bioactivity of 10 edible Korean seaweeds were screened by angiotensin converting enzyme (ACE) inhibitory and peroxynitrite assays. Among the crude extracts of selected seaweeds, including five Phaeophyta ( Ecklonia stolonifera , E. cava , Pelvetia siliquosa , Hizikia fusiforme , and Undaria pinnatifida ), four Rhodophyta ( Gigartina tenella , Gelidium amansii , Chondria crassicaulis , and Porphyra tenera ) and one Chlorophyta ( Capsosiphon fulvescens ), the ethanol extracts of E. stolonifera , E. cava , P. siliquosa , U. pinnatifida , and G. tenella exhibited significant inhibitory properties against ACE at more than 50% inhibition at a concentration of 163.93 µg/mL. Phloroglucinol 1 , eckstolonol 2 , eckol 3 , phlorofucofuroeckol A 4 , and dieckol 5 had been isolated previously, and triphlorethol-A 6 and fucosterol 7 were isolated for the first time from E. stolonifera. Also, the ACE inhibitory and peroxynitrite scavenging properties of phlorotannins 1–6 were evaluated, along with fucosterol 7 obtained from E. stolonifera . Among profound peroxynitrite scavenging compounds 1–6 , phlorotannins 3 , 4 and 5 were also determined to manifest marked inhibitory activity against ACE, with 50% inhibition concentration (IC₅₀) values of 70.82 ± 0.25, 12.74 ± 0.15, and 34.25 ± 3.56 µM, respectively.     

The effect of low inclusion levels of Antarctic krill (Euphausia superba) meal on growth performance,apparent digestibility and slaughter quality of Atlantic salmon (Salmo salar)

Two trials with Atlantic salmon (Salmo salar) were conducted to evaluate the potential of krill meal to improve feed intake. In the first experiment, after transfer to sea water, salmon smolts were fed diets added 75 or 150 g kg^?1 Antarctic krill meal in substitution for fish meal for 13 weeks. The apparent digestibility coefficient for crude protein and the majority of the amino acids was significantly lower in the feeds added krill meal (around 83.5%) than in the control diet (84.9%), whereas the digestibility of crude lipids, dry matter and energy was not significantly different among the three diets. Krill meal addition resulted in higher feed intake, which led to higher growth rates and final body weights. In the second experiment, large salmon were fed a diet containing 100 g kg^?1 krill meal for 6 weeks before slaughter. Their feed intake and growth performance were assessed, and fillet and visceral fat contents were measured. Salmon fed the 100 g kg^?1 krill meal diet tended to eat more, resulting in significantly increased growth rates, when compared to control fish. Fish fed krill meal also had a significantly lower condition factor.     

Potential Precursor of Angiotensin-I Converting Enzyme (ACE) Inhibitory Activity and Structural Properties of Peptide from Peptic Hydrolysate of Cutlassfish Muscle

ABSTRACT

Peptic hydrolysates were prepared by digesting the cutlassfish muscle protein using pepsin for 1, 3, and 6 h, and their inhibitory activity against angiotensin-I converting enzyme (ACE) was studied. The ACE-inhibitory effect of the peptic hydrolysate of cutlassfish muscle generated at the 3 h time point exhibited the strongest activity. After identifying the optimal hydrolysate, the active peptide was isolated by ultrafiltration, gel permeation, and high performance liquid chromatography (HPLC). The resulting purified peptide was characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and was identified to be a 496.44 Da pentapeptide (Phe-Ser-Gly-Gly-Glu). The ACE-inhibitory activity of the active peptide exhibited an IC₅₀ value of 0.033 ± 0.003 mg/ml. A molecular docking program was used to simulate the interaction between the peptide and ACE, which revealed that the inhibitory effect was mainly due to the hydrogen bonds between ACE and the peptide. Based on the ACE-inhibitory properties and the molecular docking study of the resulting active peptide, we demonstrated an increase in nitric oxide (NO) production in a dose-dependent manner. In conclusion, cutlassfish protein hydrolysate and the resulting active peptide could be used as active ingredients in functional food as anti-hypertensive agents.     

Experimental Cryptobia salmositica (Kinetoplastida) infections in Atlantic salmon, Salmo salar L.: cell-mediated and humoral immune responses against the pathogenic and vaccine strains of the parasite

Hatchery-reared Atlantic salmon, Salmo salar L., were vaccinated intraperitoneally (i.p.) with a live attenuated Cryptobia salmositica vaccine (either 100 000 or 5000 parasites fish⁻¹) and 4 weeks later were challenged with the parasite (either 100 000 or 5000 parasites fish⁻¹). Unvaccinated, infected salmon had high parasitaemias and were anaemic. Fish given a high dose (100 000 parasites fish⁻¹) had higher parasitaemias than fish given the lower dose. Vaccinated fish had low parasitaemias and a mild anaemia, but recovered quickly after challenge. Complement-fixing antibody increased in vaccinated fish after challenge and was highest at 2 weeks post-challenge. The cell-mediated response (both T cells and B cells) was depressed in infected fish until 4 weeks after infection. In vaccinated fish, the humoral response (i.e. B-lymphocytes) was greater than the cell-mediated response (i.e. T-lymphocytes). In contrast, infected fish had a greater cell-mediated than humoral immune response.     

Atlantic salmon (Salmo salar) postsmolts adapt lipid digestion according to elevated dietary wax esters from Calanus finmarchicus

Wax esters (WE) in copepods constitute huge natural marine lipid resources, which can contribute as future lipid source in formulated diets in aquaculture, and thereby reduce the pressure on use of marine resources at higher trophic levels. The present study was undertaken to investigate factors affecting WE digestibility, including production of bile and lipases in Atlantic salmon fed diets containing high proportions of oil derived from copepods. Individually tagged postsmolt Atlantic salmon (initial weight 250 g) were distributed into three dietary groups in triplicate tanks and fed either a fish oil supplemented diet or diets where 50% or 100% of the fish oil was replaced with oil extracted from Calanus finmarchicus . WE accounted for 30.7% or 47.7% of the lipids in these latter diets, respectively. Over the 100 day feeding period, the salmon fed the fish oil diet displayed a significantly higher specific growth rate (SGR; 0.74) than fish fed the 100% Calanus oil diet (SGR; 0.67). The apparent digestibility coefficient of total lipid and total fatty acids was significantly higher in salmon fed the fish oil and the mixed diet compared to fish fed the pure Calanus oil diet. However, the fish appeared to enhance the lipid digestive capacity by increasing bile volume and the lipolytic activity. It is concluded that the digestion of WE in Atlantic salmon is poorer than for triacylglycerols. However, the digestive capacity is increased by elevating the bile content and lipase activity. At very high levels however, WE of lipid between 37.5% and 47.7%, are there no more compensation and WE utilisation decreases.     

Inclusion of camelina meal as a protein source in diets for farmed salmonids

Camelina meal (Camelina sativa) (CM) is a potential protein source for aquaculture feeds, on account of its crude protein level (380 g kg^?1) and inclusion of most indispensable amino acids. Two experiments were conducted with rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Rainbow trout (44.9 g fish^?1) were fed diets with CM at 0 g kg^?1 (0% CM), 70 g kg^?1 (7% CM), 140 g kg^?1 (14% CM) or 210 g kg^?1 (21% CM) for 12 weeks at 14 °C in freshwater, and salmon (241.8 g fish^?1) were fed diets with CM at 0 g kg^?1 (0% CM), 80 g kg^?1 (8% CM), 160 g kg^?1 (16% CM) or 240 g kg^?1 (24% CM) for 16 weeks at 14 °C in sea water. Growth, lipid and amino acid tissue compositions were compared between species. Trout could tolerate up to 14% CM diets without affecting the growth compared to the control, while salmon fed ≥8% CM gained less weight than the control (P = 0.008). The feed conversion ratio in trout fed 21% CM was higher than the control (P = 0.002), and feed intake in salmon fed ≥8% CM was lower than the control (P = 0.006). Trout fatty acid and amino acid composition showed minimal differences between CM‐fed and control‐fed fish, while salmon showed significant alterations after feeding CM diets. Multivariate analyses emphasized differences in tissue composition between species fed CM diets.     

Growth and ontogenetic change in activities of digestive enzymes in Fennero Penaeus indicus postlarvae

The growth and enzyme response of Fennero Penaeus indicus postlarvae (PL) was assessed with natural and artificial diets for the first 2 weeks of PL development to PL14 (14 days after metamorphosis to PL1). At PL14, postlarvae reared under similar conditions were separated into two size groups based on slow growth (9.57 ± 0.46 mm) and fast growth (13.90 ± 0.28 mm), and reared for a further 63 days. Growth among postlarvae PL1 and the smaller size group at PL14 fed on artificial diet was significantly slower than that of PLs fed on Artemia nauplii or fresh mussel. In contrast, the larger size group at PL14 did not show a significant difference in growth when fed artificial diet or fresh mussel. Trypsin and amylase response was significantly related to postlarval total length ( P  < 0.001); thus, digestive enzyme response increased with size and stage of development, but did not change significantly with diet fed. The amylase–protease ratio increased from PL1 and amylase activity became higher than the activity of trypsin for 2–3 weeks after metamorphosis. It appears that during the early stages of development postlarvae are unable to digest artificial diets efficiently because of low digestive capacity. Hence, poorly digestible feed delayed development and increased size variability. For smaller PL14, poor performance on artificial diets is possibly linked to a genetically regulated constraint, but not to digestive capacity since enzyme levels were similar to those found in larger PL14. During postlarval stages digestive enzyme production seems to be developmentally cued so that for postlarvae it appears that more digestible high-energy diets are required to reduce size variability during the nursery phase.     

Efficacy of bithionol as an oral treatment for amoebic gill disease in Atlantic salmon Salmo salar (L.)

This study examined the efficacy of bithionol as an oral treatment for Atlantic salmon Salmo salar affected by amoebic gill disease (AGD). The current commercial management strategy of AGD is a costly 3 h freshwater bath. It is labour intensive and the number of baths needed appears to be increasing; hence, there is an effort to identify alternative treatments. Efficacy was examined by feeding AGD-affected Atlantic salmon twice daily to satiation with bithionol, an antiprotozoal, at 25 mg kg^− 1 feed. Three seawater (35‰, 17 °C) re-circulation systems were used each consisting of three tanks containing 32 Atlantic salmon smolts with an average (± SEM) mass of 90.4 g (± 5.2). Three feeds were examined in the trial including bithionol, plain commercial control and oil coated commercial control. Feeding commenced 2 weeks prior to exposure to Neoparamoeba spp. at 300 cells L^− 1 and continued for 28 days post-exposure. Efficacy was determined by examining gross gill score and identifying percent lesioned gill filaments twice weekly for 4 weeks post-exposure. Bithionol when fed as a two-week prophylactic treatment at 25 mg kg^− 1 feed delayed the onset of AGD pathology and reduced the percent lesioned gill filaments by 53% and halved the gill score from 2 to 1 when compared with both the plain and oil controls during an experimental challenge. There were no palatability problems observed with mean feed intake of bithionol over the trial duration with fish consuming higher levels of the bithionol diet compared to both the oil and plain controls. This study demonstrated that bithionol at 25 mg kg^− 1 feed, when fed as a two-week prophylactic treatment for Neoparamoeba spp. exposure, delayed and reduced the intensity of AGD pathology and warrants further investigation as an alternative to the current freshwater bath treatment for AGD-affected Atlantic salmon.     

Peptidase compensation in the digestive system of whiteleg shrimp Penaeus vannamei against dietary Kunitz‐type soybean trypsin inhibitor

The aim of this study was to assess the regulatory process of digestive peptidases of crustaceans in the presence of soybean trypsin inhibitor (SBTI). This naturally occurring inhibitor in soybean meal was used to inhibit the activity of digestive serine peptidases of the whiteleg shrimp Litopenaeus vannamei. In vitro, SBTI inhibited the total proteolytic activity and chymotrypsin activity by 65%. Trypsin activity was reduced by SBTI from 40% to 15% from 2 to 4 hr of incubation, which is the average time of residence of feed in the shrimp digestive system. During the bioassays, experimental groups were fed with increasing concentration of supplemental SBTI (1 g kg^?1 and 2 g kg^?1) and digestive gland and faeces of individual specimens were collected daily. At the end of the bioassay, peptidase activity of digestive gland and faeces was shown, revealing differential inhibition after feeding for 5 days. Several serine peptidases were observed in zymograms, showing a compensation effect on the digestive gland through the activation of peptidases from different catalytic type. These results provide evidence that the shrimp digestive gland can overcome the effect of SBTI by two adaptive mechanisms: synthesis of additional peptidases of the serine class and other unidentified peptidases.     

Biotin in practical fish-meal based diet for Atlantic salmon Salmo salar L. fry

Groups of Atlantic salmon fry (0.19 g initial weight) were fed a fish-meal based starter diet with different supplements of biotin (0, 0.5, 1.0 and 1.5 mg biotin kg^–1) in triplicate tanks for 18 weeks. The basal diet contained 0.3 mg total biotin kg^–1. The experimental design included a negative control diet made by replacing 10% of the fish-meal with spray-dried raw hen's egg white.
Throughout the experimental period the fish grew to about 5 g and there were no significant differences in growth and mortality among the groups of fish fed the fish-meal diets. At the end of the experiment there were no significant differences in biotin level in the liver, while whole-body biotin concentrations correlated significantly with the dietary biotin concentrations. No significant increase in pyruvate carboxylase (PC) activity was found in livers from fish given different dietary levels of biotin. The control diet with egg white resulted in severe growth reduction and increased mortality compared with the other dietary groups. The concentrations of biotin in liver and whole body were decreased in fish fed egg white. Increased levels of glycogen and reduced PC activity in the liver were observed in this group after 18 weeks.
Histology of the gills showed no differences in appearance when fish were fed fish-meal based diets while the addition of egg white resulted in hypertrophy and hyperplasia of the gill tissue and extensive fusions of the secondary gill lamellae.
The results show that there is no need for supplemental biotin in practical fish-meal based diets for Atlantic salmon fry to achieve optimal growth, survival and maximal liver PC activity.     

Dietary menadione nicotinamide bisulphite (vitamin K3) does not affect growth or bone health in first-feeding fry of Atlantic salmon (Salmo salar L.)

The aim of the present study was to elucidate if increasing levels of added vitamin K in the feed influenced fish growth, health or the incidences of bone deformities in Atlantic salmon ( Salmo salar L.) from onset of start feeding to ∼100 g in freshwater. Duplicate groups of Atlantic salmon fry (∼0.20 g) were fed eight levels (0, 2.5, 5, 7.5, 10, 15, 20 and 50 mg menadione kg⁻¹) of the vitamin K derivate menadione nicotinamide bisulphite (MNB) in a regression design for 28 weeks. All fish maintained high growth rates throughout the experiment, and showed no significant differences in specific growth rate, condition factor, whole body proximate analysis, blood coagulation time, vertebra morphology or mechanical properties of vertebrae. We found a dose-response between whole body vitamin K concentration and the dietary MNB supplementation level. Analysis of liver γ-glutamylcarboxylase activity revealed significant dose-dependent differences between groups given the 0, 10 and 50 mg MNB kg⁻¹ diets. In conclusion, Atlantic salmon seems to require low levels of dietary vitamin K, and the amount of vitamin K found naturally in the presently used feed ingredients may be enough to maintain optimal growth, health and bone strength in Atlantic salmon fry from start feeding.     

Comparative activity and stability under salinity conditions of different antimicrobial peptides isolated from aquatic animals

This study reports the in vitro activity of six antimicrobial peptides (AMPs) produced by aquatic animals (most marine invertebrates): tachyplesin (Tach), magainin (Mag), clavanin (Clav), penaeidin (Pen), mytilin (Myt) and antilipopolysaccharide factor (ALF) against marine vibrios, filamentous fungi and yeast. Their stability under salinity conditions and seawater was also examined. The results showed that Mag, Myt and especially Tach and ALF (minimum inhibitory concentration<1.5 μM) had a potent activity against all tested vibrio species, whereas Clav and Pen were ineffective (up to 50 μM). With respect to the antifungal activity, each AMP had a different potency according to the fungal species. In general terms, Tach was the most potent peptide, followed by Mag. Interestingly, Tach, Myt and ALF had a significant effect on the filamentous fungus Fusarium solani that could be pathogenic to marine organisms. All AMPs had a tendency to decrease or lose their activity at high salinity (>225 mM NaCl). Tach and Myt were the most stable peptides, maintaining significant activity under seawater salinity (450 mM). Curiously, all peptides lost their effect under seawater conditions. The results suggest that Tach, ALF and Myt are the most promising candidates for potential therapeutic use in farmed-marine species, because all have a significant and broad antimicrobial activity maintained at high salinity.     

Protein Hydrolysates and Ultrafiltration Fractions Obtained from Krill (Euphausia superba): Nutritional,Functional, Antioxidant,and ACE-Inhibitory Characterization

ABSTRACT

Krill (Euphausia superba) was hydrolyzed by proteolytic enzymes in order to produce multifunctional bioactive peptides, and their functional properties were evaluated. Krill protein hydrolysate (KPH) by pepsin with 4-h hydrolysis showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and angiotensin I converting enzyme (ACE) inhibitory activities. The solubility and foaming properties of KPH were higher than those of the unhydrolyzed krill protein at a wide range of pHs. KPH was further fractionated based on molecular weight. The 1- to 3-kDa peptide fraction exhibited the highest DPPH scavenging activity (IC₅₀ value of 0.5 mg/mL), oxygen radical absorbance capacity (497.39 ± 4.31 µM TE/mg fraction), 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid cation radical scavenging activity (48.41 ± 0.23 µM TE/mg fraction), and reducing power (110.40 ± 2.07 µM TE/mg fraction). However, the < 1-kDa peptide fraction exhibited a higher ACE inhibitory activity than that of other fractions. The 1- to 3- and < 1-kDa peptide fractions are rich in aromatic and hydrophobic amino acids, respectively.     

Preventive efficacy of sodium hypochlorite against water mold infection on eggs of chum salmon Oncorhynchus keta

ABSTRACT:   To protect chum salmon eggs from water mold infection during incubation, the eggs were treated daily with sodium hypochlorite solution (NaOCl) at 10 mg/L residual chlorine concentration for 15 min during their developmental period from fertilization to eyed stages. The number of infected eggs and number of eyed eggs were observed on day 23 of incubation. The percentage of infected eggs to total eggs was significantly lower with NaOCl treatment (1.8 : 33.4%) than in the control (11.3 : 59.3%, P  < 0.01). The percentage of eyed eggs to total eggs was significantly higher with NaOCl (85.9 : 98.6%) than in the control (66.1 : 97.5%, P  < 0.01). The antifungal activity of NaOCl resulted in improving egg survival. Accordingly, NaOCl is a useful antifungal agent against water mold infection on chum salmon eggs.     

Effect of dietary bovine lactoferrin on growth,haematology and non‐specific immune response in rainbow trout (Oncorhynchus mykiss)

The aim of this study was to investigate the effect of dietary lactoferrin (Lf) on growth, haematology and non‐specific immune response of rainbow trout Oncorhynchus mykiss (Walbaum). Fish were fed an experimental diet containing 0 (as control), 50, 100, 200 and 400 mg Lf kg^?1 diet twice daily for 8 weeks and sampled at 2, 4, 6 (immune function and growth), and 8 weeks (immune function, haematology and growth). Statistical analyses revealed no significant effects of dietary Lf on growth performance (specific growth rate, weight gain, feed conversion ratio, feed intake and condition factor) or haematological parameters (red and white blood cell count, haemoglobin, haematocrit, serum iron and total iron binding capacity [TIBC]). Among the serum non‐specific immune parameters, lysozyme activity increased significantly in fish fed 100, 200, or 400 mg Lf kg^?1 feed for 8 weeks, whereas haemolytic complement activity increased in fish fed 100 and 400 mg Lf kg^?1 diet after 6 weeks. The antiprotease activity increased in groups fed 100, 200 or 400 mg Lf kg^?1 diet after 8 weeks. However, no significant effect was observed on serum peroxidase level. It can be concluded that feeding of rainbow trout on the diet supplemented with 100 mg kg^?1 or higher for 8 weeks enhances the non‐specific immune response.     

Protein Hydrolysate Fractions from Sea Cucumber (Isostichopus badionotus) Inhibit Angiotensin-Converting Enzyme

Angiotensin-converting enzyme (ACE) inhibitory capacity was evaluated in hydrolysates from sea cucumber Isostichopus badionotus. The pepsin-pancreatin and Alcalase®-Flavorzyme® sequential systems were used. Hydrolysates were characterized in terms of degree of hydrolysis (DH), electrophoretic profile, and inhibitory activity. The Alcalase®-Flavorzyme® system produced hydrolysates with the highest DH, with that produced at 90 min exhibiting the highest ACE inhibitory action (86%). This hydrolysate was chosen for purification by ultrafiltration with five molecular weight cut-offs (< 1, 1–3, 3–5, 5–10, and > 10). The > 10-kDa fraction exhibited the highest ACE inhibitory action (80.7%) and was fractionated by gel fractionation chromatography. Of the 11 resulting fractions, IX had the highest ACE inhibitory action (73.4%) and lowest IC₅₀ value (2-μg prot/mL). Hydrolysis of sea cucumber I. badionotus with the Alcalase®-Flavorzyme® system produced hydrolysates with ACE inhibitory action. These contained a peptide with an IC₅₀ value comparable to those of synthetic ACE inhibitors.     

An in vitro method to determine phosphorus digestibility of rainbow trout Oncorhynchus mykiss (Walbaum) feed ingredients

An in vitro method was developed to assess the digestibility of phosphorus in 12 plant and animal feed ingredients for rainbow trout Oncorhynchus mykiss (Walbaum). The method simulates the gastrointestinal tract of the rainbow trout with regard to pH and gastrointestinal enzymes. Phosphorus solubility was measured after acid digestion (pH 3) with and without gastric enzymes, after alkaline digestion (pH 9) with and without intestinal enzymes, and after a two-step process involving acid and alkaline digestion. Commercially available digestive enzymes from mammals were compared with digestive enzymes from rainbow trout. Correlating in vitro digestibility with in vivo digestibility showed that acid digestion with both commercial enzymes ( r ²=0.98, P  < 0.05) and trout enzymes ( r ²=0.94, P  < 0.05) predicted the in vivo digestibility of animal feed ingredients. Alkaline digestion with both enzyme systems (commercial r ²=0.79; trout r ²=0.74, P  < 0.05) or without ( r ²=0.82, P  < 0.05) enzymes predicted the in vivo digestibility of ingredients from animal byproducts but not those from plant products. The in vitro digestibility with two enzyme steps (acid and alkaline) predicted in vivo digestibility of plant and animal ingredients ( r ²=0.79 for commercial enzymes and r ²=0.74 for trout enzymes) better than did one-step acid or alkaline digestion.