Optimization of Hydrolysis Conditions for the Production of the Angiotensin-I Converting Enzyme Inhibitory Peptides from Sea Cucumber Collagen Hydrolysates

The effects of different hydrolysis conditions on the angiotensin-I converting enzyme (ACE) inhibitory properties of giant red sea cucumber (Parastichopus californicus) collagen hydrolysates were investigated. Optimal conditions predicted by central composite rotatable design (CCDR) modeling for producing ACE inhibitory peptides were found to be 54.9°C, 1.76 h and an enzyme to substrate (E/S) ratio of 0.064. Compared with experiments performed under these optimal conditions, the predicted degree of hydrolysis (DH) and ACE inhibitory activities had error rates of 3 to 6%. Under optimum conditions, the molecular weights of collagen hydrolysates were less than 6.5 kDa. Lineweaver-Burk plots suggest that the collagen hydrolysates acted as competitive inhibitors with an inhibition constant (Ki) value of 0.706 mg/mL.     

Evaluation of the growth and survival rate of the Caribbean Sea cucumber,Isostichopus badionotus (Selenka, 1867), early juveniles produced in captivity

Isostichopus badionotus is a new commercially important sea cucumber in the Caribbean that has rapidly shown signs of overexploitation. There is no information on the growth and survival rates for wild or hatchery‐produced early juveniles. Hatchery production and determination of juvenile growth rates are essential for future aquaculture efforts for this species. In this study, daily growth rate (GR), specific growth rate (SGR), weight gain (WG), survival rate (SR), and weight–length relation (WLR) were evaluated using small (0.01 g) and large (0.25 g) hatchery‐produced early juveniles under laboratory conditions. Growth was rapid compared to other cultured sea cucumber species; maximal values of SGR and WG were obtained for small juveniles (4.58%/day and 2,191%, respectively), while maximal GR and SR were obtained for large juveniles (0.29 mm/day and 99.17%, respectively). A wide range of sizes was evident at the end of each trial. The WLR of small and large juveniles fit with a negative allometric growth trend. This study highlights the potential production of I. badionotus as early juvenile stages withstand hatchery handling well and exhibit a high SR, reaching commonly released mean sizes (>0.7 g and 25 mm) after almost 3 months (120 days postfertilization).     

Antioxidant and ACE Inhibitory Activities of Kawakawa (Euthynnus affinis) Protein Hydrolysate Produced by Skipjack Tuna Pepsin

ABSTRACT

Pepsin enzyme from skipjack tuna was extracted for the production of kawakawa (Euthynnus affinis) fish protein hydrolysate. Using ultra-fractionation, Kawakawa protein hydrolysates were separated into four different fractions, including fractioned protein hydrolysate I (FPH I) (< 1 kDa), FPH-II (1–3 kDa), FPH-III (3–10 kDa), and FPH-IV (> 10 kDa). The antioxidant activity was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, inhibition of linoleic acid oxidation, reducing power tests, and chelating activity of metal ions. Results indicated that FPH II fraction peptides had higher antioxidant activity in comparison with the other fractions, followed by FPH I. Further, the fractions were evaluated for angiotensin converting enzyme (ACE) inhibition, and IC₅₀ value ranged from 0.45 to 1.86 mg/ml with higher activity in FPH I (IC₅₀ 0.45). Finally, the amino acid profile of different fractions was analyzed. The fractions exhibited significant amounts of hydrophobic amino acids, which could perform as hydrogen donors, frustrate the free radicals, and inhibit the ACE. The recovered pepsin from the viscera was used to produce hydrolysates with good biological activities. Peptides lower than 3 KDa had antioxidant activity as positive controls and significant ACE activity. These are very important findings that could be used to conduct further research in a preclinical study of these peptides.     

Optimization of Hydrolysis Conditions for Production of Angiotensin-Converting Enzyme Inhibitory Peptides from Basa Fish Skin Using Response Surface Methodology

ABSTRACT

Basa fish skin free from fat and noncollagenous proteins was hydrolyzed by different proteinases to produce angiotensin-converting enzyme (ACE) inhibitory peptides. Protamex hydrolysates were found to have the highest ACE inhibitory activity. Response surface methodology (RSM) was applied to optimize the hydrolysis conditions of Protamex—including enzyme-to-substrate ratio, hydrolysis time, and solid (fish skin)-to-liquid (water for hydrolysis)—at a temperature of 50°C and a pH of 7.0. The mathematical model demonstrated a good fit with the experimental data. An enzyme-to-substrate ratio of 6,108 U/g, a hydrolysis time of 1.24 h, and a solid-to-liquid ratio of 1:8.05 g/mL were found to be the optimal conditions to achieve the highest value of ACE inhibitory activity. The IC₅₀ of the hydrolysates for ACE inhibitory activity was 1.417 mg/mL under these conditions.     

Angiotensin Converting Enzyme and Dipeptidyl Peptidase-IV Inhibitory,and Antioxidant Activities of a Blue Mussel (Mytilus edulis) Meat Protein Extract and Its Hydrolysates

ABSTRACT

Protein extracted from mussel processing coproducts was hydrolyzed with four different food-grade enzyme preparations and assessed for angiotensin converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibitory and oxygen radical antioxidant capacity (ORAC) activities. All hydrolysates tested showed higher activity than the intact protein. ACE and DPP-IV IC₅₀ values in the range 1.13–3.34 and 0.33–2.43 mg mL^?1, respectively, and ORAC values in the range 66.26–121.56 µmol trolox equivalents g^–1 were obtained. These results suggest that some of the mussel meat protein hydrolysates may have potential as functional food ingredients for the management of diseases such as type II diabetes and hypertension.     

Optimization of Protein Recovery During Hydrolysis of Yellowfin Tuna (Thunnus albacares) Visceral Proteins

Protein hydrolysates were prepared from yellowfin tuna viscera using Alcalase®. Response surface methodology (RSM) was applied to study the effect of independent variables, enzyme activity, temperature, and time on the protein recovery (PR) as the response. The coefficient of determination (r² = 98%) was satisfactory, and the lack of fit test showed a non-significant value for the PR model equation, indicating that the regression equation was adequate for predicting the PR under tested combinations of values. Enzyme activity at 79.9 AU/kg protein, temperature at 59.9°C, and the time of 105 min were found to be the optimum conditions to reach the highest PR (85%). Considerable soluble protein was released with increasing degree of hydrolysis (DH). The yellowfin tuna visceral hydrolysates' amino acids profiles showed high essential amino acids content, although lysine, methionine, and phenylalanine were limited. In addition, the results showed that with increasing hydrolysis time, the amino acids increased. Yellowfin tuna visceral hydrolysates were rich in flavor amino acids which can be used as a taste enhancer.     

Antioxidant and Angiotensin-Converting Enzyme Inhibitory Activities of Protein Hydrolysates Prepared from Threadfin Bream (Nemipterus spp.) Surimi By-products

Solid wastes from threadfin bream (Nemipterus spp.) surimi production composed of head and frame were hydrolyzed by various commercial proteases (Alcalase, Flavourzyme, Neutrase, Protamex, papain, and pepsin) to produce protein hydrolysates with bioactive properties. An Alcalase-hydrolyzed sample at 24.4% degree of hydrolysis (DH) displayed the highest antioxidant activity based on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP) assay, and potassium ferricyanide method. In addition, it showed an inhibitory activity toward angiotensin-converting enzyme (ACE) of 25.5%. Antioxidant activity of threadfin bream by-product hydrolysates increased with hydrolysis time and reached the highest DPPH activity after 6 h, while that hydrolyzed for 3 h showed the highest reducing power based on FRAP and potassium ferricyanide assays. In addition, ACE inhibitory activity was found to be at an optimum after 3 h of hydrolysis. The hydrolysates (1 mg/mL) also retarded oxidation of a linoleic acid emulsion system to a similar extent as 0.1 mg/mL 3-tert-butyl-4-hydroxy anisole (BHA), indicating a potential use in the food system. Protein hydrolysates from threadfin bream surimi by-products could be tailor-made to possess both antioxidant and ACE inhibitory activity through controlling DH of Alcalase-catalyzed reactions.     

Effect of diet on growth and body biochemical composition of juvenile four‐sided sea cucumber Isostichopus badionotus (Selenka, 1867)

The farming of sea cucumber is a growing industry that demands feeds that are tailored to the organism's nutritional needs. Therefore, in this study, the effect of diet composition, with and without additives, on the growth, survival, specific growth rate (SGR) and body biochemical composition of the four‐sided sea cucumber Isostichopus badionotus was evaluated over a period of 3 months. Four diets were used: D1 (Macrocystis sp. and Sargassum sp.), D2 (Macrocystis sp. and Spirulina sp.), D3 (Sargassum sp. and Spirulina sp.) and D4 (Macrocystis sp., Sargassum sp. and Spirulina sp.), and the same diets enriched with 0.22% of Algamac 2000^® (D1H, D2H, D3H and D4H) were also tested. The organisms fed the diets containing Spirulina sp. demonstrated the highest growth rates and significantly greater SGR (% per day) (p < .05), an effect that was enhanced by the food additive (D2 0.67%; D2H 0.78%; D3H 0.76%). Survival and body composition were not affected by diet type. Based on these results, the use of Spirulina sp. combined with supplements rich in PUFA content is recommended in the formulation of diets to promote the growth of I. badionotus juvenile.     

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.     

Utilization of Shrimp By-Products by Bioconversion with Medical Fungi for Angiotensin I-Converting Enzyme Inhibitor and Antioxidant

ABSTRACT

A liquid fermentation process to bioconvert shrimp by-products was developed with two species of fungi, Boletus edulis and Suillus bovinus. Angiotensin I-converting enzyme (ACE) inhibitory, antioxidant activities of bioconverted products and the deodorization effect of fungal bioconversion were determined. Two species of fungi could grow quickly in shrimp by-products medium and deodorize shrimp by-products. Bioconverted products had higher ACE inhibitory and antioxidant activities than control medium. Water extracts without crude polysaccharides from mycelia of B. edulis had the highest ACE inhibitory activity (IC₅₀ = 0.084 ± 0.011 mg/mL). Crude exopolysaccharides of B. edulis had higher antioxidant activity. Bioconversion with the fungus was proven to be a novel, practicable way to recycle shrimp by-products.     

Intra‐specific effects of sea cucumber (Apostichopus japonicus) with reference to stocking density and body size

The present studies deal with the intra‐specific effects of sea cucumber Apostichopus japonicus with unlimited food resources, especially the effects of stocking density on growth variation of the animal and energetic changes of small individuals under the stress of large individuals. The results showed that with the initial body weight of 5.12±0.09–6.11±0.26 g of sea cucumber among the densities of 5, 10, 20, 30, 40 and 50 ind./100 L, the density of 20 ind./100 L was the optimum stocking density because of its highest specific growth rate, crude protein content and crude lipid content in tissue. Individual growth variation of A. japonicus increased with the increase of stocking densities, whereas no significant differences in variation were found when the density was over 30 ind./100 L (P>0.05). The low‐weight individuals under the stress of heavy‐weight individuals exhibited obvious changes in energetics, such as lower ingestion rate, lower energy deposited as growth but higher respiration and excretion. The coefficient of variation in growth of the animals was over 70% due to the simultaneous action of aggression and maybe a factor of chemical mediator, and led to significant changes in the energetics of small‐sized individuals.     

Anti-Inflammatory,Antioxidant, and Antimicrobial Effects of Underutilized Fish Protein Hydrolysate

Antimicrobial, anti-inflammatory, and antioxidant activities of protein hydrolysates from Argentine croaker (Umbrina canosai) protein isolate (CPI) or Argentine croaker myofibrillar protein with different degrees of hydrolysis (DH: 10–20%) prepared using Alcalase or Protamex were determined. Results showed that an increase in the DH resulted in higher content of hydrophobic and aromatic amino acids (AAAs) and in a decrease in molecular weight (MW) distribution for all hydrolysates obtained. Furthermore, the enzyme and raw material used influenced the amino acid content and MW determined. Hydrolysates from CPI with a 20% DH by Alcalase had higher 2,2?-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity, metal chelation, and ferric-reducing antioxidant power (p < 0.05). All hydrolysate samples decreased the pro-inflammatory capacity. In all the evaluated microorganisms, only seven were inhibited, most being Gram-positive. Alcalase was found to exert a considerable influence on antibacterial activity. These hydrolysates are an alternative as natural antimicrobials, anti-inflammatory, and antioxidant compounds.     

Common Kilka Hydrolysis: Investigation of Functional and In Vitro Antioxidant Properties of Hydrolysates Obtained by Kiwifruit and Ginger Proteases

The functional and in vitro antioxidant properties of common kilka fish protein hydrolysates with different degree of hydrolysis (DH) obtained by kiwifruit protease (KP) and ginger protease (GP) were evaluated. The electrophoretic patterns of hydrolysates showed the presence of one major band in different DHs with a molecular weight of less than 25 kDa. Protein solubility was positively correlated with DH, and the solubility of KP and GP hydrolysates (HKP and HGP, respectively) at 15% DH was higher than other DHs (p ≤ 0.05). Higher emulsifying and foaming properties were observed in HKP over a pH range of 2–10 (p ≤ 0.05), and in vitro antioxidant activity was higher in HKP at 15% DH as seen from 2,2-diphenyl-1-picrylhydrazyl radical scavenging and ferric-reducing power. Thus, the results reveal that DH and enzyme type affects functional properties and antioxidant activity of the hydrolysates.     

Growth and potential purification ability of Nitzschia sp. benthic diatoms in sea cucumber aquaculture wastewater

The growth patterns and water treatment capacity of Nitzschia sp. benthic diatoms in different concentrations of sea cucumber aquaculture wastewater (0%, 10%, 30%, 50%, 80% and 100%) and f/2 medium were studied. Nitzschia sp. grew in different concentrations of aquaculture wastewater and showed remarkable differences in their rate of growth among the treatment groups. Nitzschia sp. grew most quickly (0.83 ind/day) and showed the greatest total chlorophyll‐a content in 30% wastewater. The total chlorophyll‐a content and growth rate of Nitzschia sp. were strongly correlated (R² > 0.98). The total lipid (TL), total protein, exopolysaccharide (EPS) and intracellular polysaccharide (IPS) contents of the diatoms were highest in 100% wastewater and showed significant differences among the experimental groups (p < 0.05). Total nitrogen (TN), ammonium‐nitrogen (NH₄‐N) (AN), nitrate nitrogen (NO₃‐N) (NN), nitrite nitrogen (NO₂‐N) (NIN) and total phosphate (TP) contents were significantly reduced after cultivation. TN uptake peaked at 54.58% in 30% wastewater. AN uptake exceeded 95% in 30% wastewater and 100% wastewater. NN uptake peaked at 56.42% in 80% wastewater, whereas TP uptake ranged from 16.80% to 27.69%. These results suggest that Nitzschia sp. biomass can be enhanced via cultivation in low‐concentration (30%) wastewater, after which their cultivation may be continued in high‐concentration (100%) wastewater, increasing their nutritional value and aiding in the removal of surplus nitrogen and phosphorus in sea cucumber aquaculture wastewater. Application of Nitzschia sp. using the recirculating wastewater‐treatment methods described has the potential to reduce environmental harm caused by sea cucumber cultivation and thus achieve sustainable aquaculture.     

Evaluation of Potential Feed Ingredients for the Juvenile Four‐sided Sea Cucumber,Isostichopus badionotus

The objective of this study was to evaluate two seaweed species, Solieria filiformis and Macrocytis piryfera, both as dry‐meal powder and predigested meal, to be used as ingredients in artificial diets for the juvenile four‐sided sea cucumber, Isostichopus badionotus. Four diets were tested in triplicate: S. filiformis dry‐meal powder and predigested meal and M. piryfera dry‐meal powder and predigested meal, mixed with 70% of beach sand. Survival exceeded 90% in all the treatments, and there were no differences in the growth of juveniles using dry‐meal powder or predigested meal. Weight gain was calculated for all the diets, and the highest values were obtained by animals fed S. filiformis. No differences between using dry‐meal powder and predigested meal were found, so these diets were excluded from the second trial. The ingestion rate was the same with both algae, but the feed conversion ratio was twice as high for M. piryfera. Solieria filiformis exhibited a number of differences in fatty acid and amino acid profiles, including higher proline, arginine, and saturated fatty acid levels. The results indicate that S. filiformis has better nutritional characteristics to be an ingredient for an artificial I. badionotus juvenile diet.     

Optimum Level of Dietary n‐3 Highly Unsaturated Fatty Acids for Juvenile Sea Cucumber,Apostichopus japonicus

A feeding trial was conducted to estimate the optimum level of dietary n‐3 highly unsaturated fatty acids (HUFAs) for juvenile sea cucumber, Apostichopus japonicas, based on growth performance and fatty acid compositions. Diets with five n‐3 HUFAs levels (0.15, 0.22, 0.33, 0.38, and 0.46%) were fed to sea cucumber juveniles (1.97 ± 0.01 g) once a day for 60 d. The sea cucumbers fed diets containing 0.22% n‐3 HUFAs showed significantly (P < 0.05) higher body weight gain, feed efficiency, and protein efficiency ratio than the sea cucumbers fed diets containing 0.15% n‐3 HUFAs, but not significantly different (P > 0.05) from those of sea cucumbers fed diets containing 0.33, 0.38, and 0.46% n‐3 HUFAs. The sea cucumbers fed diets containing 0.46% n‐3 HUFAs showed significantly (P < 0.05) higher eicosapentaenoic acid and saturated fatty acid than the sea cucumber fed diets containing 0.15% n‐3 HUFAs, but not significantly different (P > 0.05) from those of sea cucumbers fed diets containing 0.22, 0.33, and 0.38% n‐3 HUFAs. The results of growth performance and n‐3 HUFA compositions of body wall indicated that the optimum level of dietary n‐3 HUFAs for juvenile sea cucumber is between 0.22 and 0.46%.     

Recovery of Bioactive Peptides and Omega-3 Fatty Acids-Containing Phospholipids from Squid Processing By-Product Hydrolysate

ABSTRACT

The present study examined whether bioactive peptides and omega-3 fatty acids-containing phopholipids could be recovered from squid processing by-product (SPB) hydrolysate. The hydrolysate was produced at 55°C for varying times with endogenous proteases and centrifuged to yield peptides-containing supernatant and phospholipids-containing precipitate. The supernatant showed angiotensin I-converting enzyme (ACE) inhibitory activity with half maximal inhibitory concentration (IC₅₀) values decreasing with hydrolysis time from 2.11 mg (0 h) to 1.71 mg (1 h), 1.38 mg (1.5 h), and 1.34 mg (2 h). Two-hour squid hydrolysate was further fractionated to isolate the most active fraction whose molecular weight was found to be below 10 kDa with IC₅₀ of 0.32 mg. The phospholipids-containing precipitate (45.6 g/100 g oil) was analyzed for a fatty acid profile, with the levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) being 16.86 and 29.24 g/100 g oil, respectively. The ACE inhibition by hydrolysate peptides and omega-3 fatty acid recovery support the nutraceutical potential of the SPB hydrolysate.     

Optimum dietary protein and lipid levels for growth of juvenile sea cucumber Apostichopus japonicus

A feeding trial of three protein (200, 300 and 400 g kg⁻¹) and two lipid levels (20 and 100 g kg⁻¹) was conducted to determine the proper dietary protein and lipid levels for growth of juvenile sea cucumber Apostichopus japonicus. Dietary protein and lipid levels were adjusted by adding with different levels of soybean meal, squid liver oil and soybean oil, respectively. Three replicate groups of sea cucumbers (average weight of 1.3 g) were fed the experimental diets for 12 weeks. At the end of the feeding trial, survival was not affected by dietary protein and lipid levels (P > 0.05). Weight gain (WG) and specific growth rate (SGR) of sea cucumbers were significantly affected by dietary protein (P < 0.006) and lipid levels (P < 0.001). The highest WG and SGR were observed in sea cucumbers fed the 200 and 400 g kg⁻¹ protein diet with 20 g kg⁻¹ lipid (P < 0.05). WG and SGR of sea cucumbers fed the diet containing 20 g kg⁻¹ lipid were higher than those of sea cucumbers fed the 100 g kg⁻¹ lipid diets (P < 0.05) at each dietary protein level. Apparent digestibility coefficients of dry matter, crude protein, carbohydrate and gross energy of sea cucumbers fed the 20 g kg⁻¹ lipid diets were significantly higher than those of the 100 g kg⁻¹ lipid diets at 200 and 400 g kg⁻¹ protein (P < 0.05). Moisture, crude protein, crude lipid and ash contents were not significantly different among the groups. The results of this study indicate that the diet containing 200 g kg⁻¹ protein (170 g kg⁻¹ digestible protein) with 20 g kg⁻¹ lipid (13 g kg⁻¹ digestible lipid) may be sufficient for optimum growth of juvenile sea cucumber.     

Evaluation of Processing Methods on the Nutritional Quality of Sea Cucumber (Apostichopus japonicus Selenka)

The close relationship between the consumption of sea cucumber and health status stems from the nutritional and functional substances found in sea cucumber that play a key role in the prevention of different diseases. The processing methods have a great effect upon the nutritional and functional substances in sea cucumber. A superior product should not only be abundant in nutritional substances, but also require an easy and cost effective processing method. This study compared the influence of processing methods on the stability of macronutrients and micronutrients present in sea cucumber in order to recommend products that could provide high nutritional quality. The five processes investigated were hot air drying (60°C), vacuum freeze-drying (0.5 cm thickness material, 5–10 Pa vacuum degree, ?85 to ?90°C cold trap temperature, ?25°C material temperature), sun drying, double-distilled water cooking followed by sun drying, and 3.5% sodium chloride solution cooking followed by sun drying. A significant decrease of all the chemical compositions determined was shown in all the sea cucumber products treated by the five processing methods (p < 0.05). Among all the processing methods, hot air drying resulted in the highest nutritional quality of the product. Moreover, hot air drying can be operated easily with low cost.     

Substrate specificity of brain acetylcholinesterase and its sensitivity to carbamate insecticides in <Emphasis Type="Italic">Carassius auratus</Emphasis>

The substrate specificity of brain acetylcholinesterase (AChE) in adult Carassius auratus fish and its sensitivity to carbamate insecticides were investigated in vitro. The results showed that the order of four substrates hydrolyzed by brain AChE in C. auratus was acetylthiocholine iodide > β-methylthiocholine iodide > propionylthiocholine iodide > butyrylthiocholine iodide, and the maximum velocity (V _max) of AChE hydrolyzing acetylthiocholine iodide (ATCh) was the highest among the four substrates, and the V _max values were 0.067 and 0.082 mmol min⁻¹ mg⁻¹ for male and female fish respectively. But their Michaelis–Menten constants (K _m) were the lowest, only 0.071 and 0.072 mmol/l respectively. Compared with other carbamate insecticides, the sensitivity of brain AChE to carbofuran was the highest and the IC₅₀ values were 1.04 × 10⁻⁶ mol/l for females and 1.17 × 10⁻⁶ mol/l for males. The inhibitory tendencies of eserine, methomyl, and aldicarb to brain AChE were very similar, and the percentage inhibition increased with time at the concentration of 1 × 10⁻⁶ mol/l. The order of inhibition potential of the three inhibitors from the highest to the lowest was eserine, aldicarb, and methomyl.