A comparative study of the effects of high pressure on proteolytic degradation of sardine and blue whiting muscle

ABSTRACT:   High-pressure technology is used as an alternative to heat processing because of its inactivating effect on microorganisms and enzymes. However, it can also alter the structure of other muscle proteins. The present study compares the effects of high pressure (300 MPa, 7°C, 20 min) on the proteolytic degradation and alterations in the myofibrillar proteins of sardine and blue whiting muscle. Also, muscle homogenates and enzyme extracts were pressurized in order to evaluate the high-pressure effects on unprotected proteolytic enzymes outside the whole muscle structure. Peak proteolytic activity was found to occur at 55°C in both species. The peak activity pH was pH 3 for the sardine and pH 8 for the blue whiting; the main enzyme families being aspartic proteases in the former and alkaline serine proteases in the latter. Pressurization lowered activity levels at the peak activity pH and temperature in the fish muscle (by 30.8% in the sardine and by 9.5% in the blue whiting) and also slightly in the enzyme extracts (by 16.8% in the sardine and by 19.4% in the blue whiting). The electrophoretic profiles disclosed higher protein degradation in the pressurized muscle. Overall, the observed changes in proteolytic activity can be attributed not only to the effect of high pressure on the enzymes, but also and mainly, to the effect on other muscle proteins.     

Proteolytic enzyme activity of juvenile Asian sea bass, Lates calcarifer (Bloch), is increased with protein intake

The effect of high dietary protein intake on proteolytic enzyme activity of feeding juvenile Asian sea bass, Lates calcarifer (Bloch) was studied. Ninety fish [mean body weight ± standard error (SE) 304.62 ± 34.84 g] were randomly assigned to two dietary treatments, each with three replicates. In treatment 1, fish were fed by‐catch (Thunnus albacares) and in treatment 2, a formulated diet containing 50% protein. Proteolytic enzyme activity was determined in pyloric caecae and intestine at day 0, 7, 15, and 30. Initial proteolytic enzyme activity in sea bass ranged from 174 to 232 azocasein units (U^AC.) per mg of protein. After 7 days there was no significant difference in proteolytic enzyme activity of fish fed the two diets. However, a marked increase was observed in fish fed the formulated diet at day 15. After 30 days, the proteolytic enzyme activity in fish fed the formulated diet was threefold higher than that in fish fed the by‐catch diet. Fish fed the formulated diet had significantly higher total protein intake at day 7 than did fish fed by‐catch. Thereafter, a twofold weekly increase in the total protein intake was observed in both fish fed the by‐catch and formulated diets until day 30. These results suggest that a high dietary protein intake induces increased proteolytic enzyme activity in Asian sea bass.     

Comparative study of digestive enzymes in fish with different nutritional habits. Proteolytic and amylase activities

This work provides a comparative study of the proteolytic and amylase activities in six species of fish with different nutritional habits: rainbow trout (Oncorhynchus mykiss), gilthead seabream (Sparus aurata), European eel (Anguilla anguilla), common carp (Cyprinus carpio), goldfish (Carassius auratus), and tench (Tinca tinca). Trout and carp showed the highest digestive proteolytic activity. When proteolytic activity was determined in a wide range of pHs, the highest values in the digestive tract of all species were found at alkaline pHs, except in eel where activity could be detected only at acid pHs. Eel showed the lowest digestive proteolytic potential among all the species studied. With respect to amylase activity, the omnivorous species presented higher activity than did the carnivores. Among the carnivorous species, the lowest activity was found in trout. The ratio of total amylase:total proteolytic activity was higher in omnivorous fish species, the carp having the greatest value, whereas in trout this ratio was lower than one. Digestive enzyme activity declined as the incubation temperature decreased, but this trend varied depending on the fish species and the tissue analyzed.     

Secretion of hatching enzyme and its proteolytic activity in coregoninae (Coregonus albula L andC. lavaretus L) embryos

After the electrial stimulation Coregoninae embryos secreted the hatching enzyme (chorionase) within 0.1–0.5 h, and the dissolution of their chorions lasted 1.2–2.0 h, depending on embryo's developmental stage (DS 13 or DS 14) and water temperature (5.2 or 9.6–9.8°C).Crude chorionase (hatching liquid) ofCoregonus albula andC. lavaretus was collected in large quantities by means of the electric stimulation of eggs. In both species the temperature optimum of proteolytic activity of the crude chorionasc was 30°C; the activity was lost at temperatures < 3-2°C and > 35–40°C. The maximal proteolytic activity was observed at pH 8.5; a rapid decrease in enzyme activity was evident at pH < 7.0, and the activity was zero at pH 6.The temperature-activity curve of chorionase may reflect the adaptation of Coregoninae to hatching immediately after the ice cover recedes from lakes, whereas the rapid decrease of enzyme activity at pH 7 -pH 6 can affect adversely the process of hatching in acidified lakes.     

Enzymes Present in Pancreatic Extracts of the Dolphin Coryphaena hippurus

Pancreatic extract of adult dolphin Coryphaena hippurus was found to have a broad range of proteolytic activity with two peaks, one at pH 5–6 and another at pH 10, when casein was used as a substrate. Enzymes similar in activity to those of pepsin, trypsin, amylase, lipase and collagenase were detected in the extracts by the use of specific chromogenic substrates for each enzyme. The relationship between these enzyme activities, and the formulation of diets for culture of this species, is discussed.     

Post-prandial protease activity in the digestive tract of African catfish Clarias gariepinus larvae fed decapsulated cysts of Artemia

The alkaline proteolytic activity in the gut of African catfish larvae was studied during short time ranges from 30 min to 4 h after ingestion of decapsulated Artemia cysts. The variation in total protease and trypsin activities during the day was monitored during starvation, after one single meal ingestion, and during continuous feeding. In starved larvae the enzymatic activity was low and did not change in time. No significant endogenous secretion of digestive enzymes was detected. The level of alkaline proteolytic activity found in starved larvae was further considered as the basal level. In larvae fed only one meal during the day, the enzyme activity significantly increased from 3 h post-feeding up to a maximum level found 12 h after feeding. In the larvae receiving a meal every 4 h, the effect of feeding on the proteolytic activity was significantly different from the one in fish fed only once a day. The total protease activity in this dietary treatment changed according to the time of feeding and fluctuated around a constant level, which was intermediate between the maximum and the basal level. No rhythmic cycle of enzyme production in the fish was observed when the proteolytic activity was studied during a cycle of 24 h. When specific trypsin activity was measured, a similar pattern was found as with the total protease. The contribution of digestive enzymes from Artemia to the total digestion of food by the catfish larvae was calculated to be less than 1% of the total amount of the proteolytic activity measured in the larval gut.     

低鱼粉饲料中添加酶解豆粕对凡纳滨对虾生长性能和抗胁迫机能的影响

为研究在养殖过程中降低鱼粉用量的同时保持凡纳滨对虾良好的生长性能和抗逆能力,实验在含10%鱼粉的基础饲料中,分别添加酶解豆粕(PSM) 0%(A)、2.5%(B)、3.5%(C)、4.5%(D)、5.5%(E)制成5组等氮等能饲料,分别投喂初始体质量为(0.45±0.02) g的凡纳滨对虾幼虾8周,检测对虾生长性能及抗胁迫机能。结果显示,8周养殖实验结束后,各实验组对虾的终末体质量为14.65～15.38 g/尾,各组间对虾终末均重、成活率和饲料系数指标均无显著性差异;A组对虾肌肉粗蛋白质含量显著低于其他各实验组;C组、D组和E组对虾肌肉粗脂肪含量显著高于A组;各组间灰分和水分均无显著性差异;D组和E组对虾肝胰腺蛋白酶、淀粉酶、脂肪酶、血清溶菌酶和血清总超氧化物歧化酶活性(T-SOD)均显著高于A组;A组对虾血清丙二醛(MDA)含量显著高于D组和E组。人工急性感染高剂量副溶血性弧菌的胁迫实验中,A组对虾在弧菌感染48和60 h时的累积死亡率均显著高于D组对虾同期的累积死亡率;低剂量副溶血性弧菌人工急性感染后,在凡纳滨对虾鳃组织中检测Toll受体、免疫缺陷(IMD)和溶菌酶3种免疫相关基...     

Seasonal variation of muscle metabolic organization in rainbow trout (Oncorhynchus mykiss)

This study examined how muscle metabolic organization varied during an annual cycle in which rainbow trout (Oncorhynchus mykiss) were held in outdoor holding ponds in which they were exposed to natural changes in temperature (range 0.2 to 15.6°C) and photoperiod. We examined the activities of glycolytic and mitochondrial enzymes in red and white muscle to evaluate whether trout enhance their capacity for lipid and carbohydrate oxidation during cold-acclimization. When assayed at habitat temperature, the enzyme activities generally increased in spring to reach a maximum in summer followed by a decrease in the fall. This led to significantly higher activities at warm than cold periods for all enzymes measured in red muscle and all but one in white muscle. The activities at 10°C provided little evidence for compensatory adjustments of aerobic capacity. Particularly in red muscle, enzyme levels at 10°C were generally lower during cold than warm periods. The variation of enzyme activities throughout the cycle was not due to changes in protein concentration, as the same responses were observed when activities were expressed per g wet mass or per mg protein. Although the aerobic capacity did not increase with cold-acclimatization, the relative capacity for lipid oxidation was higher in winter than in summer trout. In contrast, the relative capacity for aerobic glycolysis was higher in summer than in winter trout. Thus, the metabolic capacities of trout muscle undergo seasonal reorganization.     

Plant protein and vegetable oil‐based diets modulate gilthead seabream (Sparus aurata) muscle biochemical status and proteolytic enzymes at an early postmortem stage

A 70 days feeding trial was carried out on juvenile gilthead seabream (Sparus aurata) (mean initial body weight: 362 ± 59 g) to evaluate whether the simultaneous replacement of fishmeal and fish oil by vegetable ingredients affected the muscle's proteolytic potential. Fish were fed either a fishmeal/fish oil diet, a fishmeal/vegetable oil diet, a plant protein/fish oil or a plant protein/vegetable oil diet. The use of dietary plant proteins resulted in lower sulphated glycosaminoglycans' content and lower glycogen phosphorylase activity in the muscle, while both plant proteins and vegetable oil increased muscle pH and reduced cathepsin B activity. Our results indicate that high replacement of fish meal and/or fish oil with plant ingredients has a significant impact on early postmortem metabolic processes and proteolytic potential of seabream muscle, especially when replacing both fish meal and fish oil.     

Some proteolytic properties of hatching liquid in the sea trout,Salmo trutta m. trutta L.

Proteolytic activity of sea trout hatching liquid was examined towards casein and azocazein as a function of pH and temperature. The optimum pH for caseinolytic and azocaseinolytic activities were 9.4, and 9.0, respectively. At alkaline pH the enzyme was activated by low concentrations of Zn²⁺ ions (10⁻⁵ M). Maximum proteolytic activity of the hatching liquid was observed at 25°C. Temperatures exceeding 30°C caused a rapid reduction in enzyme activity. Proteolytic activity observed at 10°C was approximately 50% of that observed at 25°C. In general, a pseudo-Arrhenius plot indicated a Q₁₀ of 1.6 between 6 and 25°C.     

钙蛋白酶抑制蛋白对猪肉肉质的影响

钙蛋白酶系统主要由钙蛋白酶(u2-calpain,CAPN1;m2-calpain,CAPN2)及钙蛋白酶抑制蛋白(calpastatin)组成,calpain是存在于细胞质中的依赖于Ca2+的钙中性蛋白酶,蛋白酶系统是主要的蛋白质降解物。calpastatin是钙蛋白酶的内源性抑制蛋白。研究表明,Calpastatin在肌原纤维蛋白降解中起着重要作用。因此,对肌肉生长和嫩度有重要影响。本文综述了钙蛋白酶抑制蛋白的结构组成,对肉嫩度影响的机理和钙蛋白酶抑制蛋白基因的定位。     

A Review of Proteotlytic Enzymes from Marine Organisms and Their Application in the Food Industry

Proteolytic enzyme are widely used as food processing aids. In recent years, proteolytic enzymes from marine organisms have been used as process aids. Other possible uses of enzymes form marine animals in the food industry are based on the unique properties of these enzymes. Examples of unique properties include ease of heat denaturation, high molecular activity at low temperatures and ability to catalyze hydrolysis of native proteins. The possible advantage of using an enzyme form a marine organism in specific applications rather than a conventional plant, animal or microbial source is discussed.     

Antioxidant Properties of Scomber japonicus Hydrolysates Prepared by Enzymatic Hydrolysis

The antioxidant properties of the Pacific chub mackerel (Scomber japonicus) muscle protein hydrolysates prepared by enzymatic hydrolysis were investigated. After enzyme hydrolysis at 50°C for 60 min, more than 80% of the S. japonicus muscle protein was hydrolyzed. The highest 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity (71.69%) occurred in whole muscle protein hydrolysates treated at 50°C for 30 min with Protamex, and the highest 2,2?-azino-bis(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS) radical-scavenging activity (95.39%) was observed in white muscle protein hydrolysates treated at 50°C for 30 min with Neutrase. The highest superoxide dismutase (SOD)-like activity (32.84%) was recorded in white muscle protein hydrolysates treated at 50°C for 120 min with Protamex. Changes in the molecular weight distribution of S. japonicus muscle proteins after enzymatic hydrolysis were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A robust and a convenient enzyme hydrolysis technique for obtaining S. japonicus muscle protein hydrolysates with useful biological activities, within a short time (<2 h) is proposed.     

Dietary modulation of digestive enzymes by the administration of feed additives to rainbow trout,Oncorhynchus mykiss Walbaum

The aim of this study was to assess the metabolic and digestive enzyme activities in rainbow trout, Oncorhynchus mykiss Walbaum fed dietary supplements. The biometric indices and the following enzymes activities were measured: acid protease and pepsin from the stomach homogenates, alkaline phosphatase (AP) from the small intestine and the brush border membrane, total proteolytic enzymes and trypsin from the small intestine and liver/pancreas. Dietary garlic induced a higher pepsin activity in the stomach than lipopolysaccharide (LPS) and ginger. Conversely, dietary ginger and LPS showed a significant difference (P > 0.05) in acid protease activity compared to the controls. There was not any significant difference observed between treated groups and the controls for hepato‐pancreas proteolytic enzyme activity, except for trypsin. AP activity increased significantly with dietary garlic, ginger and LPS compared to the controls.     

The interaction among age,thermal acclimation and growth rate in determining muscle metabolic capacities and tissue masses in the threespine stickleback,Gasterosteus aculeatus

Thermal acclimation may directly modify muscle metabolic capacities, or may modify them indirectly via effects upon physiological processes such as growth, reproduction or senescence. To evaluate these interacting effects, we examined the influence of thermal acclimation and acclimatization upon muscle metabolic capacities and tissue masses in 1 + stickleback, Gasterosteus aculeatus, in which confounding interactions between temperature and senescense should be absent. Furthermore, we examined the influence of thermal acclimation upon individual growth rate, muscle enzyme levels and tissue masses in 2 + stickleback sampled at the beginning of their final reproductive season. For 1 + stickleback, cold acclimation more than doubles mitochondrial enzyme levels in the axial muscle. Thermal acclimation did not change the condition of 1 + stickleback at feeding levels which could not maintain the condition of 2+ stickleback. Compensatory metabolic responses to temperature were not apparent in field acclimatized 1 + stickleback. The growth rate of 2 + stickleback was markedly affected by temperature: warm-acclimated fish generally lost mass even at very high levels of feeding (up to 78 enchytraid worms per day) while cold-acclimated fish gained mass. This suggests that warm temperatures accelerate the senescence of 2 + stickleback. Generally, muscle enzyme activities increased with growth rate. In axial muscle, the relationships between CS activity and growth rate differed with acclimation temperature. Independent of the influence of growth rate, CS activities were consistently higher in cold- than warm-acclimated 2 + stickleback, suggesting compensatory increases of CS activity with cold acclimation. Corresponding author; This paper is dedicated to the memory of Gerry J. FitzGerald who passed away on March 14, 1994.     

Effects of dissolved oxygen on glycolytic enzyme specific activities in liver and skeletal muscle of Fundulus heteroclitus

Many aquatic habitats are characterized by variable concentrations of dissolved oxygen (DO), and fish that occur in these habitats respond to changes in DO through behavioral, physiological, and biochemical adjustments. The goal of the present study was to measure the effects of an ecologically relevant range of DO treatments, from severe hypoxia to moderate hyperoxia, on the maximal activities of nine glycolytic enzymes during chronic exposure of the mummichog, Fundulus heteroclitus. Over the 28?days of exposure period, specific activity was significantly affected by DO for three enzymes in liver and one enzyme in white skeletal muscle, although at specific times of exposure three other muscle enzymes were affected by DO. In general, exposure of fish to severe hypoxia led to higher specific activities in liver, but lower specific activities in skeletal muscle. Exposure to hyperoxia did not elicit changes in enzyme specific activities in either tissue. Surprisingly, exposure duration had strong effects on glycolytic enzyme specific activities in both liver and white skeletal muscle, with specific activities increasing with exposure duration regardless of DO treatment. The results demonstrate that the effects of DO on enzyme specific activities were restricted to a subset of the glycolytic enzymes in liver and white skeletal muscle of F. heteroclitus and that the directions of the changes were opposite in these two tissues. These observations suggest that the mechanisms resulting in these alterations are enzyme- and tissue specific, rather than applying uniformly to all enzymes within the glycolytic pathway.     

Possible implication of metalloproteinases in post-mortem tenderization of fish muscle

ABSTRACT: It is suspected that the proteolytic breakdown of extracellular matrix proteins is responsible for the postmortem tenderization of fish muscle during chilled storage. In order to identify the type(s) of proteinases involved in this phenomenon, the effect of proteinase inhibitors, EDTA (ethylenediamine tetraacetic acid), 1,10-phenanthroline, ρ-APMSF [(ρ-amidinophenyl) methanesulfonyl fluoride hydrochloride] and E-64 [ L - trans -epoxysuccinyl-leucylamido-(4-guanidinobutane)] on tenderization was investigated by using Japanese flounder. Proteinase inhibitor solution was injected into a blood vessel in a caudal portion of live flounder and the firmness of muscle was then evaluated as a shear force value at 0 h and 6 h after death. Metalloproteinase inhibitors, EDTA and 1,10-phenanthroline, significantly suppressed postmortem tenderization. These findings suggest that metalloproteinases are candidates for proteinases involved in the postmortem tenderization of fish muscles. Although not significantly, p -APMSF, a serine proteinase inhibitor, partially suppressed muscle tenderization, which suggests that serine proteinases are also implicated in postmortem tenderization. A cysteine proteinase inhibitor, E-64, showed no effect, suggesting that cysteine proteinases are not involved.     

Diurnal variation of tryptic activity in larval stage and development of proteolytic enzyme activities of malabar grouper (Epinephelus malabaricus) after hatching

Diurnal variation in tryptic activity and developmental changes in proteolytic enzyme activities of malabar grouper larvae (Epinephelus malabaricus) were examined. Five different groups were prepared for the experiment of diurnal variation of tryptic activity in larvae: larvae were fed Thai-type rotifers Brachionus rotundiformis from the time of mouth opening, fed rotifers from 6 h after mouth opening, 12 h, 24 h and not fed rotifers (starved control). The experimental tanks were placed in temperature-controlled baths at 28 °C under 24 h light. Developmental changes in proteolytic activity of trypsin and pepsin-like enzyme were measured from hatching to 57 days after hatching (DAH).

The tryptic activity of all fed groups showed the same pattern, and the diurnal variation of tryptic activity was clearly observed from 3 to 6 DAH. The highest tryptic activities were found at 19:00, and the activities were lowest from 01:00 to 07:00. In contrast, that of non-fed larvae was low compared to the fed groups, however the diurnal variation of tryptic activity was shown same tendency to the fed groups. Interestingly, both groups (fed and non-fed) were exhibited a circadian rhythm under the 24 h light conditions and delaying of first-feeding. Tryptic activity of larvae notably increased from 40 to 45 DAH and markedly decreased at 52 DAH. In contrast to the tryptic activity, that of pepsin-like enzyme clearly increased from 47 to 51 DAH. The results suggest that a functional change of protein digestion occurs from 40 to 50 DAH related with metamorphosis in malabar grouper. These results could contribute to determining appropriate feeding schedules, such as feeding time, frequency and optimal time to change food items, in mass-scale production of the present species.     

Host-parasite interaction of the myxosporeans Kudoa paniformis Kabata & Whitaker, 1981 and Kudoa thyrsites (Gilchrist, 1924) in the muscle of Pacific whiting, Merluccius productus (Ayres): an ultrastructural study

Abstract. Kudoa paniformis and Kudoa thyrsites infect the muscle of Pacific whiting, Merluccius productus , and K. paniformis is known to cause a softened muscle texture after death of the fish. Ultrastructural studies were conducted on the host-parasite interaction between Pacific whiting and these myxosporeans at two stages of their infection: (1) unencapsulated plasmodia that contained mature and developing spores, and (2) encapsulated plasmodia that contained deteriorating spores. Unencapsulated plasmodia of both species had a microvillar border that was closely associated with the host muscle. Species-specific characteristics included the presence of multi-vesicular bodies in plasmodia of K. paniformis whereas the cytoplasm of K. thyrsites plasmodia contained an array of vesicles, microfilaments and microtubules. Differences in the predominant organelles of the plasmodia may be related to the production of different proteolytic enzymes by each species. A host response became apparent only after a plasmodium had replaced the width of an infected muscle fibre. At that time, the plasmodia of both species became encapsulated by a wall of fibroblast-like cells that were connected by numerous desmosomes. Occasional phagocytes were located at the edge of these capsules. Ultrastructural evidence suggests that the encapsulated plasmodia were not proteolytically active. Melanin deposition did not occur. However, encapsulated plasmodia with deteriorating spores contained electron-dense breakdown products.     

Partial purification and characterization of extracellular metalloproteases with caseinolytic, aminopeptidolytic and collagenolytic activities from Vibrio anguillarum

Abstract. Proteases produced by Vibrio anguillarum were isolated from culture supernatant by ultrafiltration, gel chromatography and ion exchange chromatography. The enzyme(s) were shown to be collagenolytic when assayed with native collagen substrates. In addition, the enzyme(s) hydrolysed azocasein, azocollagen, the collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg and the aminopeptidase substrate L-Leu-pNA effectively. Separation of the proteases by Mono Q ion exchange chromatography and native polyacrylamide gel electrophoresis revealed four distinct protein bands containing caseinase activity. However, only two of the bands showed aminopeptidase activity. The aminopeptidase activities could be separated from the caseinase activities by isoelectric focusing. Secreted proteases of different serotypcs of V. anguillarum showed a heterogeneous caseinolytic pattern. The molecular mass of the major enzyme was estimated at 35kDa as determined by its mobility on SDS-polyacrylamide gels. Serine protease inhibitors like PMSF, TPCK, TLCK and benzamidine had no inhibitory effects on the proteolytic activity when tested with azocasein as substrate. However, the enzyme was strongly inhibited by metal chelators like EDTA and 1, 10-phenanthroline. Also, normal salmon scrum and purified α₂-macroglobulin from salmon serum strongly inhibited the caseinolytic activity of the enzyme.